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Critical Reviews in Oncology/Hematology 92 (2014) 153–165

Bispecific antibody platforms for cancer immunotherapy
Roeland Lameris a , Renée C.G. de Bruin a , Famke L. Schneiders a ,
Paul M.P. van Bergen en Henegouwen b , Henk M.W. Verheul a ,
Tanja D. de Gruijl a , Hans J. van der Vliet a,∗
b

a Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Accepted 8 August 2014

Contents
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Currently available bispecific antibody platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Trifunctional hybrid antibodies (Triomab) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1. Catumaxomab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2. Ertumaxomab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3. FBTA05 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Single chain variable fragment (scFv) based platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Tandem scFv (TaFv). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Bispecific diabodies (bsDb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Other bispecific antibody based platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Limitations of the type of bsAb constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Immunogenicity of bispecific antibody constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Size of the construct: Pay off between circulation half-life time and tumor penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Antibody construct stability and manufacturing difficulties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perspectives related to the bsAb construct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Targeting specific lymphocyte subsets to maximize efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. ␥␦ T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Invariant natural killer T-cells (iNKT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Natural killer cells (NK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract
Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel
bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical
studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation

Abbreviations: VH , variable heavy chain domain; VL , variable light chain domain; mAb, monoclonal antibody; bsAb, bispecific antibody; scFv, single
chain variable fragment; Triomab, trifunctional hybrid antibody; TaFv, tandem single chain variable fragment; BiTE, bispecific T-cell engager; bsDb, bispecific
diabody; scDb, single chain diabody; DART, dual affinity retargeting molecule; VHH, variable domain of heavy chain-only Ab.
∗ Corresponding author. Tel.: +31 20 4441295; fax: +31 20 4444355.
E-mail address: jj.vandervliet@vumc.nl (H.J. van der Vliet).
http://dx.doi.org/10.1016/j.critrevonc.2014.08.003
1040-8428/© 2014 Elsevier Ireland Ltd. All rights reserved.

Of note. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. interactions of the Fc-portion with Fc␥receptors (Fc␥R) expressed by effector cells (e. binding to inhibitory Fc␥R on immune cells may result in internalization of the mAb-tumor target-Fc␥R complex reducing its therapeutic efficacy [4].) in a syngeneic C57BL/6 and BALB/c mouse model using (human) EpCAM expressing B16-melanoma and A20-lymphoma cells.154 R. a full-size functional mAb was engineered and termed Triomab (Trion pharma Inc. macrophages and ␥␦ T-cells) may result in CDC and ADCC and subsequent antitumor cytotoxicity and/or phagocytosis.8. hybrid hybridomas or quadromas). Following binding of the mAb to its tumor target. trastuzumab (anti-human-epidermal-growth-factor receptor 2 (Her2)) and cetuximab (anti-epidermal-growth-factor receptor (EGFR)) [1]. natural killer (NK) cells. ertumaxomab and FBTA05 which will be discussed below. ADCC has been demonstrated to significantly enhance the efficacy of various mAbs. Keywords: Bi-specific antibodies. Here. Catumaxomab Catumaxomab. By combining the halves of two distinct antibodies. dendritic cells (DC) and macrophages).and accessory cells (e. These data resulted in the development and clinical evaluation of a number of Triomabs. Lameris et al. clinical responses may be influenced by Fc␥R polymorphisms [2].1. as a low production yield owing to random association of heavy/light chains and immunogenicity caused by human anti-mouse/rat antibodies (HAMA/HARA) severely hampered clinical applicability [5]. the hybrid mouse/rat Fc-portion was able to activate Fc␥R+ accessory cells [6. we will review the main bsAb formats that are currently being developed for tumor retargeting of immune cells and discuss thus far obtained (pre-)clinical results and encountered limitations.e. Preclinical studies with a Triomab targeting epithelial cell adhesion molecule (EpCAM). hampered by circulating immunoglobulins (Ig) competing for Fc␥R binding spots on immune effector cells. Furthermore. Selective depletion of both CD4+ and CD8+ T-cells resulted in a marked loss of tumor protection and survival. Anti-cancer therapy 1.9]. All rights reserved. this platform offered a solution to the random association of heavy/light chains observed in classic quadroma technology. In the Triomab treated group a 100% survival rate. Although data are inconsistent. including catumaxomab.9]. Despite some clinical effects. we will elaborate on potential ways to take the bsAb approach forward. T-cell activation was complemented by the induction of T-cell mediated tumor lysis. Dual specific retargeting. The first bsAbs were engineered by either chemical crosslinking or exchange of different heavy chains as a result of fusion of two hybridoma cell lines (i.p. established a maximum . Furthermore. lack several of the above described limitations and can potentially induce a more powerful anti-tumor immune response. including rituximab (anti-CD20). demonstrated a significantly improved tumor cell elimination compared with the simultaneous administration of both parental antibodies. Due to species-preferential heavy/light chain pairing random association was greatly reduced.g. Furthermore. a tumor-specific mouse IgG2a and a CD3specific rat IgG2b.v. 2. 2.7]. expressed by the majority of epithelial cancers. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 and the induction of anti-tumor responses. capable of binding two targets simultaneously. Interestingly.g. cytotoxic cytokine release. was the first Triomab studied in patients. 1b).1. respectively. NK cells. demonstrated redirection and activation of T. It was not until 1995 that bsAb research was sparked again by the introduction of fused mouse-rat hybrid antibodies and tandem single-chain variable fragments (TaFv) [6. Immune effector cells. © 2014 Elsevier Ireland Ltd. Currently available bispecific antibody platforms Several clinically available therapeutic monoclonal antibodies (mAbs) can induce immune-mediated tumor cell killing through mechanisms that include complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Introduction 2. it is clear that mAbs still do not exploit the full potential of the immune system as effects are e. an anti-EpCAM-anti-CD3 Triomab. 1c). A phase-I trial of a single intravenous (i.1. only the animals injected with human EpCAM expressing tumors and Triomab treatment developed strong anti-EpCAM specific humoral immune responses [10]. and CD3 expressed by T-cells. Trifunctional hybrid antibodies (Triomab) Introduced in 1995. none of these bsAbs made it to advanced stage clinical trials. complete tumor eradication and protection against tumor rechallenge was reported.g. with additional evidence suggestive of epitope spreading.) dose of catumaxomab in patients with non-small cell lung cancer (NSCLC). we will discuss potential future developments that can be expected to take the bsAb approach successfully forward. The additive value of a functional Fc-portion was underscored by enhanced tumor protection in mice treated with a Triomab compared to a similar bsAb consisting of two chemically cross-linked fragment antigen binding (Fab) regions (F(ab)2 ) (Fig.) (Fig. and ADCC in the picomolar range [8. In vivo assessment of the Triomab injected intraperitoneally (i. and inadequate tumor-target penetration due to their relatively large size (∼150 kDa) [3]. In clinical series. Granting their therapeutic efficacy can in part be attributed to beneficial secondary immune effects. Bispecific Abs (bsAbs).

