Bispecific and bifunctional antibodies are attracting considerable interest as innovative anti-cancer therapeutics, but their ability to selectively localize at the tumor site has rarely been studied by quantitative biodistribution studies in immunocompetent animal models or in patients. Here, we describe the production of a novel bifunctional antibody, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin) fused to the extracellular portion of CD86 (co-stimulatory molecule B7.2). However, the fusion molecule was unable to target tumors in vivo. These data suggest that bispecific antibodies do not always localize on tumors and should therefore be characterized by imaging or biodistribution studie

Hemmerle, Teresa

Neri, Dario

Wulhfard, Sarah

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SHORT COMMUNICATIONA critical evaluation of the tumor-targeting propertiesof bispecific antibodies based on quantitativebiodistribution dataTeresa Hemmerle1, Sarah Wulhfard2 and Dario Neri1,31Department of Chemistry and Applied Biosciences, Institute ofPharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093Zurich, Switzerland and 2Philochem AG, c/o ETH Zu¨rich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland3To whom correspondence should be addressed.E-mail: neri@pharma.ethz.chReceived August 15, 2012; revised August 15, 2012;accepted August 16, 2012Edited by Phil HolligerBispecific and bifunctional antibodies are attracting consid-erable interest as innovative anti-cancer therapeutics, buttheir ability to selectively localize at the tumor site hasrarely been studied by quantitative biodistribution studiesin immunocompetent animal models or in patients. Here,we describe the production of a novel bifunctional antibody,consisting of the F8 antibody (specific to the alternativelyspliced EDA domain of fibronectin) fused to the extracel-lular portion of CD86 (co-stimulatory molecule B7.2).However, the fusion molecule was unable to target tumorsin vivo. These data suggest that bispecific antibodies do notalways localize on tumors and should therefore be charac-terized by imaging or biodistribution studies.Keywords: antibody F8/bispecific antibody/co-stimulatorymolecule B7.2/oncofetal fibronectin/vascular targetingIntroductionBispecific and bifunctional antibodies represent a novel classof anti-cancer biopharmaceuticals, which are developed withthe aim to activate an immune response at the tumor site,while sparing normal organs (Hammond et al., 2007;Nagorsen and Baeuerle, 2011; Pasche and Neri, 2012).Several bispecific antibody formats have been consideredand a number of products have been moved to clinical trials(Kontermann, 2005). Promising therapeutic results have re-cently been reported for clinical trials in patients withadvanced hematological malignancies (Topp et al., 2011).Similarly, the field of ‘immunocytokines’ (i.e. antibody-cytokine fusion proteins) has rapidly advanced. Severalimmunocytokines have been tested in preclinical models ofcancer and nine products have been moved to Phase I/II clin-ical trials (Kontermann, 2012; Pasche and Neri, 2012), withpromising results for the treatment of metastatic melanoma(Eigentler et al., 2011).In our lab, we have extensively studied many immunocyto-kines in quantitative biodistribution studies in tumor-bearingmice, using radioiodinated protein preparations and antibodiesof proven tumor-targeting performance. While fusion proteinsbased on certain cytokines or growth factors, such as interleu-kins (IL, e.g., IL2, IL12, IL15), tumor necrosis factor (TNF),vascular endothelial growth factor (VEGF, e.g., VEGF-120) andgranulocyte-macrophage colony-stimulating factor (GM-CSF)retained the excellent tumor-targeting properties of the parentalantibody, other immunocytokines or bifunctional proteins (e.g.those based on interferon (IFN)-g, trans-acting activator of