Bispecific antibody platforms for cancer immunotherapy.

ONCH-1878; No. of Pages 13

ARTICLE IN PRESS

Critical Reviews in Oncology/Hematology xxx (2014) xxx–xxx

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 1. 2.

3.

4. 5.

6.

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: [email protected] (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.

Please cite this article in press as: Lameris R, et al. Bispecific antibody platforms for cancer immunotherapy. Crit Rev Oncol/Hematol (2014), http://dx.doi.org/10.1016/j.critrevonc.2014.08.003


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ARTICLE IN PRESS R. Lameris et al. / Critical Reviews in Oncology/Hematology xxx (2014) xxx–xxx

and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward. © 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bi-specific antibodies; Dual specific retargeting; Immune effector cells; Anti-cancer therapy

1. Introduction

2. 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). Following binding of the mAb to its tumor target, interactions of the Fc-portion with Fc␥receptors (Fc␥R) expressed by effector cells (e.g. natural killer (NK) cells, macrophages and ␥␦ T-cells) may result in CDC and ADCC and subsequent antitumor cytotoxicity and/or phagocytosis. In clinical series, ADCC has been demonstrated to significantly enhance the efficacy of various mAbs, including rituximab (anti-CD20), trastuzumab (anti-human-epidermal-growth-factor receptor 2 (Her2)) and cetuximab (anti-epidermal-growth-factor receptor (EGFR)) [1]. Although data are inconsistent, clinical responses may be influenced by Fc␥R polymorphisms [2]. Granting their therapeutic efficacy can in part be attributed to beneficial secondary immune effects, it is clear that mAbs still do not exploit the full potential of the immune system as effects are e.g. hampered by circulating immunoglobulins (Ig) competing for Fc␥R binding spots on immune effector cells, and inadequate tumor-target penetration due to their relatively large size (∼150 kDa) [3]. Furthermore, 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]. Bispecific Abs (bsAbs), capable of binding two targets simultaneously, lack several of the above described limitations and can potentially induce a more powerful anti-tumor immune response. 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.e. hybrid hybridomas or quadromas). Despite some clinical effects, none of these bsAbs made it to advanced stage clinical trials, 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]. 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) [6,7]. Here, 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. Furthermore, we will elaborate on potential ways to take the bsAb approach forward.

2.1. Trifunctional hybrid antibodies (Triomab) Introduced in 1995, this platform offered a solution to the random association of heavy/light chains observed in classic quadroma technology. By combining the halves of two distinct antibodies, a tumor-specific mouse IgG2a and a CD3specific rat IgG2b, a full-size functional mAb was engineered and termed Triomab (Trion pharma Inc.) (Fig. 1b). Due to species-preferential heavy/light chain pairing random association was greatly reduced. Interestingly, the hybrid mouse/rat Fc-portion was able to activate Fc␥R+ accessory cells [6,8,9]. Preclinical studies with a Triomab targeting epithelial cell adhesion molecule (EpCAM), expressed by the majority of epithelial cancers, and CD3 expressed by T-cells, demonstrated redirection and activation of T- and accessory cells (e.g. NK cells, dendritic cells (DC) and macrophages). T-cell activation was complemented by the induction of T-cell mediated tumor lysis, cytotoxic cytokine release, and ADCC in the picomolar range [8,9]. 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. 1c). In vivo assessment of the Triomab injected intraperitoneally (i.p.) in a syngeneic C57BL/6 and BALB/c mouse model using (human) EpCAM expressing B16-melanoma and A20-lymphoma cells, respectively, demonstrated a significantly improved tumor cell elimination compared with the simultaneous administration of both parental antibodies. In the Triomab treated group a 100% survival rate, complete tumor eradication and protection against tumor rechallenge was reported. Selective depletion of both CD4+ and CD8+ T-cells resulted in a marked loss of tumor protection and survival. Of note, only the animals injected with human EpCAM expressing tumors and Triomab treatment developed strong anti-EpCAM specific humoral immune responses [10], with additional evidence suggestive of epitope spreading. These data resulted in the development and clinical evaluation of a number of Triomabs, including catumaxomab, ertumaxomab and FBTA05 which will be discussed below. 2.1.1. Catumaxomab Catumaxomab, an anti-EpCAM-anti-CD3 Triomab, was the first Triomab studied in patients. A phase-I trial of a single intravenous (i.v.) dose of catumaxomab in patients with non-small cell lung cancer (NSCLC), established a maximum




