pharmacodynamic and antineoplastic activity of bi 836845 ...ﬁed igf-i (cm001) and igf-ii (fm001;...

Large Molecule Therapeutics

Pharmacodynamic and Antineoplastic Activity of BI 836845,a Fully Human IGF Ligand-Neutralizing Antibody, andMechanistic Rationale for Combination with Rapamycin

Katrin Friedbichler1, Marco H. Hofmann1, Monika Kroez2, Elinborg Ostermann1, Herbert R. Lamche1,Christian Koessl1, Eric Borges1, Michael N. Pollak3, G€unther Adolf1, and Paul J. Adam1

AbstractInsulin-like growth factor (IGF) signaling is thought to play a role in the development and progression of

multiple cancer types. Todate, therapeutic strategies aimedat disrupting IGF signalinghave largely focusedon

antibodies that target the IGF-I receptor (IGF-IR). Here, we describe the pharmacologic profile of BI 836845, a

fully human monoclonal antibody that utilizes an alternative approach to IGF signaling inhibition by

selectively neutralizing the bioactivity of IGF ligands. Biochemical analyses of BI 836845 demonstrated high

affinity tohuman IGF-I and IGF-II, resulting in effective inhibition of IGF-induced activation of both IGF-IR and

IR-A in vitro. Cross-reactivity to rodent IGFs has enabled rigorous assessment of the pharmacologic activity of

BI 836845 in preclinical models. Pharmacodynamic studies in rats showed potent reduction of serum IGF

bioactivity in the absence of metabolic adverse effects, leading to growth inhibition as evidenced by reduced

body weight gain and tail length. Moreover, BI 836845 reduced the proliferation of human cell lines derived

from different cancer types and enhanced the antitumor efficacy of rapamycin by blocking a rapamycin-

induced increase in upstream signaling in vitro aswell as in human tumor xenograft models in nudemice. Our

data suggest that BI 836845 represents a potentially more effective and tolerable approach to the inhibition of

IGF signaling compared with agents that target the IGF-I receptor directly, with potential for rational

combinations with other targeted agents in clinical studies. Mol Cancer Ther; 13(2); 399–409. �2013 AACR.

IntroductionInsulin-like growth factors I and II (IGF-I and IGF-II) are

structurally related polypeptides that promote cellgrowth and survival (1). In normal physiology, IGFs havekey roles in the control of cellular proliferation and sur-vival and of organism growth (2). Their concentrations inblood are physiologically regulated and reflect hepaticproduction, but IGFs are also expressed locally in manytissues in a paracrine or autocrine manner (3). Tissue IGFbioactivity varies not only with circulating ligand concen-tration andwith local ligand production, but alsowith theconcentrations of the various serum IGF-binding proteins(4, 5).IGF-I signals bybinding to the IGF-I receptor (IGF-IR) or

tohybridsof IGF-IRand insulin receptors (INSR; refs. 1, 6).IGF-II initiates signaling via these same receptor species,

but can also activate the A isoform of the INSR (IR-A;refs. 7, 8). This is of particular interest from an oncologicperspective as IR-A is preferentially expressed in fetal andcancer cells (9, 10). In contrast with members of the EGFfamily, which are frequently amplified and/or mutation-ally activated in neoplasia, resulting in ligand-indepen-dent receptor activation, IGF-IR/INSR signaling alwaysrequires active ligands.

More than a decade of preclinical research has provideda rationale for targeting IGF signaling in the treatment ofcancer (1, 11). Oncogenic transformation ofmouse embry-onic fibroblasts, for instance, has been described to bedependent on the expression of IGF-IR (12). Expression ofIGF-II by transformed cells has been observed (13) andthis can lead to activation of IGF-IR in an autocrinemanner. Inmanypreclinical cancermodels, tumorgrowthcan be stimulated by IGF-I or IGF-II, and tumors growsubstantially slower in mice carrying a mutation thatreduces IGF ligand levels (4). Moreover, a separate lineof indirect evidence for the relevance of IGF signaling forneoplasia comes from the observation that there is con-siderable interindividual variation in circulating IGF-Ilevels, and that risk and prognosis of certain cancers arerelated to circulating IGF-I concentration (2, 14, 15). Final-ly, IGF signaling may also play a role as a resistancemechanism, which limits the effectiveness of cytotoxic ortargeted anticancer agents, including rapalogs (16–21).

Authors' Affiliations: 1Boehringer IngelheimRCVGmbH&CoKG, Vienna,Austria; 2Boehringer Ingelheim Pharma GmbH & Co KG, Biberach/Riss,Germany; and 3Departments of Medicine and Oncology, McGill University,Montreal, Quebec, Canada

CorrespondingAuthor:Paul J. Adam, Boehringer IngelheimRCVGmbH&Co KG, Dr. Boehringer Gasse 5-11, A-1121, Vienna, Austria. Phone: 43-80105-2310; Fax: 43-80105-32310; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0598

�2013 American Association for Cancer Research.

