A Cell-Penetrating Bispecific Antibody for Therapeutic...
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Therapeutic Discovery A Cell-Penetrating Bispecific Antibody for Therapeutic Regulation of Intracellular Targets Richard H. Weisbart 1 , Joseph F. Gera 1 , Grace Chan 1 , James E. Hansen 1 , Erica Li 1 , Cheri Cloninger 1 , Arnold J. Levine 2 , and Robert N. Nishimura 3 Abstract The therapeutic use of antibodies is restricted by the limited access of antibodies to intracellular compart- ments. To overcome this limitation, we developed a cell-penetrating monoclonal antibody, mAb 3E10, as an intracellular delivery vehicle for the intracellular and intranuclear delivery of antibodies constructed as bispecific single-chain Fv fragments. Because MDM2 is an important target in cancer therapy, we selected monoclonal antibody (mAb) 3G5 for intracellular transport. mAb 3G5 binds MDM2 and blocks binding of MDM2 to p53. Here, we show that the resulting 3E10-3G5 bispecific antibody retains cell-penetrating and MDM2-binding activity, increases tumor p53 levels, and inhibits growth of MDM2-addicted tumors. The use of cell-penetrating bispecific antibodies in targeted molecular therapy will significantly broaden the spectrum of accessible intracellular targets and may have a profound impact in cancer therapy. Mol Cancer Ther; 11(10); 2169–73. Ó2012 AACR. Introduction Cell membranes are impermeable to most macromo- lecules, and current approaches to the therapeutic reg- ulation of intracellular targets are therefore largely based on the use of small molecules that are capable of passive diffusion into cells. However, small-molecule inhibitors are prone to causing off-target effects that can result in significant toxicity (1). In contrast, antibodies have excellent binding specificity and minimal off-tar- get effects, but most antibodies do not penetrate living cells and are limited to targeting molecules that are secreted or located on the cell membrane. Gene therapy can be used to produce intracellular antibodies, but its use is not justified because of potential toxicity. We previously identified a rare monoclonal anti-DNA anti- body, 3E10, which penetrates living cells and localizes in the nucleus through an equilibrative nucleoside transporter without causing any apparent harm to the cell (2, 3). 3E10 and its single-chain variable fragment (3E10 scFv) have been developed as an intracellular delivery system for macromolecules (4–9). Notably, after localizing in the cell nucleus, 3E10 scFv is largely degraded within 4 hours, thus further minimizing any potential toxicity (unpublished data). 3E10 scFv has previously mediated intracellular delivery of biologi- cally active cargo proteins such as p53 and Hsp70 in vivo (7, 9), and we hypothesized that 3E10 scFv might also be capable of delivering biologically active antibody frag- ments into cells. MDM2, an E3 ubiquitin ligase that downregulates the function of p53, is associated with numerous malig- nancies and is an important target in cancer therapy. Inhibition of MDM2 increases active p53 levels and is associated with induction of senescence in human mel- anoma cell lines (10). The mAb 3G5 was previously shown to bind critical residues at the N-terminus of MDM2 that are required for binding to p53, and 3G5 is, therefore, an excellent candidate for application as a targeted inhibitor of MDM2 (11–15). However, 3G5 is unable to penetrate into cells. We therefore tested the ability of 3E10 scFv to deliver a functional single-chain variable fragment of the 3G5 antibody into tumor cells. Materials and Methods Cell lines Cell lines obtained from the American Type Culture Collection include: COS-7 monkey kidney cells; MC-7 human ovarian cancer cells that overexpress MDM2; 3T3-transformed mouse fibroblasts; and BJ primary human fibroblasts. Human melanoma cells sensitive to MDM2 inhibition were obtained from Maria S. Soengas (Melano- ma Laboratory, Spanish National Cancer Research Centre, Madrid, Spain), and included SK-MEL-103, SK-MEL-147, UACC-62, and UACC-257. These cell lines were not authenticated by our laboratory after receiving them. Authors' Affiliations: 1 Veterans Affairs Greater Los Angeles Health Care System, Sepulveda, California; 2 Institute for Advanced Study, Princeton University, Princeton, New Jersey; and 3 Department of Neurology, Uni- versity of California, Los Angeles, California Current address for J.E. Hansen: Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut. Corresponding Author: Richard H. Weisbart, Veterans Affairs Greater Los Angeles Health Care System, 161111 Plummer St., Sepulveda, CA 91343. Phone: 818-895-9422; Fax: 818-895-9423; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-12-0476-T Ó2012 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 2169 on May 16, 2018. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst August 3, 2012; DOI: 10.1158/1535-7163.MCT-12-0476-T
Transcript of A Cell-Penetrating Bispecific Antibody for Therapeutic...