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vascular endothelial growth factor (VEGF), 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]. Importantly, compared to control patients catumaxomab resulted in a significant prolongation of puncture-free survival (median 11 vs 46 days, p < 0.0001) and an increased time to next paracentesis (median 13 vs 77 days), precluding approximately 5 therapeutic paracentesis procedures. Likewise, symptoms and signs related to ascites were significantly reduced. Although a positive trend was observed in improvement of median overall survival (72 (95% CI: 61–98) versus 68 days (95% CI: 49–81), this only reached statistical significance in patients with gastric carcinoma (median 71 vs 44 days; p = 0.0313) [15]. Notably, HAMA/HARA development, which strongly correlated with clinical endpoints, was observed in the majority of patients (>70%) after the last treatment cycle and will be discussed in more detail in Section 3.1 [16]. Adverse effects (AE) were most commonly cytokine release-related (i.e. pyrexia, nausea and vomiting), manageable and generally reversible. Reported Common Toxicity Criteria (CTC) grade ≥3 AE considered to be treatment related included abdominal pain (9.6%) and lymphopenia (7.0%). Reported serious AE included ileus (3.2%) and gastric hemorrhage (0.6%) [15]. Based on these data, catumaxomab was approved by the European Medicine Agency (EMA) for the i.p. treatment of malignant ascites in patients with EpCAM-positive carcinomas where standard therapy is not available or no longer feasible.

Fig. 1. Variable heavy chain domains (VH ) are depicted in dark blue and dark orange, variable light chain domains (VL ) are depicted in light blue and light orange. Orange and blue indicate arms with different specificities. Peptide linkers are shown as gray lines. (a) mAb, monoclonal antibody; (b) Triomab, bispecific rat/mouse antibody; (c) F(ab)2 , two chemically crosslinked fragment antigen binding (Fab) regions; (d) scFv, single chain variable fragment; (e) TaFv, tandem single chain variable fragment; (f) bsDb, bispecific diabody; (g) scDb, single chain diabody; (h) DART, dual affinity retargeting molecule; (i) Heavy chain-only antibody; (j) bsVHH, bispecific variable domains of heavy chain-only Ab. (For interpretation of the references to color in this figure legend, 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. None of the patients developed HAMA or HARA within 28 days after infusion [11]. More extensive studies have evaluated i.p. administration of catumaxomab in patients with malignant ascites due to various EpCAM positive tumors. In a randomized phase II/III study, involving heavily pre-treated patients with symptomatic malignant ascites, infusions were given with an increasing dose (10–150 ␮g) on days 0, 3, 7 and 10 [12,13]. Analyses of ascites demonstrated decreased levels of

2.1.2. Ertumaxomab Ertumaxomab, an anti-Her2-anti-CD3 Triomab, 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, a mAb targeting Her2, was completely ineffective [17]. In a phase I trial, i.v. administration of 3 ascending doses (10–200 ␮g) of ertumaxomab on days 1, 7 and 13 to patients with metastatic breast cancer induced strong inflammatory reactions with clinical responses in some patients. The severity and number of AEs were dose related, with dose limiting events being immune related. Reported serious AE classified to be treatment related included fully reversible severe hypotension (5.9%), systemic inflammatory response syndrome (5.9%) and, aggravation of congestive heart failure (5.9%). CTC grade ≥3 toxicities included fully reversible lymphocytopenia (76%) and elevation of liver enzymes (47%). Of note, the number of CTC grade ≥3 toxicities and serious AE seemed to be dose related. Within 6 weeks after the first dose, HAMA/HARA were observed in 25 and 31% of patients, respectively [18]. A phase II trial was terminated prematurely due to changes in company development plans.