MolecularCancer

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Published OnlineFirst December 2, 2013; DOI: 10.1158/1535-7163.MCT-13-0598

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This preclinical evidence has motivated the develop-ment of several IGF-I receptor-targeted monoclonalantibodies (mAb; refs. 22–24). However, efficacy of anti-IGF-IR antibodies has been disappointing in clinical trialsto date (25–30). One possible explanation for these resultsis that in clinical use, drugs studied to date do not ade-quately inhibit IGF signaling, especially in the context ofthe need to block autocrine loops in IGF-dependent can-cers (31–33), particularly thosedrivenby IGF-II,which canalso signal via IR-A (34). For the alternative approach oftargeting all receptor species in the insulin/IGF tyrosinekinase receptor family using small-molecule inhibitors,the dosing of these agents must be well balanced to avoidmetabolic toxicity, and it remains to be demonstrated thatclinical use at tolerable doses leads to adequate receptorblockade in neoplastic tissue.

A third therapeutic strategy is to target the IGF ligandsrather than the receptors, aiming to abrogate signaling viaIGF-IR and IR-A aswell as their hybrid receptors, withoutimpact on circulating insulin and glucose levels and theirmetabolic functions (30). Two agents, BI 836845 andMEDI-573 (35), with this mode of action have been devel-oped. Here, we describe the pharmacologic activity of BI836845, a fully human mAb that neutralizes the activitiesof both IGF-I and IGF-II. In contrast with MEDI-573, BI836845 cross-reacts with mouse and rat IGF-I and IGF-II,allowing a comprehensive in vivo characterization of thepharmacodynamic properties of the antibody in preclin-ical rodent models.

Materials and MethodsReagents and cell lines

BI 836845 was isolated by selection of specific Fabfragment clones from the human combinatorial antibodyphage display library (HuCAL Gold; ref. 36) that bindhuman IGF-I with low nanomolar affinity, in three pan-ning cycles as previously described (37) and subsequentlysubjected to in vitro affinity maturation (38). IGF-IR anti-bodies usedwereaIR-3 (Calbiochem,No.GR11L) andonewhich was generated on the basis of sequence ID 2.13.2 ofthe patentWO02/053596A2 (termed IGF-IRmAb). Otherreagents included rapamycin (LC laboratories) and puri-fied IGF-I (CM001) and IGF-II (FM001; GroPep Biorea-gents Pty Ltd.).

Recombinant mouse cell lines expressing human IGF-IR and IR-A, respectively, were kindly provided by R.Vigneri (University of Catania, Italy). GEO cell line (orig-inally established by M. Brattain; Roswell Park CancerInstitute, Buffalo, NY and Baylar College of Medicine,Houston, TX)was obtained from S. Pepe (Universit�a degliStudi di Napoli, Italy). RD-ES [American Type CultureCollection (ATCC) #HTB-166], SK-ES-1 (ATCC, #HTB-86), and all other cell lines used were purchased fromATCC, Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Japanese Collection of ResearchBioresources Cell Bank (JCRB), or the Memorial Sloan-Kettering Cancer Center (New York, NY). Cell lineauthenticationwas conducted in-house or by the provider

using short tandem repeat PCR. Upon delivery, cell lineswere expanded and low-passage vials stored in liquidnitrogen. Experiments were carried out within 12 weeksafter resuscitation.

Biacore analysisBinding affinity (KD) was determined using a Biacore

3000 high-resolution surface plasmon resonance systemin Hepes Buffer Saline containing EDTA and SurfactantP-20 (HBS-EP) buffer þ 1 mg/mL CM-Dextran and 0.25mg/mLbovine serum albumin (BSA) as running buffer ata flow rate of 20 mL/minute. The sensor chip was coatedwith approximately 1,000 RU of an unspecific referenceantibody in flow cell 1 and approximately 1,000 RU of arabbit-anti-human Fc-g–specific antibody in flow cell 2using reagents from an amine coupling kit. The testantibody was captured by running a 1 mg/mL solutionover the sensor surfaces for 3 minutes. IGF antigens werediluted to 500, 250, 125, 62.5, and 31.3 nmol/L and mea-sured in randomorderusing 5minutes for association anddissociation. Data evaluation was performed using theBIAevaluation software, version 4.1. Biacore 3000 instru-ment; HBS-EP buffer, amine coupling kit, and BIAevalua-tion software were provided by Biacore/GE Healthcare.