Therapeutic Discovery
A Cell-Penetrating Bispecific Antibody for TherapeuticRegulation of Intracellular Targets
Richard H. Weisbart1, Joseph F. Gera1, Grace Chan1, James E. Hansen1, Erica Li1, Cheri Cloninger1,Arnold J. Levine2, and Robert N. Nishimura3
AbstractThe therapeutic use of antibodies is restricted by the limited access of antibodies to intracellular compart-
ments. To overcome this limitation, we developed a cell-penetrating monoclonal antibody, mAb 3E10, as an
intracellular delivery vehicle for the intracellular and intranuclear delivery of antibodies constructed as
bispecific single-chain Fv fragments. Because MDM2 is an important target in cancer therapy, we selected
monoclonal antibody (mAb) 3G5 for intracellular transport. mAb 3G5 binds MDM2 and blocks binding of
MDM2 to p53. Here, we show that the resulting 3E10-3G5 bispecific antibody retains cell-penetrating and
MDM2-binding activity, increases tumor p53 levels, and inhibits growth of MDM2-addicted tumors. The use
of cell-penetrating bispecific antibodies in targeted molecular therapy will significantly broaden the spectrum
of accessible intracellular targets and may have a profound impact in cancer therapy.Mol Cancer Ther; 11(10);
2169–73. �2012 AACR.
IntroductionCell membranes are impermeable to most macromo-
lecules, and current approaches to the therapeutic reg-ulation of intracellular targets are therefore largelybased on the use of small molecules that are capableof passive diffusion into cells. However, small-moleculeinhibitors are prone to causing off-target effects that canresult in significant toxicity (1). In contrast, antibodieshave excellent binding specificity and minimal off-tar-get effects, but most antibodies do not penetrate livingcells and are limited to targeting molecules that aresecreted or located on the cell membrane. Gene therapycan be used to produce intracellular antibodies, but itsuse is not justified because of potential toxicity. Wepreviously identified a rare monoclonal anti-DNA anti-body, 3E10, which penetrates living cells and localizesin the nucleus through an equilibrative nucleosidetransporter without causing any apparent harm to thecell (2, 3). 3E10 and its single-chain variable fragment(3E10 scFv) have been developed as an intracellulardelivery system for macromolecules (4–9). Notably,after localizing in the cell nucleus, 3E10 scFv is largely
degraded within 4 hours, thus further minimizing anypotential toxicity (unpublished data). 3E10 scFv haspreviously mediated intracellular delivery of biologi-cally active cargo proteins such as p53 and Hsp70 in vivo(7, 9), and we hypothesized that 3E10 scFv might also becapable of delivering biologically active antibody frag-ments into cells.
MDM2, an E3 ubiquitin ligase that downregulatesthe function of p53, is associated with numerous malig-nancies and is an important target in cancer therapy.Inhibition of MDM2 increases active p53 levels and isassociated with induction of senescence in human mel-anoma cell lines (10). The mAb 3G5 was previouslyshown to bind critical residues at the N-terminus ofMDM2 that are required for binding to p53, and 3G5 is,therefore, an excellent candidate for application as atargeted inhibitor of MDM2 (11–15). However, 3G5 isunable to penetrate into cells. We therefore tested theability of 3E10 scFv to deliver a functional single-chainvariable fragment of the 3G5 antibody into tumorcells.
Materials and MethodsCell lines
Cell lines obtained from the American Type CultureCollection include: COS-7 monkey kidney cells; MC-7human ovarian cancer cells that overexpress MDM2;3T3-transformedmousefibroblasts; andBJprimaryhumanfibroblasts. Human melanoma cells sensitive to MDM2inhibition were obtained from Maria S. Soengas (Melano-ma Laboratory, Spanish National Cancer Research Centre,Madrid, Spain), and included SK-MEL-103, SK-MEL-147,UACC-62, and UACC-257. These cell lines were notauthenticated by our laboratory after receiving them.
Authors' Affiliations: 1Veterans Affairs Greater Los Angeles Health CareSystem, Sepulveda, California; 2Institute for Advanced Study, PrincetonUniversity, Princeton, New Jersey; and 3Department of Neurology, Uni-versity of California, Los Angeles, California
Current address for J.E. Hansen: Department of Therapeutic Radiology,Yale School of Medicine, New Haven, Connecticut.
Corresponding Author:Richard H.Weisbart, Veterans Affairs Greater LosAngeles Health Care System, 161111 Plummer St., Sepulveda, CA 91343.Phone: 818-895-9422; Fax: 818-895-9423; E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-12-0476-T
�2012 American Association for Cancer Research.