bispecific rat/mouse antibody.2. (g) scDb. 7 and 13 to patients with metastatic breast cancer induced strong inflammatory reactions with clinical responses in some patients. Variable heavy chain domains (VH ) are depicted in dark blue and dark orange. Within 6 weeks after the first dose. precluding approximately 5 therapeutic paracentesis procedures. Adverse effects (AE) were most commonly cytokine release-related (i. HAMA/HARA were observed in 25 and 31% of patients.p. bispecific diabody. compared to control patients catumaxomab resulted in a significant prolongation of puncture-free survival (median 11 vs 46 days. the number of CTC grade ≥3 toxicities and serious AE seemed to be dose related. an anti-Her2-anti-CD3 Triomab. In a randomized phase II/III study. 7 and 10 [12. increased levels of activated CD4+ and CD8+ T-cells as well as an elimination of CD133+ /EpCAM+ cancer stem cells (CSC) compared to both baseline levels and non-treated control patients [14].1 [16]. 1. nausea and vomiting). (d) scFv. Analyses of ascites demonstrated decreased levels of 2. manageable and generally reversible. administration of catumaxomab in patients with malignant ascites due to various EpCAM positive tumors. In a phase I trial.R. two chemically crosslinked fragment antigen binding (Fab) regions. treatment of malignant ascites in patients with EpCAM-positive carcinomas where standard therapy is not available or no longer feasible. A phase II trial was terminated prematurely due to changes in company development plans. was observed in the majority of patients (>70%) after the last treatment cycle and will be discussed in more detail in Section 3. (For interpretation of the references to color in this figure legend. Ertumaxomab Ertumaxomab.v. administration of 3 ascending doses (10–200 ␮g) of ertumaxomab on days 1. The severity and number of AEs were dose related.0%). single chain variable fragment. (h) DART. (j) bsVHH.1.e. the reader is referred to the web version of this article.) tolerated dose (5 ␮g with 40 mg dexamethasone) and reported favorable survival in several patients with advanced-stage disease surviving past 26 months. single chain diabody. i. (f) bsDb. (e) TaFv. (c) F(ab)2 . Importantly. 3. which strongly correlated with clinical endpoints. this only reached statistical significance in patients with gastric carcinoma (median 71 vs 44 days. Orange and blue indicate arms with different specificities.6%) [15]. dual affinity retargeting molecule. monoclonal antibody. More extensive studies have evaluated i. was completely ineffective [17]. (a) mAb. Of note. symptoms and signs related to ascites were significantly reduced. Reported serious AE classified to be treatment related included fully reversible severe hypotension (5. p = 0.2%) and gastric hemorrhage (0. catumaxomab was approved by the European Medicine Agency (EMA) for the i. systemic inflammatory response syndrome (5. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 155 vascular endothelial growth factor (VEGF). Likewise. Peptide linkers are shown as gray lines. (b) Triomab.0001) and an increased time to next paracentesis (median 13 vs 77 days). tandem single chain variable fragment. was demonstrated to initiate effective immune mediated cytotoxicity against Her2 expressing tumor cell lines in vitro and to efficiently lyse low Her2 expressing target cells where trastuzumab. p < 0.9%).6%) and lymphopenia (7.p. a mAb targeting Her2. infusions were given with an increasing dose (10–150 ␮g) on days 0. with dose limiting events being immune related. Based on these data. Although a positive trend was observed in improvement of median overall survival (72 (95% CI: 61–98) versus 68 days (95% CI: 49–81). pyrexia.9%). (i) Heavy chain-only antibody. respectively [18]. . aggravation of congestive heart failure (5. involving heavily pre-treated patients with symptomatic malignant ascites. Reported serious AE included ileus (3. Notably. variable light chain domains (VL ) are depicted in light blue and light orange.0313) [15].13]. HAMA/HARA development. bispecific variable domains of heavy chain-only Ab. Fig.9%) and. Lameris et al. None of the patients developed HAMA or HARA within 28 days after infusion [11]. Reported Common Toxicity Criteria (CTC) grade ≥3 AE considered to be treatment related included abdominal pain (9. CTC grade ≥3 toxicities included fully reversible lymphocytopenia (76%) and elevation of liver enzymes (47%).