Cell-based IGF-IR and IR-A phosphorylation assaysMouse embryonic fibroblast cell linesderived from IGF-

IR–deficient mice and engineered to overexpress humanIGF-IR or human IR-A were used to measure IGF bioac-tivity, defined here as the ability of a sample (serum,plasma, cell culture media) to stimulate receptor phos-phorylation as determined by ELISA. The cell lines weremaintained in Dulbecco’s modified Eagle medium sup-plemented with 10% heat-inactivated FBS, 1 mmol/Lsodium pyruvate, 0.075% sodium bicarbonate, nonessen-tial amino acids (Gibco), and 0.3 mg/mL puromycin at37�C and 5% CO2. The ELISA was performed followingstandard procedures. Briefly, 10,000 cells/well were seed-ed on 96-well plates followed by a 24-hour incubation.Cells were starved in medium with 0.5% FBS overnightbefore a dilution range of BI 836845 or IGF-IR antibodieswas added to the cells, followed by either IGF-I (20ng/mL), IGF-II (100 ng/mL), or a human serum pool(20%). Cellswere incubated for 30minutes at 37�Cand 5%CO2 and subsequently fixed in 4% formaldehyde/PBS (20minutes, room temperature), quenched (1.2 wt% hydro-gen peroxide in wash buffer, 30 minutes, room temper-ature), and blocked (5% BSA in wash buffer, 60 minutes,room temperature with agitation). Subsequently, cellswere incubated with primary antibody [phospho-IGF-IR-b (tyr1135/1136)/insulin receptor-b (tyr1150/1151)antibody; Cell Signaling Technology #3024, 1:1,000 inblocking buffer, incubation overnight at 4�C withagitation] and secondary antibody (anti-rabbit immuno-globulin G goat immunoglobulins conjugatedwith horse-radish peroxidase; Dako 0#P0448, 1:500 in blocking bufferfor 60 minutes at room temperature with agitation)followed by substrate solution (tetramethylbenzidine;

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Bender MedSystems #BMS406.1000; for 5 to 20 minuteswith agitation). Finally, the reaction was stopped byadding 1mol/L phosphoric acid, and the absorbance wasread using a photometer (OD 450 nm, OD 650 nm asreference).

Rat studiesSix to 8-week-old male rats (Crl:WI[Han]) were treated

intravenously onceweekly for 13weekswith 20, 60, or 200mg/kg of BI 836845 and compared with vehicle-treatedanimals. The treatment phase was followed by a 12-weekrecovery phase. Plasma sampleswere taken at time pointsbefore, during, and after treatment. Rat body weight wasmeasured twice weekly throughout the study and taillength at the end of the treatment period.

Xenograft studiesTumors (<100 mm3) were established from cultured

GEO or RD-ES cells (5� 106 inMatrigel) by subcutaneousinjection into the right flanks of female BomTac:NMRI-Foxn1nu mice. Animals were randomized (n ¼ 7/group)and treated with vehicle (0.5% Natrosol) or rapamycin(5 mg/kg) intraperitoneally (i.p.) once a day for 5 conse-cutive days per week, twice weekly with BI 836845 (100mg/kg), or a combination of both agents. Tumor volumeswere determined three times a week using a digitalcaliper. Body weights of the mice were measured dailyas an indicator of tolerability of the compounds. Fourhours after the last treatment (day 20), serumwas sampledand RD-ES tumors were explanted, lyzed in Bio-Plex CellLysis Kit (Bio-Rad #171-304012) containing 2 mmol/Lphenylmethylsulfonylfluoride, and analyzed for AKTphosphorylation by Western blotting (n ¼ 4/treatmentgroup). Rapamycin treatment of non–tumor-bearingmicefor one week and subsequent serum sampling were per-formed similarly.

Additional materials and methodsTwo-dimensional (2D) cell viability assays, Western

blot, and quantitative real-time PCR (qRT-PCR) analyses(primers/probes listed in Supplementary Table S2), anddetermination of total rat serum IGF-I levels and ex vivoIGF-I bioactivity are described in the SupplementaryMaterials and Methods.

Statistical analysisStatistical analysis was performed using the GraphPad

Prism software (GraphPad Software Inc.). Control (pre-treatment or vehicle-treated animals), and treatmentgroups were compared in one-way ANOVA followed bya Tukey post test if not indicated otherwise. P values lessthan 0.05 (�), 0.01 (��), and 0.001 (��)were considered to bestatistically significant.

ResultsBiochemical characterization of BI 836845BI 836845 is a fully human mAb selected from a na€�ve

phage display library and affinity optimized for IGF-I

binding. As shown in Supplementary Table S1, surfaceplasmon resonance analysis demonstrated that in addi-tion to the high affinity for human IGF-I (0.07 nmol/L), BI836845 also shows high affinity for human IGF-II (0.8nmol/L). BI 836845was also shown to strongly cross-reactwith mouse and rat IGF-I and IGF-II (SupplementaryTable S1). No binding to human insulin was detected atup to 100-fold higher antibody concentrations (data notshown).