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2.1.3. FBTA05 FBTA05, an anti-CD20-anti-CD3 Triomab, demonstrated effective CD20+ lymphoma killing in vitro. A pilot study involving patients with recurrent/refractory non-Hodgkin lymphoma (NHL) or chronic lymphatic leukemia (CLL) treated with i.v. FBTA05 followed by donor lymphocyte infusion or peripheral blood stem cell transplantation showed a prompt but transient response in several patients [19]. A phase I/II study is ongoing (NCT01138579). 2.2. 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. Advances in genetic engineering have provided a vast amount of such antibody like constructs, including small antibody fragments. One such fragment, termed single-chain Fv (scFv), is made by the association of the variable parts of heavy (VH ) and light (VL ) chains through a peptide linker (Fig. 1d) [20]. Two formats based on scFvs have been studied extensively, namely tandem scFv (TaFv) and bispecific diabodies (bsDb). 2.2.1. Tandem scFv (TaFv) By covalent bonding of two scFvs with a flexible peptide linker in a tandem orientation, a bispecific TaFv can be formed (Fig. 1e). It is expected that the flexible orientation between the two binding domains enhances simultaneous target ligation, while their small size (∼55–60 kDa) allows rapid tumor penetration [3]. One type of TaFv, made by fusing an anti-CD3 to an antitumor associated antigen (TAA) scFv via a 5 residue peptide linker (GGGGS), has been extensively studied. These TaFv, termed bispecific T-cell engagers (BiTE) (Micromet Inc.) can effectively redirect and activate polyclonal T-cells in vitro resulting in a highly cytotoxic response at pico- to femtomolar concentrations in the absence of co-stimulation [21,22]. 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, which is 2–4 times lower than ratios needed for Triomabs [23]. Target cell lysis involved the formation of tight cytolytic synapses between target- and effector cells and the subsequent release of cytotoxic effector molecules (e.g. perforin and granzyme B) [24]. As T-cell activation depended on multivalent binding of BITEs due to their low affinity for CD3, activation occurred in a strictly target dependent fashion [25]. 2.2.1.1. Blinatumomab. Blinatumomab, being the first and most advanced BiTE in clinical studies, is derived from murine scFvs targeting human CD19 and CD3. Several clinical studies evaluated its efficacy in various types of Bcell NHL and leukemia. Complete (CR) and partial tumor responses (PR) were demonstrated upwards of a dose of 15 ␮g/m2 /24 h, given as continuous infusion over 4 or 8

weeks. Importantly, 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 [26,27]. Based on results in B-NHL, a phase II study was initiated in patients with persistent/relapsed minimal residual disease (MRD) positive B-cell acute lymphatic leukemia (B-ALL). Patients were treated with a continuous 4-week i.v. infusion at a dose of 15 ␮g/m2 /24 h every 6 weeks with responders being permitted to receive up to 3 consolidation cycles. Sixteen out of 20 patients became MRD-negative at the end of the first cycle, 12 of which had molecular refractory disease to previous treatment [28]. Long term follow-up (median 33 months) demonstrated a hematologic relapse-free survival in 12 out of 20 patients [29]. In all 20 patients T-cells declined rapidly but recovered within days to above baseline levels, with a significant percentage of reappearing CD8+ and CD4+ T-cells transiently expressing the early activation marker CD69. Bcell counts dropped below 1 cell/␮L and did not recover until after therapy cessation. The decline in B-cell counts most likely resulted from redirected cell lysis [30]. Similar results were reported in an interim analysis of a phase II trial in relapsed/refractory B-ALL. In this study the starting dose was reduced (5 ␮g/m2 /24 h in the first week) to prevent AE. Seventeen out of 25 patients achieved CR or CR with partial hematological recovery [31]. Most common AEs with blinatumomab included flu-like symptoms during the first days of treatment which resolved under ongoing infusion. AEs correlated with peak levels of activated CD8+ and CD4+ T-cells and with increased serum cytokines levels. Commonly reported CTC grade ≥3 AE in the different studies included lymphopenia (up to 77%) and leukopenia (up to 47%). More serious AEs, although completely reversible, included neurological AE (e.g. seizures and encephalopathy in up to 4.8% and 8.6% of patients, respectively) presumably caused by released cytokines and frequently resulted in treatment interruption or discontinuation. Also, cases of hemophagocytic lymphohistiocytosis (HLH) presumably caused by blinatumomab treatment have been described [26–30,32]. Mitigating measures, including lower starting doses, a double-step dose increase and glucocorticoid administration, were protective and allowed for treatment continuation or resumption [26–30]. Multiple trials designed to determine the optimal dose, treatment schedule and clinical efficacy of blinatumomab are ongoing (NCT01741792, NCT01471782, NCT01207388 and NCT01466179). 2.2.1.2. Alternative TaFv constructs. TaFv constructs targeting EpCAM and prostate specific membrane antigen (PSMA) have entered phase I clinical trials in epithelial and prostate cancer, respectively (NCT00635596 and NCT01723475). A multitude of other TaFvs is being evaluated pre-clinically, including constructs targeting EGFR [33], carcinoembryonic antigen (CEA) and CD33. Of interest, an anti-prostate specific cell antigen (PSCA)-anti-CD3 TaFv




