Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting,December 11–15, 2016, San Diego,CA

Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. “Antibodies to watch in 2017” and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.     

Antibody engineering and therapeutics conference: The annual meeting of the antibody society,Huntington Beach,CA, December 7–11, 2014

The 25^th anniversary of the Antibody Engineering & Therapeutics Conference, the Annual Meeting of The Antibody Society, will be held in Huntington Beach, CA, December 7–11, 2014. Organized by IBC Life Sciences, the event will celebrate past successes, educate participants on current activities and offer a vision of future progress in the field. Keynote addresses will be given by academic and industry experts Douglas Lauffenburger (Massachusetts Institute of Technology), Ira Pastan (National Cancer Institute), James Wells (University of California, San Francisco), Ian Tomlinson (GlaxoSmithKline) and Anthony Rees (Rees Consulting AB and Emeritus Professor, University of Bath). These speakers will provide updates of their work, placed in the context of the substantial growth of the industry over the past 25 years.     

An optimized antibody-single-chain TRAIL fusion protein for cancer therapy

Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.     

Inhibition of Cancer Cell Adhesion,Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFβ) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFβ. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvβ1, αvβ3 and αvβ5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.     

Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single‐chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti‐PCNA scFvs were screened in vitro after being tagged with dimeric glutathione‐S‐transferase. Anti‐PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error‐prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C‐terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide‐conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody‐based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd.     

Improving biophysical properties of a bispecific antibody scaffold to aid developability

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic.     

A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells

To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced k_off, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30⁺ target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin’s lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells.     

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.     

Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing C_H1 with Cα and C_L with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the C_H1/C_L and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.     

Biodistribution studies with tumor-targeting bispecific antibodies reveal selective accumulation at the tumor site

Bispecific antibodies are proteins that bind two different antigens and may retarget immune cells with a binding moiety specific for a leukocyte marker. A binding event in blood could in principle prevent antibody extravasation and accumulation at the site of disease. In this study, we produced and characterized two tetravalent bispecific antibodies that bind with high affinity to the alternatively-spliced EDB domain of fibronectin, a tumor-associated antigen. The bispecific antibodies simultaneously engaged the cognate antigens (murine T cell co-receptor CD3 and hen egg lysozyme) and selectively accumulated on murine tumors in vivo. The results, which were in agreement with predictions based on pharmacokinetic modeling and antibody binding characteristics, confirmed that bispecific antibodies can reach abluminal targets without being blocked by peripheral blood leukocytes.