g. Single chain variable fragment (scFv) based platforms Some of the mentioned limitations of whole antibodies can be overcome by reducing antibodies to their minimal binding domains. FBTA05 followed by donor lymphocyte infusion or peripheral blood stem cell transplantation showed a prompt but transient response in several patients [19]. 12 of which had molecular refractory disease to previous treatment [28]. These TaFv. 2. One type of TaFv. A multitude of other TaFvs is being evaluated pre-clinically. including small antibody fragments. FBTA05 FBTA05. Importantly. given as continuous infusion over 4 or 8 weeks. NCT01471782. has been extensively studied. 1e).8% and 8. demonstrated effective CD20+ lymphoma killing in vitro. carcinoembryonic antigen (CEA) and CD33. Video microscopy revealed that BiTEs allowed serial killing of target cells by engaged T-cells with complete elimination of target cells at effector-to-target ratios of 1:5.2. Similar results were reported in an interim analysis of a phase II trial in relapsed/refractory B-ALL. NCT01207388 and NCT01466179). an anti-CD20-anti-CD3 Triomab.and effector cells and the subsequent release of cytotoxic effector molecules (e. respectively (NCT00635596 and NCT01723475). termed bispecific T-cell engagers (BiTE) (Micromet Inc. which is 2–4 times lower than ratios needed for Triomabs [23]. Sixteen out of 20 patients became MRD-negative at the end of the first cycle. infusion at a dose of 15 ␮g/m2 /24 h every 6 weeks with responders being permitted to receive up to 3 consolidation cycles. Mitigating measures. a double-step dose increase and glucocorticoid administration. Seventeen out of 25 patients achieved CR or CR with partial hematological recovery [31]. 2.to femtomolar concentrations in the absence of co-stimulation [21. Complete (CR) and partial tumor responses (PR) were demonstrated upwards of a dose of 15 ␮g/m2 /24 h.156 R.27]. One such fragment. is derived from murine scFvs targeting human CD19 and CD3.1.2. Of interest. Most common AEs with blinatumomab included flu-like symptoms during the first days of treatment which resolved under ongoing infusion. Blinatumomab. while their small size (∼55–60 kDa) allows rapid tumor penetration [3]. TaFv constructs targeting EpCAM and prostate specific membrane antigen (PSMA) have entered phase I clinical trials in epithelial and prostate cancer. including constructs targeting EGFR [33]. an anti-prostate specific cell antigen (PSCA)-anti-CD3 TaFv . Bcell counts dropped below 1 cell/␮L and did not recover until after therapy cessation.2.22]. termed single-chain Fv (scFv). In this study the starting dose was reduced (5 ␮g/m2 /24 h in the first week) to prevent AE. activation occurred in a strictly target dependent fashion [25]. although completely reversible. respectively) presumably caused by released cytokines and frequently resulted in treatment interruption or discontinuation.3.1. included neurological AE (e. Tandem scFv (TaFv) By covalent bonding of two scFvs with a flexible peptide linker in a tandem orientation.6% of patients. A phase I/II study is ongoing (NCT01138579). Based on results in B-NHL.v. a phase II study was initiated in patients with persistent/relapsed minimal residual disease (MRD) positive B-cell acute lymphatic leukemia (B-ALL). Advances in genetic engineering have provided a vast amount of such antibody like constructs.1.1. Multiple trials designed to determine the optimal dose. seizures and encephalopathy in up to 4. is made by the association of the variable parts of heavy (VH ) and light (VL ) chains through a peptide linker (Fig. Patients were treated with a continuous 4-week i. perforin and granzyme B) [24]. Several clinical studies evaluated its efficacy in various types of Bcell NHL and leukemia. being the first and most advanced BiTE in clinical studies.2. In all 20 patients T-cells declined rapidly but recovered within days to above baseline levels.g. treatment schedule and clinical efficacy of blinatumomab are ongoing (NCT01741792. 2. including lower starting doses. More serious AEs. As T-cell activation depended on multivalent binding of BITEs due to their low affinity for CD3. Target cell lysis involved the formation of tight cytolytic synapses between target.32]. A pilot study involving patients with recurrent/refractory non-Hodgkin lymphoma (NHL) or chronic lymphatic leukemia (CLL) treated with i. continuous infusion was required to ensure sustained effective serum levels due to a very short serum half-life time of less than 2 h due to renal excretion [26. AEs correlated with peak levels of activated CD8+ and CD4+ T-cells and with increased serum cytokines levels. Long term follow-up (median 33 months) demonstrated a hematologic relapse-free survival in 12 out of 20 patients [29]. a bispecific TaFv can be formed (Fig. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 2. with a significant percentage of reappearing CD8+ and CD4+ T-cells transiently expressing the early activation marker CD69. Alternative TaFv constructs. Lameris et al. 1d) [20]. 2.2. made by fusing an anti-CD3 to an antitumor associated antigen (TAA) scFv via a 5 residue peptide linker (GGGGS). Also. cases of hemophagocytic lymphohistiocytosis (HLH) presumably caused by blinatumomab treatment have been described [26–30.) can effectively redirect and activate polyclonal T-cells in vitro resulting in a highly cytotoxic response at pico. The decline in B-cell counts most likely resulted from redirected cell lysis [30]. Commonly reported CTC grade ≥3 AE in the different studies included lymphopenia (up to 77%) and leukopenia (up to 47%).v. Blinatumomab. namely tandem scFv (TaFv) and bispecific diabodies (bsDb).1. were protective and allowed for treatment continuation or resumption [26–30]. Two formats based on scFvs have been studied extensively. It is expected that the flexible orientation between the two binding domains enhances simultaneous target ligation.