To measure IGF bioactivity (IGF-IR or IR-A kinaseactivating activity) in serum, plasma, or cell culturemedia, we developed a cell-based assay utilizingmouse fibroblasts engineered to express human IGF-IR or IR-A, in which receptor phosphorylation can bequantified by ELISA. This assay was initially used todetermine the potency and effectiveness of BI 836845 inneutralizing recombinant IGF-I and IGF-II. As shownin Fig. 1A and B, BI 836845 inhibited both IGF-I (20ng/mL) and IGF-II (100 ng/mL)-induced IGF-IR phos-phorylation with an IC50 of 90 ng/mL (0.6 nmol/L) and1,120 ng/mL (7.5 nmol/L), respectively. In the sameassay, two IGF-IR mAbs were less effective with respectto IGF-I–induced activation and displayed only weakeffects on IGF-II–induced activation with more than30% of IGF-II–induced IGF-IR phosphorylation remain-ing even at an antibody concentration of 100 nmol/L(Fig. 1B). Figure 1C demonstrates that BI 836845 alsopotently inhibited IGF-II (100 ng/mL)-induced IR-Aactivation with an IC50 of 815 ng/mL (5.4 nmol/L),unlike IGF-IR–targeted antibodies that had no effect.When cell culture medium containing 20% humanserum was used to stimulate receptor phosphorylation,this could be completely neutralized by BI 836845 withan IC50 of 246 ng/mL (1.6 nmol/L). In contrast, an IGF-IR–targeted antibody showed only partial inhibition ofIGF bioactivity (Fig. 1D).

Pharmacodynamic effects of BI 836845 in vivoThe cross-reactivity of BI 836845 to mouse and rat

IGF-I and IGF-II has allowed a comprehensive preclin-ical characterization of the pharmacodynamic proper-ties of the antibody in these species. Pharmacodynamiceffects of BI 836845 were studied in 6- to 8-week-oldrats treated once weekly for 13 weeks with 20, 60, or 200mg/kg of the antibody (last treatment on day 85).Treatment with BI 836845 at all dose levels was welltolerated and resulted in a clear reduction in plasma IGFbioactivity as determined ex vivo by the bioassaydescribed above (Fig. 2A). These effects of BI 836845on plasma IGF bioactivity were seen despite a corre-sponding increase in total IGF-I plasma levels. Figure 2Bshows that treatment with BI 836845 resulted in elevat-ed total IGF-I from day 3 onward, with up to 25-foldincreases seen for the groups treated with 60 and 200mg/kg. Following cessation of treatment, a dose-depen-dent return of both plasma IGF bioactivity (Fig. 2A) andtotal IGF-I levels (Fig. 2B) toward control levels wasseen by day 169. A dose-dependent effect of BI 836845

Preclinical Properties of an IGF Ligand-Neutralizing mAb

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treatment was observed on rat body weight gain (Fig.2C). Compared with controls, the 60 and 200 mg/kgdoses showed a similar reduction in body weight gain,which was less pronounced for the 20 mg/kg dose level(Fig. 2C). To confirm that the effect of BI 836845 on bodyweight was due to a reduction in body growth, taillength was monitored. Figure 2D shows that there wasa dose-dependent reduction in tail length comparedwith vehicle controls on day 85 of the study. In additionto these findings, we noted that essentially all organweights were reduced. Moreover, we observed a reduc-tion in secondary spongiosa of bones, where IGF-I isknown to play an important role (ref. 39; data notshown).

Antineoplastic activity of BI 836845 in cancermodelsBI 836845 was assayed for in vitro growth inhibitory

activity in cancer-derived cell lines grown in 10% FBS.The proliferation of several cell lines derived from

different cancer types, including non–small cell lungcancer (NSCLC), small cell lung cancer (SCLC), Ewingsarcoma, and multiple myeloma, was found to bepotently inhibited by BI 836845 (low nmol/L EC50

values; Table 1). Approximately one third of theresponsive cell lines showed elevated levels of IGF-Iand/or IGF-II mRNA expression compared with medi-an expression of 625 cell lines (examples are shownin Fig. 3A), indicating that BI 836845 is capable ofattenuating in vitro proliferation of cell lines showingautocrine ligand production. The colorectal carcinomacell line GEO showed a particularly high level of IGF-IIexpression (50-fold over median; Fig. 3B), which wasassociated with constitutive IGF pathway activation intumors in vivo (Fig. 3C). In addition, we assessedmRNA expression of INSR and its variants by qRT-PCR and identified expression of both INSR splicevariants in GEO cells (Supplementary Fig. S1A). Incontrast, the Ewing sarcoma cell line RD-ES exclusively