was recently successfully humanized without compromising its in vitro and in vivo characteristics. Humanization may prohibit HAMA associated AE [34]. Efforts have also focused on adding a third binding site to TaFv, thereby creating triple bodies (scTb). Indeed, an anti-CD16-anti-CD33-anti-CD19 scTb demonstrated enhanced lysis of CD33-CD19-positive leukemic cells in vitro, compared to both an anti-CD16-anti-CD19 and an anti-CD16-CD33 TaFv [35]. 2.2.2. Bispecific diabodies (bsDb) In contrast to TaFvs, bsDbs are formed by non-covalent association of two scFvs, consisting of a VH and VL domain from different parent Abs, connected with a very short peptide linker precluding intra-chain domain interactions (Fig. 1f). bsDbs have their variable domains orientated in an opposed fashion and are very rigid and small (∼60 kDa) [36]. Because individual chains are not covalently associated, VH and VL binding interactions remain a limiting factor in the stability of the molecule, possibly resulting in disintegration, dimerization and aggregate formation. This can, at least in part, be prevented by introducing an additional peptide linker between the scFv, forming a single chain bsDb (scDb) (Fig. 1g) [37–39]. Alternatively, an increased stability can be achieved by introducing a disulfide bond between the C-terminus of each scFv, resulting in a Dual-Affinity ReTargeting (DART) molecule (Fig. 1h). DART molecules have an extended storage and serum stability, do not form aggregates and demonstrate potent in vitro and in vivo activity [40]. Notably, by varying the length of the additional peptide linker in scDbs, tetramers and tandem bsDbs can be formed [37,41–43] with higher affinity, a reduced target celldissociation rate and improved storage stability. Furthermore, in vivo experiments showed higher serum retention compared to bsDbs [41], possibly due to their increased affinity and size (∼114 kDa) reducing renal clearance. Their increased size may however decrease effective tumor penetration. 2.2.2.1. Pre-clinical studies. 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]. Similar results were reported for an anti-CD19-anti-CD3 [44,45] and an anti-EGFR-anti-CD3 bsDb [46]. Direct comparison of an anti-EGFR–anti-CD3 bsDb with its TaFv equivalent indicated an equal binding affinity, but enhanced in vitro cytotoxicity in favor of the TaFv [47]. As the compact structure of bsDbs might hinder cross-linking, it is interesting to note that rearrangement of the variable domains (e.g. varying the VH -linker-VL order) can lead to superior in vivo results when compared to the TaFv equivalent, though this in vivo superiority may also result from the enhanced stability of bsDbs at 37 ◦ C [48]. The potency of DARTs was demonstrated in an in vitro side-by-side comparison of blinatumomab and a DART

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bearing identical CD3 and CD19 Fv-domains. The DART molecule outperformed the BiTE molecule with respect to both the induction of B-cell lysis and stimulation of T-cell activation, with T-cell activation and proliferation occurring solely in a target-cell dependent manner in both formats. Based on identical recognition specificities and similar molecular masses, differences between the DART and BiTE platform thus resulted from structural differences, e.g. 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]. While bsDbs yielded promising results in pre-clinical studies, with some apparently outperforming TaFv, no clinical data have yet been reported. 2.3. Other bispecific antibody based platforms Although a number of alternative bispecific constructs designed for tumor targeting of effector cells have been engineered, to date only a few have been evaluated in preclinical studies [20,50]. These include, but are not limited to, engineered human monospecific IgG1 and IgG2 subtypes that allow, under appropriate redox conditions, heavy chain exchange to form full-length bsAb [51], an anti-CD3 scFv fused to a high-affinity monoclonal T-cell receptor (mTCR) specific for a tumor-associated MHC-1 antigen (ImmTAC) [52,53], a fusion protein consisting of a scFv targeting CEA and the human UL16 binding protein 2 (ULBP2), which as an NKG2D ligand can be used for targeting NKG2D-expressing immune effector cells [54] and, a scFv fused to the N- and C-terminus of a trimerizing scaffold domain, resulting in a bispecific hexavalent trimerbody [55]. In, 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 [56,57]. These platforms are not further discussed as limited preclinical efficacy data and no clinical efficacy data are presently available.