1g) [37–39]. This can. Similar results were reported for an anti-CD19-anti-CD3 [44. under appropriate redox conditions. heavy chain exchange to form full-length bsAb [51]. dimerization and aggregate formation. forming a single chain bsDb (scDb) (Fig.g. Based on identical recognition specificities and similar molecular masses.1. a reduced target celldissociation rate and improved storage stability. Lameris et al. at least in part.53].45] and an anti-EGFR-anti-CD3 bsDb [46]. to date only a few have been evaluated in preclinical studies [20. be prevented by introducing an additional peptide linker between the scFv.and rat-derived mAbs have been associated with short serum half-life time upon repeated administration due to the formation of neutralizing HAMA/HARA and the related susceptibility to trigger a cytokine release syndrome (CRS) in patients. 3. 2. Pre-clinical studies. differences between the DART and BiTE platform thus resulted from structural differences. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 was recently successfully humanized without compromising its in vitro and in vivo characteristics. where up to 89% of patients . with some apparently outperforming TaFv. in the level of fixation between the two opposing binding sites. The observed higher association rate and target affinity of DARTs may have resulted in prolonged intercellular contacts creating a more robust cytotoxic T-cell response [49]. Direct comparison of an anti-EGFR–anti-CD3 bsDb with its TaFv equivalent indicated an equal binding affinity.1. tetramers and tandem bsDbs can be formed [37. engineered human monospecific IgG1 and IgG2 subtypes that allow.50].57]. varying the VH -linker-VL order) can lead to superior in vivo results when compared to the TaFv equivalent. Their increased size may however decrease effective tumor penetration. with T-cell activation and proliferation occurring solely in a target-cell dependent manner in both formats. in vivo experiments showed higher serum retention compared to bsDbs [41]. by varying the length of the additional peptide linker in scDbs. addition fusion conjugates consisting of an anti-TAA coupled to an MHC class I molecule containing a selected antigenic peptide have been shown to be able to effectively redirect and activate T-cells in vivo [56. The DART molecule outperformed the BiTE molecule with respect to both the induction of B-cell lysis and stimulation of T-cell activation. which as an NKG2D ligand can be used for targeting NKG2D-expressing immune effector cells [54] and. These platforms are not further discussed as limited preclinical efficacy data and no clinical efficacy data are presently available.2. an increased stability can be achieved by introducing a disulfide bond between the C-terminus of each scFv.2. bsDbs are formed by non-covalent association of two scFvs. Other bispecific antibody based platforms Although a number of alternative bispecific constructs designed for tumor targeting of effector cells have been engineered. As the compact structure of bsDbs might hinder cross-linking. Notably. i.3. resulting in a Dual-Affinity ReTargeting (DART) molecule (Fig.g. it is interesting to note that rearrangement of the variable domains (e. a scFv fused to the N. The potency of DARTs was demonstrated in an in vitro side-by-side comparison of blinatumomab and a DART 157 bearing identical CD3 and CD19 Fv-domains. Though humanization is generally thought to limit anti-drug antibody (ADA) formation. dosing of Triomabs was limited by immunological AE and. do not form aggregates and demonstrate potent in vitro and in vivo activity [40]. 1h). though this in vivo superiority may also result from the enhanced stability of bsDbs at 37 ◦ C [48]. connected with a very short peptide linker precluding intra-chain domain interactions (Fig.R. While bsDbs yielded promising results in pre-clinical studies. but enhanced in vitro cytotoxicity in favor of the TaFv [47]. Alternatively. 2. Because individual chains are not covalently associated. Indeed. e. Efforts have also focused on adding a third binding site to TaFv. 1f). Immunogenicity of bispecific antibody constructs Historically. no clinical data have yet been reported. but are not limited to. HAMA/HARA formation may restrict repetition of treatment cycles. resulting in a bispecific hexavalent trimerbody [55].v. consisting of a VH and VL domain from different parent Abs. possibly due to their increased affinity and size (∼114 kDa) reducing renal clearance. VH and VL binding interactions remain a limiting factor in the stability of the molecule. a fusion protein consisting of a scFv targeting CEA and the human UL16 binding protein 2 (ULBP2). These include. compared to both an anti-CD16-anti-CD19 and an anti-CD16-CD33 TaFv [35].2. an anti-CD16-anti-CD33-anti-CD19 scTb demonstrated enhanced lysis of CD33-CD19-positive leukemic cells in vitro.41–43] with higher affinity. as was demonstrated by the anti-TNF-␣ mAb adalimumab. murine. Bispecific diabodies (bsDb) In contrast to TaFvs. as discussed. Furthermore. Humanization may prohibit HAMA associated AE [34]. thereby creating triple bodies (scTb).and C-terminus of a trimerizing scaffold domain. an anti-CD3 scFv fused to a high-affinity monoclonal T-cell receptor (mTCR) specific for a tumor-associated MHC-1 antigen (ImmTAC) [52. An anti-PSMA-anti-CD3 bsDb proved to be potent for CD3+ T-cell tumor targeting specifically inducing lysis of PSMA-expressing prostate cancer cells in vitro and inhibiting human prostate cancer growth in xenografted mice [42]. Indeed. DART molecules have an extended storage and serum stability. bsDbs have their variable domains orientated in an opposed fashion and are very rigid and small (∼60 kDa) [36]. 2. possibly resulting in disintegration. In. Limitations of the type of bsAb constructs 3. it cannot completely circumvent this.2.

