Bispecific antibodies for cancer therapy

Bispecific antibodies for cancer therapy
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   1 Current Opinion in Drug Discovery & Development 2009 12 (2): © Thomson Reuters (Scientific) Ltd ISSN 1367-6733 Bispecific antibodies for cancer therapy Patrick Chames* & Daniel Baty Address Institut de Biologie Structurale et Microbiologie, Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS UPR 9027, GDR 2352, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France Email: *To whom correspondence should be addressed The 2 last decades have seen the emergence of monoclonal antibodies as therapeutics. Nine monoclonal antibodies have been approved for cancer therapy. However the efficiency of these molecules is far from optimal, and antibody engineering is actively used to improve them. Because of their ability to simultaneously bind two different antigens, bispecific antibodies are unique, and their wide potential as retargeting reagent has been demonstrated over the years. However their use as therapeutics has been restrained by manufacturing challenges. Several new recombinant formats have changed the situation. Innovative molecules have led to impressive preclinical and clinical results, and hold great promise. This review presents an overview of the most  promising candidates. Keywords  Bispecific, cancer therapy, immunotherapy, retargeting, single domain antibodies Abbreviations ADCC  antibody-dependent cell-mediated cytotoxicity, bsAb  bispecific antibody, BiTE  bispecific T-cell engager, dAb  domain antibody, Db  diabody, DC  dendritic cell, FcR   Fc receptor, IFN  interferon, Ig  immunoglobulin, IL  interleukin,  PBMC  peripheral blood mononuclear cell, scFv  single chain Fv, TaFv  tandem scFv   2 Introduction Monoclonal antibodies (mAbs) are endowed with exquisite specificities, and since their discovery in 1975, they have raised many expectations for the development of novel treatments, particularly in cancer therapy [1]. However, all of these molecules had to be extensively optimized and engineered before they were able to deliver approved therapeutic molecules [2]. Nine mAbs are currently approved for cancer therapy, and hundreds are at different stages of clinical development [3]. Despite the enthusiasm for developing mAbs for therapy, most mAbs show limited efficacy, and so there is great potential for further improvement in this research area. Most mAbs function either by directly blocking a ligand or a receptor, sometimes leading to apoptosis, or as an adaptor molecule capable of recruiting effector cells from the immune system through interaction between their Fc portion and various Fc receptor (FcR) bearing cells. Consequently, considerable efforts are spent in the optimization of the Fc/FcR interaction [4]. Bispecific antibodies: Concepts, formats and limitations   However, as hypothesized in 1985, one solution for many mAb shortcomings might be the creation of bispecific antibodies (bsAbs), which would be capable of the simultaneous binding of two different targets and thus also capable of delivering a great variety of payloads to cancer cells [5]. BsAbs, however, do not exist in nature. Despite numerous attempts and various proposed formats, the production BsAbs in significant quantities remains, to a certain extent, a challenge. Three main approaches to the large-scale production of bsAbs have been described. The first was the quadroma technology, involving a somatic fusion of two different hybridoma cell lines [6]. Because of the random pairing of the two heavy and two lights chains of the antibody, the use of sophisticated purification procedures to isolate the bsAb was required. This inefficient, time- and resource-consuming approach led to the production of small amounts of bsAb that, although were sufficient for characterization, were insufficient for clinical requirements. Numerous studies have demonstrated the potential of such bsAbs but have also highlighted important shortcomings, including the high immunogenicity of such murine molecules and a high toxicity, which was attributed to the presence of an Fc portion capable of stimulating various FcR-bearing cells [7]. As a consequence, quadromas are no longer favored, with one notable exception: Lindhofer et al   demonstrated that the fusion of a murine immunoglobulin (Ig)G2a-producing hybridoma and a rat IgG2b-producing hybridoma may lead to preferential heavy/light chain pairing and easy separation of the homo- and heterodimers of heavy chains [8]. This approach can thus efficiently produce bispecific forms of whole IgG. Moreover, the resulting hybrid Fc portion of the resulting antibody has been shown to efficiently bind to activating human FcRs [9]. These molecules, called triomabs, are thus considered to be trispecific, and have produced exciting results in clinical trials (see below) [10]. The second approach that was developed to produce bsAbs is