Figure 1. Effect of BI 836845 or receptor-targeted mAbs on recombinant IGF or human serum-induced receptor phosphorylation. In mouse cellsengineered to express human IGF-IR, BI 836845 (up to 15 mg/mL; 100 nmol/L) effectively inhibited IGF-IR phosphorylation induced by (A) IGF-I(20 ng/mL) and (B) IGF-II (100 ng/mL), with an IC50 of 90 ng/mL (0.6 nmol/L) and 1120 ng/mL (7.5 nmol/L), respectively. Two IGF-IR–targetedmAbs were much less effective at inhibiting IGF-II activity. C, in IR-A–expressing cells, BI 836845 blocked IGF-II (100 ng/mL)-induced IR-Aphosphorylation with an IC50 of 815 ng/mL (5.4 nmol/L), whereas IGF-IR–targeted antibodies showed no effect. D, BI 836845 potently neutralizedserum IGF bioactivity in 20% pooled human serum (IC50 of 1.6 nmol/L), whereas an IGF-IR mAb showed only partial inhibition. Results presentedare representative of three independent experiments.

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expressed the IR-A variant (Supplementary Fig. S1C),in line with previously published findings (40). In bothcell lines, no insulin mRNA expression was detectable(Supplementary Fig. S1A and S1C), and INSR mRNAlevels were significantly lower than those of IGF-IR(Supplementary Fig. S1B and S1D). This difference wasalso reflected at the protein level in RD-ES cells in vitroand in vivo (Supplementary Fig. S1E). The GEO cell linewas established as a subcutaneous xenograft model inimmunodeficient (nude) mice to determine whether BI836845 could inhibit the in vivo growth of a tumor withhigh IGF-II expression. Figure 3D shows that treatmentwith 100 mg/kg of BI 836845 twice weekly resulted in54% tumor growth inhibition (TGI) after 27 days oftreatment.

BI 836845 enhances the antineoplastic efficacy ofrapamycin

Previous studies have shown improved efficacy whencombining IGF-IR mAbs with rapamycin (41, 42). Wewere therefore interested in assessing whether the com-bination of an IGF ligand-neutralizing antibody withrapamycin would be an effective antineoplastic strate-gy. We therefore tested the combined effect of BI 836845and rapamycin on the viability and signaling of two BI836845-sensitive Ewing sarcoma-derived cell lines, SK-ES-1 and RD-ES. Cell viability studies in vitro demon-strated that the combination of BI 836845 and rapamycinwas more potent and effective than either single agent atinhibiting proliferation of both cell lines (Fig. 4A and C;P < 0.001 at concentrations �12.33 and �0.1 nmol/L,

Figure 2. Comprehensive preclinical characterization of the pharmacodynamic properties of BI 836845 in growing rats. Intravenous treatment of growingmale rats with 20, 60, or 200 mg/kg of BI 836845 (n � 24/group) once weekly for 13 weeks followed by a 12-week recovery period showed (A) ahighly significant reduction in plasma IGF bioactivity during the treatment period for all dose levels relative to untreated controls (P < 0.0001).At the end of the recovery phase (day 169), a dose-dependent increase toward baseline was observed. B, BI 836845 treatment led to elevated totalIGF-I levels in the plasma, which returned to baseline during the recovery period. During treatment all doses showed highly significant effects(P < 0.0001) compared with control animals, with the peak increase for 60 and 200 mg/kg being greater than that seen with 20 mg/kg. Treatment withBI 836845 at all dose levels resulted in a significant reduction of (C) body weight gain (P < 0.0001) and (D) tail length in growing male rats at day85. The Kruskal–Wallis test with Dunn post hoc test was performed for statistical evaluation of differences in tail lengths. �, P < 0.05; ��, P < 0.01; and��, P < 0.001 (D).






respectively). To investigate the molecular mechanismbehind the improved efficacy seen when both agentswere combined, we assessed AKT phosphorylationlevels (serine 473; pAKT) as a biomarker reflecting theconsequences of rapamycin-induced relaxation of aninhibitory feedback loop (43). Treatment of SK-ES-1(Fig. 4B) and RD-ES cells (Fig. 4D) with 100 nmol/Lrapamycin resulted in increased pAKT compared withuntreated cells. When BI 836845 was combined withrapamycin, phosphorylation of AKT was inhibited,indicating that the feedback increase in pAKT in the

presence of rapamycin is dependent on IGF ligand-driven signaling (Fig. 4B and D). The inhibitory effectson cell proliferation of the combination versus eachsingle agent were tested in 60 cancer cell lines (Supple-mentary Fig. S2A). In 13 out of 17 cell lines derived fromdifferent cancer types, the effects on proliferation wereconsistent with the effects on AKT phosphorylation(Supplementary Fig. S2B).