In support of this. as with all whole mAbs. Both superior tumor penetration and target retention may result in a synergistic effect on tumor destruction. Concerns have been raised about ongoing T-cell activation during BiTE-treatment.63] thereby limiting the intended efficacy of tumor targeting of these immune cells. Despite these drawbacks.65]. indicating low immunogenicity [27.40. Similar reductions in cytotoxicity have been reported for other serum half-life time extending strategies [64. Of note. possibly due to steric hindrance. Size of the construct: Pay off between circulation half-life time and tumor penetration The Fc-domain of Abs enhances the serum half-life time (to >10 days) through neonatal-FcR mediated recycling by e. mammalian cells are often needed as a host.42. serum levels of small-sized bsAb constructs might not necessarily reflect efficacy. small bsAb fragments exhibit improved tumor penetration and display a more homogeneous distribution within tumors. Linkage of an anti-CEA-anti-CD3 scDb to an albumin-binding domain resulted in a prolonged half-life time and a ∼5-fold increase in accumulation in xenografted CEA+ tumors in mice. in this setting reduced cytotoxicity was observed [65]. As indicated. though the observed continued B-cell suppression during treatment essentially reflects preserved T-cell function [30].g. Lameris et al. especially solid and bulky tumors may require strong and homogeneous tumor penetration provided by small sized constructs.59]. Although tentative. Moreover.g. Small sized bsAb fragments (50–60 kDa). also allow for easy extravasation. nevertheless prolonged “exposure time” is often considered critical for optimal therapeutic efficacy [58]. Though long-term stability of TaFvs may require additional engineering [40. 3. species restricted heavy-light chain paring in Triomabs resulted in a ∼3.58. Compared to large IgG (150 kDa) molecules. continuous infusion of BiTEs was required to maintain stable plasma levels and maximize clinical efficacy [26]. TaFv) molecules tend to form insoluble aggregates in bacteria due to incorrect folding and therefore require production in mammalian cells for high production yields [7.28]. inactive homodimers can be produced alongside the active heterodimers. renal filtration and degradation due to a glomerular filtration barrier (GBM) threshold of ∼40–50 kDa [3.45].64]. but since two different polypeptide chains are expressed within one cell. Nevertheless. These properties greatly facilitated production and clinical development [6]. or linkage to antiCD16 Ab fragments [64]. scDb are 2-3-fold more stable than TaFvs in human plasma at 37 ◦ C [70].e.g. hence absence of the Fc-tail may result in swift clearance from the circulation.e. TaFvs and bsDbs) and it is believed that they are therefore less immunogenic [38]. Due to their enhanced tumor penetration. By eliminating the Fc-region Abs are reduced in size to their minimal binding domain (e. rapid clearance from the circulation may reduce non-specific (off-target) cytotoxicity due to favorable tumorto-blood ratios. By introducing an additional peptide linker (as in scDbs) or disulfide bond (as in DARTs) homodimerization can be prevented. 3. simultaneous targeting of Fc␥R+ cells and T-cells may also result in cross-linkage and activation of these cells in the absence of target antigen [17. Indeed. Hence various methods have been successfully deployed to extend the serum half-life time of small-sized constructs including PEGylation. bsDbs lack this disadvantage. Fc␥R may also trigger CRS.21.41]. scFv can be expressed in bacteria. Indeed. Nevertheless. in this case lack of ADA might also have been due to effective B-cell depletion. such as TaFv and bsDb. the combination of an anti-CD19-anti-CD3 and an anti-CD19-anti-CD16 (Fc␥RIIIA) bsDb had a synergistic effect [61]. disulfide bonds reduce yields in bacteria [37. Antibody construct stability and manufacturing difficulties As mentioned. it has been shown for various mAbs that both ADA and CRS can actually promote tumor destruction [16. patients treated with (murine mAbderived) blinatumomab. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 developed neutralizing human anti-human Abs (HAHA).38. due to their multivalent nature bsAb fragments tend to have high avidity with prolonged target retention [3]. HAMA development in patients with malignant ascites treated with catumaxomab correlated with prolonged puncture-free survival [16]. as was observed in in vitro studies with catumaxomab. furthermore. CRS and HLH) [32]. At present no information is available on the impact of such continuous infusions on either treatment costs or patient quality of live. Other tri-specific constructs that cross-link effector cells may exhibit similar limitations.60].5-fold higher production yield compared to conventional quadromas and allowed for a single purification step. The in vivo stability of catumaxomab appeared favorable. Biological properties of scDb may however be strongly affected by even modest . with several patients developing uncontrolled immune activation (i. targeting the Fc␥R can enhance tumor destruction through ADCC. endothelial cells.38.68]. Interestingly. increasing cost and production time [67]. While scDb can be expressed in bacteria.2. developed no HAMA and had stable steady-state levels over the course of treatment. with up to 100% being immunologically active after three days in ascites [66]. However.69]. BsDbs may be even less immunogenic due to their very compact size [37.3. immune-related AEs of mAbs cannot always be prevented [38. Indeed. persistent T-cell activation may induce anergy.158 R. HAMA development may also simply reflect a less suppressed immune system. Taking the above into consideration. Similarly an amplified anti-tumor response was seen after adding a functional Fc-tail to a scDb [62]. fusion to human albumin (covalently or using albumin-binding domains). As direct ligation of effector cells through e. but tandem (i. N-glycosylation.

