BioDrugs https://doi.org/10.1007/s40259-018-0299-9 REVIEW ARTICLE Expanding the Boundaries of Biotherapeutics with Bispecifc Antibodies Bushra Husain1 · Diego Ellerman1 © The Author(s) 2018 Abstract Bispecifc antibodies have moved from being an academic curiosity with therapeutic promise to reality, with two molecules being currently commercialized (Hemlibra­ ® and Blincyto­ ®) and many more in clinical trials. The success of bispecifc antibodies is mainly due to the continuously growing number of mechanisms of actions (MOA) they enable that are not accessible to monoclonal antibodies. One of the earliest MOA of bispecifc antibodies and currently the one with the largest number of clinical trials is the redirecting of the cytotoxic activity of T-cells for oncology applications, now extending its use in infective diseases. The use of bispecifc antibodies for crossing the blood–brain barrier is another important application because of its potential to advance the therapeutic options for neurological diseases. Another noteworthy application due to its growing trend is enabling a more tissue-specifc delivery or activity of antibodies. The diferent molecular solutions to the initial hurdles that limited the development of bispecifc antibodies have led to the current diverse set of bispecifc or multispecifc antibody formats that can be grouped into three main categories: IgG-like formats, antibody fragment-based formats, or appended IgG formats. The expanded applications of bispecifc antibodies come at the price of additional chal- lenges for clinical development. The rising complexity in their structure may increase the risk of immunogenicity and the multiple antigen specifcity complicates the selection of relevant species for safety assessment. Key Points 1 Introduction The success of bispecifc antibodies is mainly due to the Monoclonal antibodies (mAbs) have revolutionized thera- new biologies they enable. peutics for a broad range of disease indications [1]. In oncology, they can work through a variety of mechanisms Some promising applications (i.e., improved tissue deliv- of action [2]; for example, directly blocking the interaction ery) still ofer ample potential for further growth. between an angiogenic factor and its receptor [3], or blocking The additional binding specifcities present a more dif- the mitogenic interaction of two membrane tyrosine kinases fcult scenario for safety assessment experiments. [4]. In addition, mAbs can also induce antibody-dependent Immunogenicity in patients may provide a critical barrier cellular cytotoxicity (ADCC), antibody-dependent cellular for developing highly engineered antibody formats. phagocytosis (ADCP), and complement-mediated cytotoxic- ity (CMC) [5], or stimulate the patient’s immune system to produce an anti-tumor response [6]. Similar to the advance- ment in the therapeutics feld propelled by mAbs, the advent of bispecifc antibodies is greatly expanding the therapeu- tic potential of antibodies in general. Bispecifc antibodies are able to target two diferent epitopes simultaneously; these can either be on the same antigen or on two difer- ent antigens. In some applications, bispecifc antibodies do * Diego Ellerman not provide a functional advantage over the combination of [email protected] the corresponding monospecifc antibodies; however, they 1 Protein Chemistry Department, Genentech Inc., ofer a simpler and more cost-efective development path South San Francisco, CA 94080, USA than two individual drugs, which would require two separate Vol.:(0123456789) B. Husain, D. Ellerman manufacturing processes [7] as well as fling on the safety need to achieve freedom to operate without infringing on of each antibody component separately [8]. However, the intellectual property. In parallel with the increase in diver- biggest impact of bispecifc antibodies in the pharmaceutical sity in bispecifc antibody formats, the number of patents feld is due to the fact that they enable novel mechanisms of fled for bispecifc antibodies has been growing in recent action not accessible to monospecifc antibodies. This type years [15]. In spite of the variety in antibody formats, some of bispecifc antibodies has been referred to as ‘obligatory fundamental overarching criteria should be considered when bispecifcs’ [9] as the mechanisms of action depend on the developing a bispecifc antibody. They are discussed below. physical connection of the antibodies with the two distinct specifcities. The novel biologies enabled by bispecifc and multispecifc antibodies are the main thrust for their success, 3 Considerations for Selecting a Bispecifc and thus the central focus of this review. In the following Antibody Format sections, we briefy review the diferent antibody formats in the context of design