An RD-ES xenograft model was used to determinewhether the additive effects of BI 836845 and rapamycinseen in vitro could be recapitulated in vivo. The combi-nation of BI 836845 (twice weekly, 100 mg/kg i.p.) andrapamycin (5 � weekly, 5 mg/kg i.p.) was more effec-tive (TGI 93%) at inhibiting tumor growth than eithersingle agent (rapamycin TGI 75%, BI 836845 TGI 53%)after 20 days of treatment (Fig. 5A). Single-agent treat-ments were significantly different from control treat-ment from day 6 onward (P < 0.05 for rapamycin andP < 0.01 for BI 836845 on day 6), whereas combinationtreatment differed significantly from controls from day 3onward (P < 0.05 on day 3). Differences between single-agent treatments and combination treatment were sta-tistically significant from day 8 onward (P < 0.05 on day8). Further analyses of pAKT levels in the RD-ES tumorsshowed that rapamycin increased pAKT levels, and thatthis increase could be inhibited by BI 836845 (Fig. 5B).IGF bioactivity in the serum of RD-ES tumor-bearingmice was increased upon rapamycin treatment com-pared with control mice (Fig. 5C). BI 836845 treatmentalone, and in combination with rapamycin, completelyinhibited IGF bioactivity in plasma (Fig. 5C). To inves-tigate the contribution of RD-ES tumors to the elevated

Figure 3. Activity of BI 836845 incancer-derived cell lines in thepresence of autocrine IGF ligands.A, IGF-I, IGF-II, and IGF-IRexpression data of selectedsensitive cancer cell lines and (B) ofcolorectal GEO cells (probe setsNM_000618_at, NM_000612_at,and NM_000875_at of Exon chipdataset of 625 cell lines). Medianexpression was calculated from allcell lines in dataset. C, 50-fold overmedian IGF-II mRNA expression inthe GEO cell line was associatedwith constitutive pathwayactivation in explanted GEOxenograft tumors as assessed byp-Tyr1135/1136-IGF-IR and p-Ser473-AKTWestern blot analyses(n ¼ 2); the respective bandsshown were cropped from thecomplete blots. D, intraperitonealadministration of 100 mg/kg of BI836845 twice weekly for 27 dayssignificantly inhibited the growth ofGEO xenograft tumors by 54% (P <0.01); two-tailed t test was used forstatistical evaluation.

Table 1. Activity of BI 836845 in cancer-derivedcell lines

Cancer indication Cell lineEC50 mg/mL(nmol/L)

Multiple myeloma NCI-H929 2 (13)Multiple myeloma LP-1 0.06 (0.4)Ewing sarcoma TC-71 0.7 (5)Ewing sarcoma SK-ES-1 3 (20)Ewing sarcoma RD-ES 1.8 (12)Neuroblastoma TC-177 1 (7)Small cell lung cancer NCI-H128 6.8 (45)Non–small cell lung cancer NCI-H2122 15 (100)

NOTE: The effect of BI 836845 on viability of cancer-derivedcell lines was tested in 2D assays. EC50 values of selectedcell lines inhibited by BI 836845 at a low nmol/L concentra-tion are shown.

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levels of plasma IGF bioactivity, we treated non–tumor-bearing mice similarly and found that rapamycininduced a significant increase in plasma IGF bioactivitycomparable with that seen in the RD-ES tumor-bearingmice (Fig. 5D).

DiscussionBI 836845, a fully human mAb, blocks the activation of

IGF-IR and IR-A by binding to and neutralizing both IGFligands. The preclinical data presented in this study con-tributed to the rationale for a clinical trial program, andphase I studies are now in progress.The mechanism of action of BI 836845 is distinct from

that of IGF-IR–targeted antibodies, which to date haveproved disappointing in clinical studies (30). By bindingto and neutralizing the IGF ligands, BI 836845 blocksactivation not only of the IGF-IR, but unlike anti-IGF-IR

antibodies also reduces IGF-II–stimulated activation ofIR-A. This may be an important advantage, as the expres-sion of this receptor variant is often increased in tumorcells (7, 8, 10, 44) and may play a role in autocrine IGF-II–mediated growth stimulation (34, 40). In addition, someIGF-IR antibodies have been shown to be weak inhibitorsof IGF-II–driven IGF-IR signaling, which is thought to bedue to differences in the binding sites of IGF-I and IGF-IIon the IGF-I receptor (45).