based on the use of heterobifunctional chemical reagents. The three major problems associated with chemical cross-linking are: (i) product homogeneity,   3 leading to poor yields and complicated purification steps; (ii) possible inactivation of the molecules or poor stability; and (iii) toxicity associated with the presence of Fc portions. Nevertheless, such molecules, such as CD20Bi and Her2Bi, have been evaluated in the clinic [11]. To decrease toxicity, F(ab') can be produced by enzymatic digestion and mild reduction of the two parental IgGs, followed by heterodimerization via thiol-reactive heterobifunctional reagents. Such artificial bispecific F(ab')2 have been extensively studied, and some molecules have reached the clinic, although none of them have been approved so far. [12]. The implementation of recombinant antibody techniques has revolutionized the bsAb field, leading to a third approach. More precisely the ability to use single-chain FV (scFv) fragments (antibody variable domains linked by a flexible peptide linker) has been a source of inspiration [13]. Two formats have been intensively studied. Tandem scFvs (TaFv) consist of two scFv fragments linked via an extra peptide linker. This 50-kDa molecule, which is difficult to produce in Escherichia coli  , is well expressed by mammalian cells and is expected to confer a good flexibility to each scFv fragment. This format has been used to create bispecific T-cell engager molecules (BiTE), which are extremely potent bsAbs that have shown impressive results in clinical trials [14]. By reducing the length of the peptide linker between variable domains so that the domains cannot assemble, it is possible to force the pairing of domains from two different polypeptides, leading to compact bsAbs called diabodies (Dbs) [15]. These molecules can be expressed at high yields in bacteria, and have been shown by crystallography experiments to adopt several conformations [16]. The Db format has been improved further by the addition of an extra peptide linker between the two polypeptides in order to further decrease the number of homodimers formed, leading to a fragment called a single chain diabody (scDb) [1]. Numerous studies have demonstrated the potency of these formats, although the reduced flexibility of the two binding sites might prove to be a limitation in some cases. In addition to TaFv and Db, several other scFv-based formats have been proposed which use various heterodimerization motifs including the human CH1/C !  domains and CH3 domain pairs that have been elegantly engineered to favor heterodimerization [17]. More recently, an approach called Dock-and-Lock was used to create trivalent bispecific molecules [18]; this method relies on the natural interaction between the 44 C-terminal residues of the regulatory unit of human protein kinase A, which spontaneously dimerize and bind to a 17-residue motif derived from the A-kinase anchoring protein. This trimerization motif has been stabilized by the introduction of cysteine residues into both domains, leading to the formation of covalent disulfide bonds. Such 150-kDa trivalent molecules have been successfully used for pretargeting strategies (see below) [18]. Recently, a new format consisting of fusions of an extra variable domain to each N-terminus of an IgG (ie, VH1-VH2-CH1-Fc and VL1-VL2-CL) was described [19]. The resulting bispecific molecules, called dual-variable-domain antibodies, have been shown to fold correctly and to exhibit pharmacokinetics similar to those of the   4 parental mAbs [19]. Steric constraints have to be expected for one of the binding sites, although this antibody format has been shown to successfully target soluble targets. Finally, recent years have seen the emergence of domain antibodies (dAbs), which were first engineered from mouse VH domains [20], and later from human variable domains [21]. DAbs are also found in nature in some isotypes of camelids and sharks [22,23]. These fragments are compact, extremely stable, and do not need domain pairing, and so represent ideal building units for the generation of more complex molecules including multispecific or multivalent molecules. For example, bispecific tandem dAbs can be produced efficiently using flexible linkers and such molecules have demonstrated excellent production yields and stability [24]. Interestingly, their modularity has been used to create a linker-free bsAb by directly fusing two llama-derived dAbs to a human CH1/C !  