considerations for the diferent applica- 3.1 Developability tions. More in-depth reviews on the structural aspects of the diferent antibody formats can be found in previous excel- Generation of therapeutic antibodies with a reasonable cost lent reviews [9–11]. In addition to an up-to-date review of requires that the engineered proteins express at high lev- the growing applications of bispecifc antibodies, we also els. This is perhaps the earliest critical attribute that novel discuss actual challenges and potential future hurdles for the platforms need to fulfll in order to be viable. Reduction of clinical advancement of bispecifc antibodies. the cost of goods is a consideration that is also driving the adoption of IgG-like formats compatible with expression in a single cell (some of these are discussed later in the text) 2 Origin and Evolution of Bispecifc or the exploration of processes that allow expression in a Antibody Formats single fermentor [16]. Another important aspect is the puri- fcation strategy, as a platform that requires a very complex The current success of bispecifc antibodies is only possible purifcation scheme would be disadvantageous over other because the initial limitation for their efcient production alternatives with a simpler process. Associated with purifca- has been overcome. The main problem for producing bispe- tion, the proper characterization of the desired product and cifc antibodies using wild-type IgG sequences was that the potential contaminant species is important to guarantee the random association of chains led to the combinatorial asso- robustness of the production process and the quality of the ciations of the two heavy and light chains, creating up to ten fnal product. Depending on the format, the potential forma- diferent products. Early attempts to produce bispecifc anti- tion of diferent unwanted species could be more difcult to bodies relied on the conjugation of antibody fragments or on detect and quantify, presenting a bigger challenge for analyt- the fusion of two diferent hybridomas to generate a quad- ical groups. For example, single-cell expression of a bispe- roma. These approaches were suitable for research purposes cifc IgG with two diferent heavy chains and two diferent but not for clinical applications as the source material was light chains, although it simplifes downstream process and not easily scalable [12]. The earliest engineering solution to reduces costs, may lead to the formation of contaminants the random association of the heavy chains without using with very similar biochemical properties to the intended linkers or chemical conjugation was the so-called ‘knobs- product and may require advanced methods for their detec- into-holes’ strategy, where two sets of sterically comple- tion [17]. The chemical and physical stability of the pro- mentary mutations in the CH3 domain alter the association tein is also important, as poor stability may compromise interface such that heterodimerization is greatly favored over the activity as well as increase the risk of immunogenicity. the formation of homodimers [13]. Another early strategy Physical stability has been a limitation, for example, for the to produce bispecifc antibodies and overcoming the initial development of some single-chain variable fragment (scFv)- limitations was the fusion of antibody fragments. The frst containing bispecifc antibody formats due to their intrinsic described fragment-base format was the so-called ‘diabody’ propensity to aggregate [18]. Several approaches have been [14], where two diferent chains with crossed-over variable developed to overcome this limitation, such as introducing domains (VHA-VLB/VLA-VHB) associate to form the bispe- a stabilizing disulfde bond, grafting the complementarity cifc molecule. As the benefts of bispecifc antibodies have determining regions (CDRs) onto a stable framework, CDR become clearer, these two initial strategies have evolved into engineering [19], or by swapping kappa and lambda frame- an ever-growing plethora of related formats [9, 10]. The work regions [20]. In one published example, the use of remarkable explosion in diversity of all bispecifc antibody scFvs with frameworks preselected for increased stability formats has been fueled not only by the pursuit of more translated into better biophysical properties for the corre- efcacious and/or more developable formats, but also by the sponding mAb-scFv bispecifc antibody [21]. Introducing Bispecifc Antibodies novel functions in an IgG usually requires signifcant modi- efector functions could be required for some applications; fcations of the IgG structure that often reduces the stability for example, antibodies against infectious agents mediat- of the resulting protein. For example, one