The rodent models presented here are informative notonly with regards to in vivo efficacy but also to tolera-bility of BI 836845, as this antibody blocks the bioactivityof both human and rodent IGF ligands. Long-termadministration of BI 836845 to rats was well toleratedat doses sufficient to reduce somatic growth. Growthinhibition of rats was associated with a substantialdecrease of serum IGF bioactivity, a relevant pharma-codynamic endpoint. This was achieved in the presence

Figure 4. Combination potential of BI 836845 with rapamycin. In SK-ES-1 cells, (A) treatment with the combination of BI 836845 and rapamycin for5 days resulted in a significant reduction of cell viability and (B) treatment with 100 nmol/L rapamycin for 24 hours increased AKT Ser473-phosphorylation,which was inhibited by the addition of 100 nmol/L BI 836845. C, cell viability and (D) pAKT levels of RD-ES cells were altered similarly. For cell viabilityassays (A and C), samples were measured in triplicates. Results of both cell lines are representative of two independent experiments each. Statisticalanalysis was performed using a Mixed Model for Repeated Measurements with SAS 9.2 (SAS Institute Inc.). AKT Ser473-phosphorylation (B and D) wasnormalized to GAPDH loading control. The results are representative for three independent experiments; respective bands shown were croppedfrom the complete blots. ��, P < 0.001.






of significantly elevated total IGF-I levels in the plasmafollowing BI 836845 treatment. A plausible explanationfor the increase in total serum IGF-I relates to a com-pensatory increase in growth hormone secretion, re-sulting in increased hepatic IGF-I expression (1).Although we did not observe a significant increase ingrowth hormone levels in BI 836845-treated rats, thisdoes not exclude this mechanism, as the pulsatile natureof growth hormone secretion limits the utility of singlegrowth hormone measurements (data not shown). Asimilar phenomenon has been observed with the anti-

VEGF ligand-targeting approach of bevacizumab,where following treatment serum VEGF levels havebeen found to be increased, presumably as a result ofan increase in VEGF synthesis and/or a decrease inVEGF clearance caused by complex formation betweenVEGF and the antibody (46).

In human cancers, autocrine expression of IGF-I or IGF-II is not a rare event (2). It is unlikely that this represents arandom deregulation of gene expression. Rather, a malig-nant clone that expresses both a growth factor and itsreceptor will likely have a proliferative advantage that

Figure 5. Enhanced in vivo antitumor efficacy of BI 836845 in combination with rapamycin in the RD-ES xenograft model. A, treatment of mice bearingRD-ES tumors with BI 836845 (twice weekly, 100 mg/kg i.p.) or rapamycin (5 � weekly, 5 mg/kg i.p.) led to a TGI of 53% and 75%, respectively,compared with control animals (0.5% Natrosol, 10 mL/kg i.p. daily). Combining these agents improved efficacy (93% TGI). Median tumor volumesare shown in the plot. n ¼ 7/treatment group. For statistical analysis, tumor volumes were log transformed to stabilize the variance over the time courseand analyzed utilizing MMRM. B, effect of BI 836845, rapamycin (P < 0.05), and the combination of BI 836845 and rapamycin on pAKT levels inexplanted RD-ES tumors (n ¼ 4/group) as determined by densitometric analysis of Western blot bands. Phosphorylated AKT values were normalizedto total AKT values and are expressed as% pAKT/AKT. IGF bioactivity was increased by rapamycin in serum samples of (C) RD-ES tumor-bearing(n ¼ 4/group; P < 0.01) and (D) non–tumor-bearing mice (n ¼ 4/group; P < 0.005). C, treatment with BI 836845 showed a complete and potentneutralization of serum IGF bioactivity relative to control samples and also inhibited the rapamycin-induced increase of IGF bioactivity (reduction tobaseline levels of the assay). �, P < 0.05; ��, P < 0.01; and ��, P < 0.001.

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will be selected for. Thus, autocrine expression of IGFmay indicate a cancer that is responsive to and/or depen-dent on IGF-IR activation (1). However, this does notnecessarily imply that such tumors will be responsive toany particular IGF-targeting strategy. Interruption of anautocrine loop in vivo requires a high drug concentrationin the tumor, and it is conceivable that pharmacokinetic ortolerability issues may limit efficacy of IGF-targetingtherapies even for the subset of cancers that depend onIGF stimulation. Therefore, it was of interest to observethat BI 836845 was capable of significantly inhibiting notonly in vitro proliferation of IGF-secreting cell lines butalso the in vivo growth of a colorectal cancer model thatexpresses high levels of IGF-II. Despite the importantpharmacodynamic information that has been generatedfrom the profiling of BI 836845 in rats and mice, thetranslational limitation of using rodent models for study-ing IGF physiology is that they express far less IGF-II thanhumans (47), and this needs to be taken into considerationwhen interpreting such studies in rodents.Besides being potential drivers of tumor growth in