including CD4+ .73]. This can. if not all. (ii) have a high serum and storage stability. 159 evaluated for immune effector cell retargeting. Lameris et al. Furthermore. (vii) be non-immunogenic.R. However. For example. 4. finally. Targeting of CD3 results in the recruitment of a wide range of T-cells. (v) have high target-cell affinity to ensure tumor retention. multiple studies have demonstrated that treatment with anti-TAA-anti-CD3 bsAbs can result in the induction of an overall effective proinflammatory antitumor immune response and in clinical responses. but also Tregs resulting in an increased secretion of the immunosuppressive cytokine IL-10. whereas Th 2-cells and most notably CD4+ CD25+ Foxp3+ regulatory T-cells (Tregs) may promote tumor growth. Specific VHHs are easily retrieved after panning of a phage-displayed rearranged VHH -gene pool cloned from an immunized camelid. but carries a risk as was strikingly demonstrated by TGN1412. of these ideal characteristics. due to their distinctive three-dimensional structure [3. currently used bsAb constructs can induce retargeting of immune effector cells and can result in therapeutic responses. and can inexpensively be produced in bacteria. and include strictly target dependent effector cell activation as a means of improving safety [59]. Still. mouse/human albumin [79] or Fc␥R.g. . These variable domains of heavy chain-only Abs (VHH) or Nanobodies (Ablynx Inc. As of yet it remains unclear whether tumor targeting of all CD3-expressing T-cells has significant clinical impact. The relatively small size (∼15 kDa) permits easy linkage of VHH into dimers (∼35 kDa). a monospecific superagonistic CD28-mAb that induced a life threatening cytokine storm in healthy volunteers [81]. be prevented by fusion to a VHH targeting e. Based on discussed limitations. including cryptic and not otherwise easily accessible epitopes. bsVHHs may prove to be very efficient. Tregs have been recognized for their deleterious effect in malignancy by inducing immunosuppression. CD8+ .78]. Perspectives related to the bsAb construct As outlined. aggregation and degradation problems often encountered with scFvs. naturally occurring in the family of Camelidae. yet (iv) have a sufficiently long half-life time to induce a therapeutic effect. yet improvements to overcome encountered difficulties are required and appear feasible. Redirected Tregs suppressed the proliferation and cytokine production of CD4+ effector T-cells both in vitro and in vivo and abrogated the antitumor effector function of redirected Th -cells thereby promoting tumor growth in vivo [87]. including llama. Owing to their size VHH are rapidly cleared from the circulation.) (Fig. (iii) be small (50–60 kDa or less) to allow for rapid and homogeneous tumor penetration. with CD4+ T-cells. being recruited at a later stage [21. most bsAbs target the uniform CD3-portion of the TCR complex. however. VHH are usually humanized before clinical application [75] with preliminary clinical data thus far reporting no ADA development [80]. All previously discussed bsAb constructs lack several of these characteristics to some extent. T-helper 1 (Th 1-)cells are considered to be proinflammatory cells that play an important role in tumor immunity. whereas more deep and homogeneous tumor penetration was observed using the antiEGFR-anti-albumin VHH (Ab-construct binding to 100% vs 60% of tumor cells) [79]. Targeting specific lymphocyte subsets to maximize efficacy In order to effectively recruit and activate all T-cell subsets and induce tumor cell lysis. diversified and specific repertoire of Ag binding sites as conventional Abs but also allow specificity to unique epitopes. As yet no bispecific VHH has been 5. Future studies will have to determine if these properties will translate to the clinic. Bio-distribution studies in mice with a radiolabeled bivalent anti-EGFR-anti-albumin VHH demonstrated an extended half-life time. 1i). VHH amino acid sequences share high homology with the human type 3 VH domain (VH 3). / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 variations in their composition (e. Despite these drawbacks. tumor resident T-cells were sufficient to induce substantial tumor reduction in a NOD/SCID xenotransplanted mouse model treated with BiTEs [69].69].72]. Blood clearance was similar to that of the anti-EGFR mAb cetuximab. The variable domain of heavy chain-only Abs (Fig. several ideal characteristics of bsAbs can be recognized: they should be (i) easy to manufacture at relatively low cost. trimers (∼50 kDa) or multimers that retain rapid and homogeneous tumor penetration [75–78]. are prevented due to their single domain nature. Solubility. differences in linker lengths or the order of variable domains) [38]. ␥␦ T-cells and different immunoregulatory and immunosuppressive T-cell subsets. camel and dromedary [71. may fulfill many. in order to maximize clinical effect and minimize potential AE it seems more attractive to restrict tumor targeting to specific effector cell subsets with known antitumor effects. 1j) share the same large. Recently it has been demonstrated that an anti-PSCA-antiCD3 scDb not only effectively re-directed and activated effector T-cells. Targeting T-cells is attractive due to their destructive potential. including Tregs. most likely accounting for their low immunogenicity [72. yeast or mammalian cells [74]. but (vi) safeguard strictly target-dependent activation and. however based on the above listed properties. This may be explained by an initial recruitment of CD8+ effector T-cells. Measures to preclude such tragedies are required.g. High Treg numbers have been detected systemically and in the tumor microenvironment in patients with various types of cancer and correlate with poor survival [82–86].