3. Limitations of the type of bsAb constructs 3.1. Immunogenicity of bispecific antibody constructs Historically, murine- 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. Indeed, as discussed, i.v. dosing of Triomabs was limited by immunological AE and, HAMA/HARA formation may restrict repetition of treatment cycles. Though humanization is generally thought to limit anti-drug antibody (ADA) formation, it cannot completely circumvent this, as was demonstrated by the anti-TNF-␣ mAb adalimumab, where up to 89% of patients



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developed neutralizing human anti-human Abs (HAHA). As direct ligation of effector cells through e.g. Fc␥R may also trigger CRS, immune-related AEs of mAbs cannot always be prevented [38,58,59]. Despite these drawbacks, it has been shown for various mAbs that both ADA and CRS can actually promote tumor destruction [16,60]. Indeed, HAMA development in patients with malignant ascites treated with catumaxomab correlated with prolonged puncture-free survival [16]. Although tentative, HAMA development may also simply reflect a less suppressed immune system. As indicated, targeting the Fc␥R can enhance tumor destruction through ADCC. Indeed, the combination of an anti-CD19-anti-CD3 and an anti-CD19-anti-CD16 (Fc␥RIIIA) bsDb had a synergistic effect [61]. Similarly an amplified anti-tumor response was seen after adding a functional Fc-tail to a scDb [62]. However, as was observed in in vitro studies with catumaxomab, 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 [17,63] thereby limiting the intended efficacy of tumor targeting of these immune cells. Other tri-specific constructs that cross-link effector cells may exhibit similar limitations. By eliminating the Fc-region Abs are reduced in size to their minimal binding domain (e.g. TaFvs and bsDbs) and it is believed that they are therefore less immunogenic [38]. In support of this, patients treated with (murine mAbderived) blinatumomab, developed no HAMA and had stable steady-state levels over the course of treatment, indicating low immunogenicity [27,28]. Of note, in this case lack of ADA might also have been due to effective B-cell depletion. BsDbs may be even less immunogenic due to their very compact size [37,42,45]. Concerns have been raised about ongoing T-cell activation during BiTE-treatment, with several patients developing uncontrolled immune activation (i.e. CRS and HLH) [32]. Moreover, persistent T-cell activation may induce anergy, though the observed continued B-cell suppression during treatment essentially reflects preserved T-cell function [30]. 3.2. 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.g. endothelial cells; hence absence of the Fc-tail may result in swift clearance from the circulation. Small sized bsAb fragments (50–60 kDa), such as TaFv and bsDb, also allow for easy extravasation, renal filtration and degradation due to a glomerular filtration barrier (GBM) threshold of ∼40–50 kDa [3,38,64]. Indeed, continuous infusion of BiTEs was required to maintain stable plasma levels and maximize clinical efficacy [26]. At present no information is available on the impact of such continuous infusions on either treatment costs or patient quality of live, nevertheless prolonged “exposure time” is often considered critical

for optimal therapeutic efficacy [58]. Hence various methods have been successfully deployed to extend the serum half-life time of small-sized constructs including PEGylation, N-glycosylation, fusion to human albumin (covalently or using albumin-binding domains), or linkage to antiCD16 Ab fragments [64]. 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. Nevertheless, in this setting reduced cytotoxicity was observed [65], possibly due to steric hindrance. Similar reductions in cytotoxicity have been reported for other serum half-life time extending strategies [64,65]. Compared to large IgG (150 kDa) molecules, small bsAb fragments exhibit improved tumor penetration and display a more homogeneous distribution within tumors; furthermore, due to their multivalent nature bsAb fragments tend to have high avidity with prolonged target retention [3]. Both superior tumor penetration and target retention may result in a synergistic effect on tumor destruction. Taking the above into consideration, especially solid and bulky tumors may require strong and homogeneous tumor penetration provided by small sized constructs. Due to their enhanced tumor penetration, serum levels of small-sized bsAb constructs might not necessarily reflect efficacy. Interestingly, rapid clearance from the circulation may reduce non-specific (off-target) cytotoxicity due to favorable tumorto-blood ratios. 3.3. Antibody construct stability and manufacturing difficulties As mentioned, species restricted heavy-light chain paring in Triomabs resulted in a ∼3.5-fold higher production yield compared to conventional quadromas and allowed for a single purification step. The in vivo stability of catumaxomab appeared favorable, with up to 100% being immunologically active after three days in ascites [66]. These properties greatly facilitated production and clinical development [6]. Nevertheless, as with all whole mAbs, mammalian cells are often needed as a host, increasing cost and production time [67]. scFv can be expressed in bacteria, but tandem (i.e. TaFv) molecules tend to form insoluble aggregates in bacteria due to incorrect folding and therefore require production in mammalian cells for high production yields [7,21,41]. bsDbs lack this disadvantage, but since two different polypeptide chains are expressed within one cell, inactive homodimers can be produced alongside the active heterodimers. By introducing an additional peptide linker (as in scDbs) or disulfide bond (as in DARTs) homodimerization can be prevented. While scDb can be expressed in bacteria, disulfide bonds reduce yields in bacteria [37,38,40,68]. Though long-term stability of TaFvs may require additional engineering [40,69], scDb are 2-3-fold more stable than TaFvs in human plasma at 37 ◦ C [70]. Biological properties of scDb may however be strongly affected by even modest