tran-scription (TAT) peptides, calmodulin or dual cytokine fusions)completely abrogated tumor targeting at pharmacologicallyrelevant concentrations, while being fully immunoreactive invitro and displaying good pharmaceutical quality in biochem-ical assays (e.g. sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and gel-filtration analysis)(Pasche and Neri, 2012).To our knowledge, there is only one report in the literaturedescribing biodistribution studies of a bispecific antibody(Stork et al., 2009). Unfortunately, the study provides littleinformation about the tumor-targeting properties of theproduct, as it was performed in immunocompromised mice.In principle, a bispecific antibody capable of avid binding tocirculating leukocytes (e.g. T-cells) should have problems ex-travasating and reaching tumor cells outside the vascularcompartment.Performing biodistribution studies with bispecificantibodies is technically challenging, as it is difficult to dem-onstrate that both antibody moieties display full immunoreac-tivity. Indeed, in certain bispecific antibody formats, variabledomains may swap pairing, thus leading to a set of mole-cules with reduced immunoreactivity (Little and Kipriyanov,2007; Molhoj et al., 2007). Furthermore, it is difficult tomeasure the immunoreactivity towards antigens, which arenot available as pure protein, and which cannot be used inaffinity chromatography procedures (e.g. CD3 components).In order to generate and characterize the properties of abifunctional antibody molecule, with avid binding to T-cellsand to a validated tumor-associated antigen, we fused the F8antibody (specific to the alternatively spliced EDA domainof fibronectin, a marker of tumor angiogenesis; Rybak et al.,2007; Villa et al., 2008) with the extracellular portion ofmurine CD86 (B7.2; Sharpe and Freeman, 2002). B7.2 bindsto CD28 on T-cells with a dissociation constant Kd ¼ 20 mM(Collins et al., 2002). When avidity effects come into play,due to the multivalent display of CD28 and CD86 on the cellmembrane, the functional affinity of the interaction increases.Similarly, anti-CD3 antibody (Dreier et al., 2002) moietieswith Kd values in the 0.1 mM range have extensively beenused for the preparation of bispecific antibodies (Kipriyanovet al., 1999; Brischwein et al., 2006).# The Author 2012. Published by Oxford University Press. All rights reserved.For Permissions, please e-mail: journals.permissions@oup.com851Protein Engineering, Design & Selection vol. 25 no. 12 pp. 851–854, 2012Published online September 12, 2012 doi:10.1093/protein/gzs061The binding properties of B7.2-based bispecific fusionproteins can be readily tested by affinity chromatography,using the tumor-associated antigen or murine CTLA-4immobilized on resin (Eagar et al., 2002).In our study, we observed that the F8 antibody efficientlytargets tumors in vivo, but completely loses its tumor-targeting ability when fused to murine B7.2. These datasuggest that the analysis of the tumor-targeting properties ofbispecific and bifunctional antibodies by quantitative biodis-tribution studies should represent an essential activity inproduct development strategies, since therapeutic perform-ance correlates with the ability of the biopharmaceutical tolocalize at the site of disease (Carnemolla et al., 2002; Halinet al., 2002b; Wagner et al., 2008).MethodsCell lines and mouse tumor modelHEK 293 cells (CRL-1573, ATCC) were cultured inFreestyle 293 expression medium (Gibco) containing 0.1%Pluronic F68 (Sigma) in a shaker incubator at 378C and 5%CO2. The murine teratocarcinoma F9 cell line (CRL-1720,ATCC) was cultured as described before (Pasche et al.,2011). Female 129SvPas and Balb/c nude mice wereobtained from Charles River (Germany).Cloning and protein expressionThe gene structure for SIP(F8) and for the F8 diabody (Villaet al., 2008) has been previously