including multiple myeloma (MM). nasal submucosal administration of ␣GalCer-pulsed APCs combined with intra-arterial infusion of activated iNKT via tumor-feeding arteries produced objective responses in 5 out of 10 patients. invariant natural killer T-cells (iNKT). Reciprocal interactions between DC and iNKT can reverse defects in the iNKT population.107]. Invariant natural killer T-cells (iNKT) iNKT represent a distinct population of lymphocytes characterized by a (semi-)invariant TCR.and granzyme mediated mechanisms) and secrete pro-inflammatory cytokines (e. of which there are both host and microbe-derived counterparts. in vitro results indicate rehabilitation of iNKT function after stimulation with monocyte derived DC (moDC) pulsed with the agonistic CD1d ligand ␣-galactosylceramide (␣-GalCer) and exogenous IL12 [104. NHL. Lameris et al. The number of infiltrating iNKT in extirpated tumor tissue correlated with clinical outcome [112].105]. once regarded an evolutionary redundant T-cell subset en route to extinction. produce a diversified set of cytokines and chemokines to regulate both immune and nonimmune cells. resulted in clinical responses [90]. Although less clear than in mice. Unlike conventional T-cells. V␥9V␦2 T-cells have been shown to be able to recognize and eliminate malignant cells from multiple tumors types. have been demonstrated to hold a unique position in the immune system. ADCC). It was shown that sustained activation of iNKT at the tumor site could be induced after systemic treatment with ␣-GalCer loaded soluble CD1d-molecules fused to an anti-tumor scFv.e. clinical studies evaluating administration of ␣-GalCer-pulsed moDC with or without adoptive transfer of ex vivo expanded iNKT have reported objective tumor regressions in several patients [108–112]. iNKT recognize (glyco-)lipids presented by non-polymorphic CD1d molecules and rapidly secrete a wide range of cytokines upon stimulation thereby inducing effector (e. NK cells become highly cytotoxic (e. iNKT from patients with advanced cancer display quantitative and qualitative defects and circulating numbers correlate with patient survival [100–103]. Moreover. Their role in tumor immunosurveillance is underscored by epidemiological studies correlating disease prognosis with .1.90].g. ␥␦ T-cells. aminobisphosphonates or mAbs combined with IL-2. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 Several specific immune effector cell subsets that can be considered attractive candidates for tumor targeting (i. aminobisphosphonate compounds (e. Upon activation. Indeed. extensive preclinical and early clinical data underscore the important role of iNKT in tumor immunosurveillance and indicate beneficial effects with low toxicity in cancer treatment. these functional V␥9V␦2 T-cell defects are reversible [90].g.95]. Importantly. iNKT contribute to immune surveillance in early-stage tumors and chemically induced cancers and play a pivotal role in controlling different forms of cancer in mice [100. respectively. Quantitative and qualitative defects in the V␥9V␦2 T-cell population are noted in various malignancies [90] and negatively impact disease-free survival. it was reported that butyrophilin (BTN) 3A1 is required for the presentation of pAg to V␥9V␦2 T-cells [91. typified by isopentenyl pyrophosphate (IPP) and hydroxymethyl-but-2-enyl-pyrophosphate (HMBPP).e. and natural killer cells (NK)) are discussed below.g. They can directly lyse stressed or infected cells.and colon cancer. prostate-. MHC class I chain-related genes (MIC) A and B) – “induced self” – and by Fc␥RIIIA ligation (i. NK and CTL) cell activation in an IFN-␥ dependent manner [97–99].160 R. In stressed/malignant cells pAg production is frequently upregulated allowing discrimination from normal tissue. e. adoptive transfer of V␥9V␦2 T-cells following ex vivo expansion by pAg.92]. Although clinical data are scarce. and can present antigens for ␣␤ T-cell priming [88]. Natural killer cells (NK) NK cells represent a major subset of innate cytotoxic lymphoid cells tightly regulated by inhibitory and activating signals sensed via cell surface receptors. zoledronic acid) sensitize target cells to V␥9V␦2 Tcell killing by promoting the intracellular accumulation of endogenous IPP by inhibiting mevalonate metabolism [89. through perforin. preliminary findings clearly indicate that exploiting the natural abilities of V␥9V␦2 T-cells in cancer immunotherapy is feasible and carries low toxicity. Activation can be triggered by a lack of inhibitory signals delivered by MHC class I molecule engagement – “missing self” – via ligation of the activating receptor NKG2D by stress-induced molecules (e. Observed toxicities were minor and limited to transient flu-like symptoms [94. V␥9V␦2 T-cells constitute the predominant ␥␦ T-cell subset in human peripheral blood.g. In summary. in ovarian carcinoma [93].g. 5.g. IFN-␥) [113.101]. therefore redirecting this invariant subset potentially constitutes a valuable approach. V␥9V␦2 T-cells can be activated and expanded by non-peptidic pyrophosphate antigens (pAg).2. Potent tumor inhibition of aggressive tumor grafts expressing the targeted antigen was observed in mice [106. Furthermore. γδ T-cells ␥␦ T-cells. 5. Strikingly. Recently. 5. In patients with recurrent HNSCC. Indeed. in vitro V␥9V␦2 T-cell mediated lysis of hepatic tumor cell lines was significantly enhanced by an anti-EpCAM-anti-CD3 BiTE [96].3. renal cell. In patients with various metastatic cancers treatment with zoledronic acid and IL-2 promoted the differentiation of peripheral blood V␥9V␦2 T-cells toward an effector/memory-like phenotype with augmented numbers correlating with arrested disease progression.114]. In patients with various malignancies.