variations in their composition (e.g. differences in linker lengths or the order of variable domains) [38].

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evaluated for immune effector cell retargeting, however based on the above listed properties, bsVHHs may prove to be very efficient. Future studies will have to determine if these properties will translate to the clinic.

4. Perspectives related to the bsAb construct As outlined, currently used bsAb constructs can induce retargeting of immune effector cells and can result in therapeutic responses, yet improvements to overcome encountered difficulties are required and appear feasible. Based on discussed limitations, several ideal characteristics of bsAbs can be recognized: they should be (i) easy to manufacture at relatively low cost, (ii) have a high serum and storage stability, (iii) be small (50–60 kDa or less) to allow for rapid and homogeneous tumor penetration, yet (iv) have a sufficiently long half-life time to induce a therapeutic effect, (v) have high target-cell affinity to ensure tumor retention, but (vi) safeguard strictly target-dependent activation and, finally, (vii) be non-immunogenic. All previously discussed bsAb constructs lack several of these characteristics to some extent. The variable domain of heavy chain-only Abs (Fig. 1i), naturally occurring in the family of Camelidae, including llama, camel and dromedary [71,72], may fulfill many, if not all, of these ideal characteristics. These variable domains of heavy chain-only Abs (VHH) or Nanobodies (Ablynx Inc.) (Fig. 1j) share the same large, diversified and specific repertoire of Ag binding sites as conventional Abs but also allow specificity to unique epitopes, including cryptic and not otherwise easily accessible epitopes, due to their distinctive three-dimensional structure [3,73]. Specific VHHs are easily retrieved after panning of a phage-displayed rearranged VHH -gene pool cloned from an immunized camelid, and can inexpensively be produced in bacteria, yeast or mammalian cells [74]. Solubility, aggregation and degradation problems often encountered with scFvs, are prevented due to their single domain nature. The relatively small size (∼15 kDa) permits easy linkage of VHH into dimers (∼35 kDa), trimers (∼50 kDa) or multimers that retain rapid and homogeneous tumor penetration [75–78]. Owing to their size VHH are rapidly cleared from the circulation. This can, however, be prevented by fusion to a VHH targeting e.g. mouse/human albumin [79] or Fc␥R. Bio-distribution studies in mice with a radiolabeled bivalent anti-EGFR-anti-albumin VHH demonstrated an extended half-life time. Blood clearance was similar to that of the anti-EGFR mAb cetuximab, 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]. VHH amino acid sequences share high homology with the human type 3 VH domain (VH 3), most likely accounting for their low immunogenicity [72,78]. Still, VHH are usually humanized before clinical application [75] with preliminary clinical data thus far reporting no ADA development [80]. As yet no bispecific VHH has been

5. Targeting specific lymphocyte subsets to maximize efficacy In order to effectively recruit and activate all T-cell subsets and induce tumor cell lysis, most bsAbs target the uniform CD3-portion of the TCR complex. Targeting T-cells is attractive due to their destructive potential, but carries a risk as was strikingly demonstrated by TGN1412, a monospecific superagonistic CD28-mAb that induced a life threatening cytokine storm in healthy volunteers [81]. Measures to preclude such tragedies are required, and include strictly target dependent effector cell activation as a means of improving safety [59]. Targeting of CD3 results in the recruitment of a wide range of T-cells, including CD4+ , CD8+ , ␥␦ T-cells and different immunoregulatory and immunosuppressive T-cell subsets. T-helper 1 (Th 1-)cells are considered to be proinflammatory cells that play an important role in tumor immunity, whereas Th 2-cells and most notably CD4+ CD25+ Foxp3+ regulatory T-cells (Tregs) may promote tumor growth. For example, Tregs have been recognized for their deleterious effect in malignancy by inducing immunosuppression. 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]. Recently it has been demonstrated that an anti-PSCA-antiCD3 scDb not only effectively re-directed and activated effector T-cells, but also Tregs resulting in an increased secretion of the immunosuppressive cytokine IL-10. 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]. Despite these drawbacks, 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. This may be explained by an initial recruitment of CD8+ effector T-cells, with CD4+ T-cells, including Tregs, being recruited at a later stage [21,69]. Furthermore, tumor resident T-cells were sufficient to induce substantial tumor reduction in a NOD/SCID xenotransplanted mouse model treated with BiTEs [69]. As of yet it remains unclear whether tumor targeting of all CD3-expressing T-cells has significant clinical impact. However, 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.