described. F8-B7.2 containsthe extracellular domain of murine B7.2 (gene from SourceBioScience) sequentially fused to the diabody (Pasche et al.,2011).The fusion protein was expressed using transient gene ex-pression in HEK-293 cells (Backliwal et al., 2008) and puri-fied by protein A chromatography. The purified protein wasanalyzed by SDS-PAGE before and after deglycosylationwith PNGase F (NEB), size exclusion chromatography(Superdex200 10/300GL, GE Healthcare), BIAcore on aEDA antigen-coated sensor chip and immunofluorescencestaining of F9 tumor sections as previously described(Pasche et al., 2011).Biodistribution studiesThe in vivo targeting performance was assessed by quantita-tive biodistribution studies as described before (Pasche et al.,2011) and 15 mg of radioiodinated F8-B7.2 was injected intothe tail vein of tumor-bearing mice. Mice were sacrificed atdifferent time points after injection. Experiments were per-formed under a project license granted by the Veterinaeramtdes Kantons Zurich (169/2008).Fig. 1. Cloning, expression and characterization of F8-B7.2. (a) Schematic representation of the cloning strategy of F8-B7.2. Lead Seq, leader sequencepeptide; VH, VL, variable heavy and light chain. (b) Domain assembly of Sip (F8), diabody (F8) and F8-B7.2. (c) SDS-PAGE analysis of purified F8-B7.2.MW, molecular marker; 1, F8-B7.2; 2, deglycosylated F8-B7.2; NR, R, non-reducing and reducing conditions. (d) Gel filtration analysis of affinity-purifiedF8-B7.2. 1, Thyroglobulin 669 kDa; 2, BSA 67 kDa; 3, b-lactoglobulin 35 kDa. (e) Immunofluorescence analysis of murine F9 tumors. Tumor sections werestained with a biotinylated F8-B7.2 (green, Alexa488) and an anti-CD31 antibody (red, Alexa594). Magnification 20, scale bars ¼ 100 mm. (f ) BIAcoreanalysis of purified F8-B7.2 on an EDA-coated sensor chip. (g) BIAcore analysis of F8-B7.2 on a CTLA-4-Fc-coated sensor chip. (h) Immunoreactivity of125I-SIP (F8) and 125I-F8-B7.2 on EDA-resin and on CTLA-4-Fc-resin: 1, 125I-SIP (F8) on CTLA-4-Fc-resin (n ¼ 3); 2, 125I-SIP (F8) on EDA-resin (n ¼ 3); 3,125I-F8-B7.2 on CTLA-4-Fc resin (n ¼ 3); 4, 125I-F8-B7.2 on EDA-resin (n ¼ 3).T.Hemmerle et al.852Immunoreactivity testingCNBr-activated sepharose (GE Healthcare) was coupled toEDA or to murine CTLA-4-Fc. The retention of radioiodi-nated F8-B7.2 on resin was analyzed by affinity chromatog-raphy, as described (Pasche et al., 2011).Results and discussionThe fusion protein F8-B7.2 was expressed in non-covalenthomodimeric ‘diabody’ format (Fig. 1a and b), leading to fullyimmunoreactive products with bivalent (i.e. avid) tumorbinding and long residence time on neoplastic lesions in vivo(Pasche and Neri, 2012). The corresponding F8 antibody, indiabody format and in Small Immune Protein (SIP) format(Fig. 1b), has been extensively studied in biodistribution experi-ments (Villa et al., 2008). F8-B7.2 was purified to homogeneityby affinity chromatography and was well behaved inSDS-PAGE (Fig. 1c and d). The fusion protein displayed ahigh degree of N-linked glycosylation, as evidenced bySDS-PAGE analysis before and after N-glycanase treatment.F8-B7.2 stained neo-vascular structures in F9 murine tumors(Fig. 1e) and displayed full retention of immunoreactivity inBIAcore (Fig. 1f and g) and in affinity chromatography proce-dures with immobilized EDA domain of fibronectin or immobi-lized murine CTLA-4-Fc fusion (Fig. 1h).When tested in biodistribution studies in immunocompe-tent (Fig. 2a) and in nude mice (Fig. 2b) carrying subcutane-ously grafted F9 tumors, a radioiodinated preparation ofF8-B7.2 failed to preferentially localize on the tumor 24 hafter intravenous (i.v.) injection, while the F8 antibody inSIP format (Fig. 2a and b) and in diabody format (Villaet al., 2008) efficiently targeted the neoplastic lesions.Biodistributions at earlier time points (e.g. 15 min after i.v.injection; Fig. 