certain subsets of cancer, IGFs may contribute to resis-tance mechanisms limiting the efficacy of other anti-cancer agents. Previous studies have shown a potentialfor IGF targeting in rational combinations, such as IGF-IR and IGF ligand antibodies (48), or combinations ofIGF-IR mAbs with rapamycin to more efficiently blockangiogenesis (41, 42). We studied the combination of BI836845 with rapamycin in view of these prior preclin-ical and clinical data indicating that the efficacy ofrapalogs may be limited because inhibition of mTORcomplex (mTORC)-1 results in AKT activation dueto IGF-IR–dependent interference with negative feed-back loops constraining signaling downstream of theIGF-IR (21, 43, 49, 50). Our in vitro and in vivo findingsprovide evidence that the activity of rapamycin can beimproved by coadministration of BI 836845 by abolish-ing AKT activation, suggesting a rational combinationthat deserves clinical evaluation. Apart from theknown cell-autonomous actions (43, 49), we observeda novel homeostatic response to mTORC1 inhibition atthe whole organism level, characterized by increases inserum IGF-IR activating activity, a phenomenonobserved in nude mice in the presence or absence ofxenograft tumors. This response was clearly attenuatedby BI 836845, suggesting that rapamycin-induced AKTactivation is dependent on IGF ligand activity (mech-anistic model depicted in Supplementary Fig. S3). Inter-estingly, a similar effect was seen in a study usingvascular smooth muscle cells (51). However, the precisedefinition of the mechanism by which rapamycinincreases serum IGF bioactivity requires additionalinvestigation and is an active area of research. Similar-ly, the evaluation of angiogenesis and proliferationmarkers, such as CD34 and Ki67, will be importantbecause enhanced antiangiogenic effects may also playa role in explaining the improved efficacy of the com-bination (48).

Despite the recent clinical setbacks with agents tar-geting the IGF-IR (30), the data presented here supportthe differentiated therapeutic concept of IGF-I/II ligandneutralization, which warrants clinical investigation.Preclinical antiproliferative activity was observed withBI 836845 both in vitro and in vivo, and long-termexposure at levels sufficient to abolish IGF serum bio-activity and strongly attenuate somatic growth was notassociated with significant toxicity in rats. The resultspresented also provide clues that may be relevant to thedesign of phase II clinical trials. Presuming that dosingof BI 836845 will not be limited by toxicity, a crucialissue will be defining a phase II dose. Our preclinicalstudies have identified measurable pharmacodynamicendpoints such as serum IGF bioactivity that will beuseful for defining the pharmacokinetic/pharmacody-namic (PK/PD) relationship in humans. With regards torational rather than arbitrary drug combinations, ourfindings indicate that coadministration of BI 836845with rapalogs deserves consideration, and with respectto potential selection criteria for tumors that may beparticularly responsive, autocrine expression of IGF-I orIGF-II may define a subpopulation of interest (52).

Disclosure of Potential Conflicts of InterestMichael N. Pollak has commercial research grants from Pfizer and

Novo Nordisk and is a consultant/advisory board member of BoehringerIngelheim, Pfizer, Novo Nordisk, Sanofi Aventis, Bristol Myers Squibb,and Axelar. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: K. Friedbichler, M.H. Hofmann, E. Borges,G. Adolf, P.J. AdamDevelopment of methodology: P.J. AdamAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Friedbichler, M.H. Hofmann, M. Kroez,E. Ostermann, H.R. LamcheAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): K. Friedbichler, M.H. Hofmann, M. Kroez,H.R. Lamche, C. Koessl, E. Borges, M.N. Pollak, G. Adolf, P.J. AdamWriting, review, and/or revision of themanuscript:K. Friedbichler, M.H.Hofmann, C. Koessl, M.N. Pollak, G. Adolf, P.J. AdamAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):Study supervision: E. Borges, G. Adolf, P.J. Adam

AcknowledgmentsThe authors thank Ilse Apfler, Ulrike Nagl, Margit Zehetner, Astrid

Jeschko, Stefan Fischer, Brenda Rohrhan-Demeties, Romana Ranftl (allBoehringer Ingelheim RCV GmbH & Co KG), Sarah Grundler, AnitaBurghardt, Elvira J€ager-Arndt, and Melanie Radzio (all Boehringer Ingel-heim Pharma GmbH & Co KG) for their excellent technical assistance,Volker Krzykalla and Dietmar Zellner (Boehringer Ingelheim PharmaGmbH & Co KG) for their support with statistical data evaluation, andRobert Rauchenberger (MorphoSys AG, Martinsried/Planegg, Germany)for his support with affinity optimization.

Grant SupportThis work was financially supported by Boehringer-Ingelheim.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 29, 2013; revised November 15, 2013; accepted November18, 2013; published OnlineFirst December 2, 2013.






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2014;13:399-409. Published OnlineFirst December 2, 2013.Mol Cancer Ther Katrin Friedbichler, Marco H. Hofmann, Monika Kroez, et al. Rationale for Combination with RapamycinHuman IGF Ligand-Neutralizing Antibody, and Mechanistic Pharmacodynamic and Antineoplastic Activity of BI 836845, a Fully

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pharmacodynamic and antineoplastic activity of bi 836845 ...ﬁed igf-i (cm001) and igf-ii (fm001;...

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