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Several specific immune effector cell subsets that can be considered attractive candidates for tumor targeting (i.e. ␥␦ T-cells, invariant natural killer T-cells (iNKT), and natural killer cells (NK)) are discussed below. 5.1. γδ T-cells ␥␦ T-cells, once regarded an evolutionary redundant T-cell subset en route to extinction, have been demonstrated to hold a unique position in the immune system. They can directly lyse stressed or infected cells, produce a diversified set of cytokines and chemokines to regulate both immune and nonimmune cells, and can present antigens for ␣␤ T-cell priming [88]. V␥9V␦2 T-cells constitute the predominant ␥␦ T-cell subset in human peripheral blood. V␥9V␦2 T-cells can be activated and expanded by non-peptidic pyrophosphate antigens (pAg), of which there are both host and microbe-derived counterparts, typified by isopentenyl pyrophosphate (IPP) and hydroxymethyl-but-2-enyl-pyrophosphate (HMBPP), respectively. Furthermore, aminobisphosphonate compounds (e.g. zoledronic acid) sensitize target cells to V␥9V␦2 Tcell killing by promoting the intracellular accumulation of endogenous IPP by inhibiting mevalonate metabolism [89,90]. Recently, it was reported that butyrophilin (BTN) 3A1 is required for the presentation of pAg to V␥9V␦2 T-cells [91,92]. In stressed/malignant cells pAg production is frequently upregulated allowing discrimination from normal tissue. Indeed, V␥9V␦2 T-cells have been shown to be able to recognize and eliminate malignant cells from multiple tumors types, including multiple myeloma (MM), NHL, prostate-, renal cell- and colon cancer. Quantitative and qualitative defects in the V␥9V␦2 T-cell population are noted in various malignancies [90] and negatively impact disease-free survival, e.g. in ovarian carcinoma [93]. Importantly, these functional V␥9V␦2 T-cell defects are reversible [90]. 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. Observed toxicities were minor and limited to transient flu-like symptoms [94,95]. In patients with various malignancies, adoptive transfer of V␥9V␦2 T-cells following ex vivo expansion by pAg, aminobisphosphonates or mAbs combined with IL-2, resulted in clinical responses [90]. Strikingly, 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]. Although clinical data are scarce, preliminary findings clearly indicate that exploiting the natural abilities of V␥9V␦2 T-cells in cancer immunotherapy is feasible and carries low toxicity.

5.2. Invariant natural killer T-cells (iNKT) iNKT represent a distinct population of lymphocytes characterized by a (semi-)invariant TCR. Unlike conventional T-cells, iNKT recognize (glyco-)lipids presented by non-polymorphic CD1d molecules and rapidly secrete a wide range of cytokines upon stimulation thereby inducing effector (e.g. NK and CTL) cell activation in an IFN-␥ dependent manner [97–99]. 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 [100,101]. Moreover, iNKT from patients with advanced cancer display quantitative and qualitative defects and circulating numbers correlate with patient survival [100–103]. Reciprocal interactions between DC and iNKT can reverse defects in the iNKT population. Indeed, 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 [104,105]. 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. Potent tumor inhibition of aggressive tumor grafts expressing the targeted antigen was observed in mice [106,107]. Although less clear than in mice, 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]. In patients with recurrent HNSCC, 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. The number of infiltrating iNKT in extirpated tumor tissue correlated with clinical outcome [112]. In summary, 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, therefore redirecting this invariant subset potentially constitutes a valuable approach. 5.3. 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. 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.g. MHC class I chain-related genes (MIC) A and B) – “induced self” – and by Fc␥RIIIA ligation (i.e. ADCC). Upon activation, NK cells become highly cytotoxic (e.g. through perforin- and granzyme mediated mechanisms) and secrete pro-inflammatory cytokines (e.g. IFN-␥) [113,114]. Their role in tumor immunosurveillance is underscored by epidemiological studies correlating disease prognosis with