Lim SH. [4] Vaughan AT. et al. V␥9V␦2-TCR. J Immunol 1999. Wollenberg B. Implications for a single-step purification of bispecific antibodies.23(Sep (9)):1126–36. Baty D. Additionally.155(Jul (1)):219–25. Thierfelder S.118]. Adoptively transferred ex vivo expanded and activated autologous NK cells proved to be safe. stability.6:1. their conserved receptors preclude the need for individualized treatment. Kufer P. the fusion protein ULBP2anti-CEA scFv induced an effective anti-tumor response and NK activation in a syngeneic colon carcinoma mouse model [54]. Iriyama C. several bsAb constructs (e. Concluding remarks Advances in bsAb engineering have marked a new era of Ab based cancer treatment and have resulted in an array of constructs shown to be effective in inducing target cell 161 killing by engaged effector cells. J Immunol 1995.g. Reisbach G. . Dobrovic A.12(Apr (4)):278–87. As noted.v. Williams EL. and tumor penetration as well as with respect to the specific immune effector cell subset to be targeted are required. and grant 14-0343 from the Association for International Cancer Research (AICR). J Hematol Oncol 2013. at least ex vivo [115]. Importantly. (pre-)clinical trials have demonstrated results that are not just an enhancement of those accomplished by conventional mAbs. Schmitt B. A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer. IL-2 [116. 90700309 from The Netherlands Organization for Health Research and Development (ZonMw). References [1] Scott AM. however no significant clinical responses were observed. Bispecific antibodies for cancer therapy: the light at the end of the tunnel? MAbs 2009. Wolchok JD. [3] Holliger P. Mocikat R. Given the role NK cells play in first-line defense against malignancies. [2] Mellor JD.118]. targeting invariant/conserved lymphocyte subsets through their highly conserved receptor (e. Proc Natl Acad Sci USA 1995. ADCC could be significantly enhanced through Fc-tail modifications resulting in increased Fc␥RIIIA affinity. grant VU 2010-4728 from the Dutch Cancer Society (KWF). Inhibitory FcgammaRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity. further improvements of the constructs with respect to increasing their half-life.R. Irving HR.120]. Lameris et al. the expanding interest in this field will result in multiple compounds entering clinical trials in the near future shedding light on these issues. Redirection of adoptively transferred NK cells by bsAbs targeting tumor cells and NK cells may therefore prove to be highly effective. Chan CH. Beers SA. Blood 2014. NK-receptors or Fc␥RIIIA) allow for redirection of specific lymphocyte subsets with intrinsic anti-tumor efficacy and can prevent the simultaneous redirection of immunosuppressive T-cells. Brown MP.g. Lang S. NK cells were shown to contribute to mAb based cancer treatment though Fc␥RIIIA (CD16A) mediated recognition and elimination of mAb coated tumor cells. but represent a whole new therapeutic repertoire and embody a leap forward in terms of therapeutic efficacy. Chaubal S. Fc␥RIIIa ligation) and/or homing to tumors may be required for the induction of more robust anti-tumor responses [116. Triomab. / Critical Reviews in Oncology/Hematology 92 (2014) 153–165 tumor infiltrating NK cells and diminished cytotoxicity with an increased risk of cancer development [114–116]. Similarly. Riethmuller G. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. et al. [6] Lindhofer H. Old LJ. Nat Biotechnol 2005. at least in vivo [117].1(Nov (6)):539–47. Steipe B. To break selftolerance associated with autologous NK. possibly limiting their therapeutic efficacy. Though proven safe. Indeed. Nevertheless. [7] Mack M. A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. dampened NK cell responses through tumor derived soluble NKG2D-ligands were reversible upon cross-linking of tumor and NK cells by an anti-CD30-anti-CD16A bsAb construct. iNKT-TCR.123(Jan (5)):669–77. Antibody therapy of cancer. whether new formats such as VHH can overcome shortcomings of existing constructs and what impact redirection of different effector cell subsets will have on clinical outcome. 6. Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing. Engineered antibody fragments and the rise of single domains.92(Jul (15)): 7021–5. anti-CD19-anti-CD16 bsDb/TaFv and an anti-CD33-anti-CD16 TaFv) are capable of enhancing tumor destruction through effective Fc␥R-mediated redirection and activation of NK cells [10. Acknowledgments This work is supported by grant no. In conclusion. Hudson PJ. adoptive transfer of allogeneic NK cells has been investigated as an alternative. a substantial increase in host Tregs was reported after combined IL-2 and NK cell therapy [119].163(Aug (3)):1246–52. NK cell therapy has thus far induced modest and inconsistent clinical responses across various cancer types and it has been argued that additional activation stimuli (e. [5] Chames P. Nat Rev Cancer 2012. Undoubtedly. An enhanced leukemia clearance rate was reported in patients with poor-prognosis acute myeloid leukemia (AML) with successful in vivo NK cell expansion after haploidentical related donor infusion and daily i.g.61. Conflict of interest statement All authors have no conflicts of interest to declare. Of note. Zalcberg JR. [8] Zeidler R. Future research will reveal to what extent bsAbs will impact cancer treatment. Moreover. their therapeutic use has been widely explored in clinical trials.

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