heterodimerization motif, thereby creating a Fab-like bispecific antibody fragment termed bsFab [Cornillon, A. et al  , unpublished]. These bispecific molecules are easily produced, extremely stable, and do not suffer from the usual limitations experienced by linker-based molecules, including production issues, poor stability, aggregation that can favor immunogenicity, and proteolytic degradation. Pharmacokinetics of bispecific antibodies Several of the methods for producing bsAbs described above generate bispecific antibody fragments of a small size (eg, 30 kDa for TaFv). Although a small size is clearly an advantage in terms of tumor penetration, it is also a potential problem because small molecules administered through intravenous injection are rapidly eliminated from the circulation by renal clearance. Moreover, several bsAb formats do not possess an Fc portion, and thus do not bind to the neonatal Fc receptor (FcRn), which is a receptor expressed by endothelial cells and responsible for the very long serum half-life of IgGs [25]. Several solutions have been designed to overcome the rapid clearance of of bsAbs. Addition of PEG to bsAbs has been shown to improve the serum half-life [26], but this often alters the binding affinities of bsAbs [27], which might affect tumor targeting. Two other methodologies designed to reduce the clearance of bsAbs are based on the interaction between human serum albumin (HSA) and FcRn. HSA, as is the case with IgG, enables recycling of the bsAb by endocytosis, thereby leading to reduced renal clearance and extended serum half-life. Indeed, a direct fusion of a bsAb to HSA can significantly improve its kinetics, as has been demonstrated for anti-CEA x CD3 Dbs [28]. The fusion of a Fab fragment to HSA-binding peptides has been shown to increase the serum half-life of the Fab fragment by 10- to 15-fold, leading to superior in vivo  efficacy of the fragment compared to the full IgG [29]. This general strategy has not yet been applied to bsAb but should be applicable with success to TaFv and Db. Similarly, small dAbs (llama and human) that bind strongly to serum albumin have been isolated. These dAbs have an extended serum half-life matching that of serum albumin itself [30], and can be fused to other antibody fragments to create bsAbs with favorable kinetics [31].   5 New strategies enabled by bispecific antibodies (Sub) Dual targeting The inhibition of signaling pathways is one of the main modes of action of therapeutic antibodies. Combinations of small-molecule-based treatments can lead to additive or even synergistic effects [32], block redundant signaling pathways and minimize the possibility of escape from therapy, but are often not adopted in the clinic because of issues with high toxicity. However, human or humanized mAbs are often very well tolerated and are thus very good candidates for combination therapies. Consequently, several companies are actively testing combinations of mAbs, including Genentech Inc with trastuzumab (anti-Her2) plus bevacizumab (anti-VEGF), and Immunomedics Inc with epratuzumab (anti-CD22) plus rituximab (anti-CD20) [33]. However, the combination of therapeutic antibodies faces several limitations, including intellectual property issues and the high costs involved in research, manufacturing and regulatory affairs. An elegant answer to these difficulties might be the simultaneous inactivation of two targets by a single (bispecific) antibody, as exemplified by two studies [34,35] that successfully simultaneously targeted epidermal growth factor (EGF) and insulin-like growth factor (IGF) receptors, demonstrating that simultaneous targeting is more efficient than monotherapies against the same targets. (Sub) T-cell retargeting Perhaps the most obvious applications of bsAb are in T-cell retargeting. Cytotoxic T-cells are considered the most potent killer cells in the immune system. These T-cells are abundant, can efficiently proliferate upon activation, can kill multiple times [36], and can efficiently infiltrate tumors but do not express Fc "  receptors. The concept of applying the novel bsAb methodology to enable T-cells to kill tumor cells more potently emerged in the 1980s [5]. BsAbs directed against both a tumor marker and CD3 have the potential to redirect and activate any circulating T-cells against tumor cells. However, T-cells have a major drawback. Without a secondary signal provided by the interaction between CD28 and one of its ligands, such as B7, T-cells are not fully activated and may even become anergic [37]. The first anti-CD3 bsAbs were therefore applied in combination with anti-CD28 antibodies, leading to mixed results [38]. Other alternatives are being explored, such as the massive ex vivo expansion (> 300 x 10 9 ) and activation of patient's T-cells using low concentrations of anti-CD3 and interleukin (IL)-2, followed by reinjection of these polyclonally activated T-cells decorated with an anti-CD3 bsAb [11]. Interestingly, activated T-cells armed with Her2/neu bsAbs have been demonstrated to be resistant to activation-induced cell death and are able to proliferate and kill tumor cells repeatedly [39]. Surprisingly, a small recombinant bsAb format appears to bypass the need for costimulation. Baeuerle and collaborators are developing a murine TaFv BiTE [40]. An anti-CD19 x CD3 BiTE exhibited very efficient
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