2c) revealed that the majority of the proteinhad already been excreted via the hepatobiliary route, whilenever reaching the cognate antigen in vivo, which is localizedon the abluminal side of tumor blood vessels (Rybak et al.,2007; Villa et al., 2008).The selective localization of antibody-fusion proteins tothe site of disease in vivo is strictly dependent on the pay-loads that are attached to the antibody. Our group observedthat highly charged payloads (e.g. Calmodulin (Melkkoet al., 2002), VEGF-164 (Halin et al., 2002a) and HIV-1TAT peptides (Niesner et al., 2002)), bulky payloads (Gafneret al., 2006) and payloads prone to receptor trapping whenadministered at low doses (e.g. IFNg; Ebbinghaus et al.,2005) can completely abrogate tumor targeting.Our data suggest that it is not obvious that a bifunctionalprotein with avid binding to circulating leukocytes wouldefficiently extravasate and reach a tumor-associated antigenin vivo, even if the parental antibody displays good tumor-targeting properties. In the case of F8-B7.2, the tumor-targetingprocess does not take place even in immunocompromised miceand is prevented by the rapid clearance from blood through theliver. Due to its heavily glycosylated nature, the fusion proteinis subject to binding by carbohydrate-recognizing receptors onhepatocytes and the resulting efficient and fast blood clearancerate prohibits the targeting to the site of disease.Some bispecific and bifunctional antibodies have demon-strated a dramatic therapeutic activity in certain clinical trialsand it is possible that even small amounts of therapeuticprotein, delivered at the site of disease, may mediate a potentanti-tumoral immune response. However, there is ample evi-dence that efficient tumor localization of bifunctional anti-bodies correlates with therapeutic performance and aquantitative biodistribution study of targeting should repre-sent an essential requirement for future product developmentin this field.AcknowledgementsThis work was supported by the Swiss National Science Foundation, theETH Zu¨rich, the European Union (ADAMANT Project), the Swiss CancerLeague, the Swiss-Bridge Foundation and the Stammbach Foundation.Contribution: T.H. conceived the experiments, produced the protein andperformed biodistribution studies and wrote the manuscript; S.W. developedTGE methodologies for protein expression, helped in biodistribution studiesand corrected the manuscript; D.N. conceived the experiments, supervisedthe experimental work and wrote the manuscript.Conflict of interest: Dario Neri is a co-founder and shareholder of Philogen(www.philogen.com), the company which owns the F8 antibody.Fig. 2. Biodistribution study of radioiodinated F8-B7.2. Mice bearingsubcutaneous F9 teratocarcinoma were injected i.v. with 15 mg radiolabeledprotein. (a) Immunocompetent 129SvPas mice injected with 125I-SIP (F8)(black bar, n ¼ 5) or with 125I-F8-B7.2 (gray bar, n ¼ 4). Mice weresacrificed after 24 h. (b) Nude mice injected with 125I-SIP (F8) (black bar,n ¼ 5) or with 125I-F8-B7.2 (gray bar, n ¼ 4). Mice were sacrificed after24 h. (c) Nude mice injected with 125I-F8-B7.2 (n ¼ 4). Mice were sacrificedafter 15 min. Organs were excised and radioactivity counted, expressingresults as percent of injected dose per gram of tissue (%ID/g+SE).The tumor-targeting properties of bispecific antibodies853ReferencesBackliwal,G., Hildinger,M., Hasija,V. and Wurm,F.M. (2008) BiotechnolBioeng, 99, 721–727. First published on 2007/08/08, doi: 10.1002/bit.21596.Brischwein,K., Schlereth,B., Guller,B., Steiger,C., Wolf,A., Lutterbuese,R.,Offner,S., Locher,M., Urbig,T., Raum,T., et al. 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A critical evaluation of the tumor-targeting properties of bispecific antibodies based on quantitative biodistribution data

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