tumor infiltrating NK cells and diminished cytotoxicity with an increased risk of cancer development [114–116]. Additionally, NK cells were shown to contribute to mAb based cancer treatment though Fc␥RIIIA (CD16A) mediated recognition and elimination of mAb coated tumor cells. ADCC could be significantly enhanced through Fc-tail modifications resulting in increased Fc␥RIIIA affinity, at least in vivo [117]. Given the role NK cells play in first-line defense against malignancies, their therapeutic use has been widely explored in clinical trials. Adoptively transferred ex vivo expanded and activated autologous NK cells proved to be safe, however no significant clinical responses were observed. To break selftolerance associated with autologous NK, adoptive transfer of allogeneic NK cells has been investigated as an alternative. 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.v. IL-2 [116,118]. Of note, a substantial increase in host Tregs was reported after combined IL-2 and NK cell therapy [119], possibly limiting their therapeutic efficacy. Though proven safe, 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.g. Fc␥RIIIa ligation) and/or homing to tumors may be required for the induction of more robust anti-tumor responses [116,118]. As noted, several bsAb constructs (e.g. Triomab, 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 [10,61,120]. Moreover, 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, at least ex vivo [115]. Similarly, the fusion protein ULBP2anti-CEA scFv induced an effective anti-tumor response and NK activation in a syngeneic colon carcinoma mouse model [54]. Redirection of adoptively transferred NK cells by bsAbs targeting tumor cells and NK cells may therefore prove to be highly effective. In conclusion, targeting invariant/conserved lymphocyte subsets through their highly conserved receptor (e.g. V␥9V␦2-TCR, iNKT-TCR, 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. Importantly, their conserved receptors preclude the need for individualized treatment.

6. 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

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killing by engaged effector cells. Indeed, (pre-)clinical trials have demonstrated results that are not just an enhancement of those accomplished by conventional mAbs, but represent a whole new therapeutic repertoire and embody a leap forward in terms of therapeutic efficacy. Nevertheless, further improvements of the constructs with respect to increasing their half-life, stability, and tumor penetration as well as with respect to the specific immune effector cell subset to be targeted are required. Future research will reveal to what extent bsAbs will impact cancer treatment, 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. Undoubtedly, the expanding interest in this field will result in multiple compounds entering clinical trials in the near future shedding light on these issues.

Conflict of interest statement All authors have no conflicts of interest to declare.

Acknowledgments This work is supported by grant no. 90700309 from The Netherlands Organization for Health Research and Development (ZonMw), grant VU 2010-4728 from the Dutch Cancer Society (KWF), and grant 14-0343 from the Association for International Cancer Research (AICR).

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Biography Hans J. van der Vliet is a medical oncologist at the Department of Medical Oncology of the VU University Medical Center and Cancer Center Amsterdam. His translational research focuses on cancer immunotherapy with a special emphasis on conserved immunoregulatory and immune effector cell subsets.


Bispecific antibodies in cancer immunotherapy.

Bispecific antibodies for viral immunotherapy.

Targeted cancer immunotherapy via combination of designer bispecific antibody and novel gene-engineered T cells.

Novel antibody-based proteins for cancer immunotherapy.

Immunotherapy of cancer with antibody.

Utilizing the BiTE (bispecific T-cell engager) platform for immunotherapy of cancer.

A novel bispecific antibody, BiSS, with potent anti-cancer activities.

Cellular and Antibody Based Approaches for Pediatric Cancer Immunotherapy.

Circulating protein and antibody biomarker for personalized cancer immunotherapy.

Human c-erbB-2 proto-oncogene product as a target for bispecific-antibody-directed adoptive tumor immunotherapy.

Antibody-based immunotherapy for ovarian cancer: where are we at?

A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy.

Bispecific antibody: a tool for diagnosis and treatment of disease.

Amgen's bispecific antibody puffs across finish line.

Opposites attract in bispecific antibody engineering.

Co-targeting cancer stem-like cells and bulk cancer cells with a bispecific antibody.

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

natural anti-Gal antibody reaction.

NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy.

Boosting Cancer Immunotherapy with Anti-CD137 Antibody Therapy.

Immunotherapy for cancer.

Immunotherapy for bladder cancer.

Immunotherapy for Colorectal Cancer.

Fc gamma RI (MoAb 32).