ReviewMechanism of action of therapeutic monoclonal antibodies: Promises and pitfalls of in vitro and in vivo assays
Highlights
► The major known mechanisms of action of therapeutic mAbs are summarized. ► The methods and problems with cell death and proliferation assays are reviewed. ► The assays and pitfalls of immune mediated activities of antibodies are reviewed. ► Whether in vitro and in vivo assays of antibodies predict clinical efficacy is discussed.
Introduction
Antibodies have been used as therapeutics already in the late 19th century in the form of patient or animal derived sera to treat infectious diseases. With the invention in the 1970’s of the method to produce monoclonal antibodies (mAbs), the idea of a magic bullet, in particular against tumour specific antigens in addition to infectious diseases, started to take a strong hold [1]. However the field required the contribution of molecular biology before a successful anti-tumour mAb, the anti-CD20 rituximab for the treatment of B-non Hodgkin’s lymphoma, reached the clinic [2]. The development of therapeutic mAbs has since progressed very fast, so that present antibody based therapeutics include unconjugated mAbs, antibody drug conjugates (ADC1), antibody based radioconjugates (ARC), bispecific antibodies (BsAb) recognising two different antigens, Ab fragments and Fc fusion proteins [3], [4]. The antibodies usually derive from mouse, rat or human sources, although recently exotic sources such as camel, lama and sharks have gained interest, due to the fact that these species produce IgG antibodies made of the sole dimerized heavy chain. Thus the antigen binding Fab fragments of these antibodies are natural single chain molecules that conveniently can be linked to other peptides [5]. The clinical use of antibodies has also been extended to other diseases than tumours and infections, in particular autoimmune and inflammatory diseases.
Thanks to the modular structure of IgGs, which are made of well defined, self assembling immunoglobulin domains that compose both the antigen binding sites (Fab) and immune modulating Fc region, antibodies and antibody fragments can be relatively easily modified by genetic engineering to generate novel chimeric molecules or antibody fragments linked to each other or to other molecules [6], [7]. This has led to the production of a plethora of different antibody based compounds, showing one or more functional activities in addition to that of antigen binding, depending upon the desired use of the final product.
All these elements mean that for any given antigen, a virtually unlimited number of different antibody formats can be created in the laboratory, designed to have optimal functional activity according to the intended use of the drug. Thus, antibodies have been designed to recognise for example a tumour antigen and to have optimised FcγRIII binding for enhanced antibody dependent cytotoxicity (ADCC), with the hope of generating an effective molecule to control the tumour in vivo. Differently, a BsAb can be planned to bind to two different cytokines or two pro-angiogenic factors, in order to control inflammation or tumour angiogenesis, respectively. Other bispecifics are designed to bind to and bring together a tumour cell and an effector cell, such as a cytotoxic T cell or NK cell, in order to augment specific tumour killing activity, by the so-called “redirected” killing effect. Finally antibodies or their fragments can be linked to different drugs or toxins to create a variety of ADCs.
Thus the plethora of different antibody formats and functional capabilities bring new challenges to the laboratories [3], [4]. Indeed these molecules are not small chemical drugs, but complex biological molecules, which have multiple and often complex mechanism of actions, as we will see below, and measurement of these biological activities require rather specialised techniques. The main question is therefore how to select at a preclinical stage, as early as possible, the best antibody based drug candidates, on the basis of in vitro activity and in vivo animal models.
In this review we will summarise the major assays that have been employed to assess antibody function in vitro and in vivo in small laboratory animals. We will point out to some of the pitfalls that are linked to these assays and suggest some ways to overcome at least in part these limitations. We will restrict the discussion on anti-tumour antibodies, since these can rely on both direct effects, via Fab binding to cell surface tumour antigens, and immune mediated effects. In contrast, antibodies directed against soluble molecules such as angiogenic factors and cytokines have usually only a neutralising activity, which is relatively more simple to measure, a least in vitro, and will therefore not be further discussed here. Furthermore we will not discuss the methods of measuring antibody binding, PK or PD, even though these obviously strongly impact final antibody efficacy in vivo.
Section snippets
Direct effects of antibodies: inhibition of proliferation and induction of cell death
Whereas the Fc portion of antibodies activate immune mediated mechanisms, the Fab portion of antibodies have antigen binding activity that can therefore either neutralise the antigen or activate signalling pathways within the target cells. Indeed several unconjugated therapeutic antibodies have been designed to bind to growth factor receptors or cell surface signalling molecules and are able to either inhibit proliferation or induce direct cell death. This is the case for example of daclizumab,
In vivo assay
Therapeutic antibodies are particularly difficult to study in a meaningful way in mice. First of all, the antibodies are often specific for the human target and may not recognise the murine antigen. In other cases it may recognise murine antigen but there may be differences in affinity, which may have an impact on activity. In the case of lack of recognition, the most commonly used models are xenograft, i.e. the inoculation of a human tumour cells, either as cell lines or primary tumour samples
Conclusions
In vitro and in vivo assays of antibody-based therapeutics, in spite of the limitations associated with some of these assays as illustrated above, have been fundamental towards defining the major mechanisms of action and resistance of these drugs. This has contributed to the design of a plethora of novel more effective immunotherapeutics, amongst which are several unconjugated mAbs, ADCs and bispecific molecules with a very promising clinical activity [4], [30], [62]. Nonetheless, we would like
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