MolecularCancer
Therapeutics
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Design, expression, andpurificationof the3E10-3G5bispecific antibody
3G5 hybridoma was obtained from Arnold J. Levine,Princeton University (Princeton, NJ). 3G5 Vk and VHwere cloned by reverse transcription-PCR from hybrid-oma RNA with degenerate primers designed to identifymouse immunoglobulin variable region genes, and 3G5scFvwas constructed asdescribedpreviously (4).Variableregion heavy and light chains were attached with a(GGGGS)3 linker. The Fv fragments were connected witha linker composed of CH1 sequences combined with aswivel sequence (6). 3E10-3G5 bispecific scFv cDNA wasconstructed in pPicZaA between the EcoRI and XbaI clon-ing sites in frame with the C-terminal myc-his6 tag. Plas-midswere transfected into X-33 cells, and a high-secretingclonewas identified asdescribed previously (6). Bispecificantibody was purified from X-33 supernatant by metalchelation chromatography on Ni-NTA-Agarose. The bis-pecific antibody was shown to be stable at 4�C for 3months.
Cell penetration assayCOS-7 cells were incubated with control media, media
containing 10 mmol/L 3E10 scFv, or media containing10 mmol/L 3E10-3G5 for one hour. Media was thenremoved from the cells, and cells were washed, fixed,and stained with an anti-myc antibody as described pre-viously (3).
In vitro assays of 3E10-3G5 cytotoxicityCells were grown in Dulbecco’s Modified Eagle’s
Medium with 10% fetal calf serum. Adherent cells wereremoved with EDTA and distributed in 96-well platesovernight in the presence ofmedium alone, 3E10, or 3E10-3G5. Growthwas evaluated after 3 days by counting cells.Results were expressed as percent total cell number (rel-ative to control) � SD.
In vivo assays of 3E10-3G5 cytotoxicityAnimal studies were done under a protocol approved
by the VeteransAffairs Institutional Animal Care andUseCommittee. Nude mice (nu/nu) were obtained from TheJackson Laboratory (Bar Harbor, Maine). Six nude micewere injected subcutaneously with 1 � 106 UACC-257cells and observed (control). Six nude mice were injectedsubcutaneouslywith 1� 106UACC-257 cells on day 1 andtreated with intraperitoneal injections of 1.0 mg 3E10-3G5scFv on days 1 through 4. Tumor volume (mm3) wasmeasured in mice that developed tumors, and animalswere euthanized when tumors exceeded 2,000 mm3 or atthe termination of the experiment on day 22.
Western blot assaysUACC-257 tumors were excised, and tumor tissue was
lysed in 2% SDS. Protein (20 mg) from each tumor waselectrophoresed in a 4 to 20% polyacrylamide gradientgel and then transblotted to a nylon membrane. Westernblotswereprobedwithantibodies top53,MDM2,andactin.
Statistical analysesSignificant differences in tumor growth were deter-
mined by Student t test.
Results3E10-3G5 retains theMDM2-bindingactivity of 3G5and the cell-penetrating activity of 3E10
A 3E10-3G5 bispecific antibody composed of the sin-gle-chain variable fragments of the cell-penetrating3E10 antibody and the anti-MDM2 3G5 antibody wasproduced as a secreted protein by Pichia pastoris X-33cells transfected with pPicZaA containing the bispecificscFv cDNA (Fig. 1A). 3E10-3G5 was purified from yeastsupernatant by metal chelation chromatography on Ni-NTA-Agarose as described previously (ref. 6; Fig. 1B).Purified 3E10-3G5 was used as a probe for MDM2 in aWestern blot assay on lysates from MC-7 cells over-expressing MDM2, and was found to recognize andbind MDM2 similar to the full 3G5 antibody and withsimilar binding specificity (Fig. 1C). MC-7 cells wereselected as a convenient source of MDM2. Next, 3E10-3G5 was applied to COS-7 cells in culture and wasobserved to penetrate into the cells and localize in nucleisimilar to 3E10 scFv alone (Fig. 1D). COS-7 cells over-express hENT-2 and served as a convenient model cellto show cellular penetration by the bispecific scFv.These results show that the 3E10-3G5 bispecific anti-body retains the cell-penetrating activity of 3E10 scFvand the MDM2-binding activity of 3G5 scFv.
Figure 1. The 3E10-3G5 bispecific antibody retains the MDM2-bindingactivity of 3G5 and the cell-penetrating activity of 3E10. A, schematic ofthe 3E10-3G5 bispecific antibody. B, purified 3E10-3G5 visualized bySDS-PAGE and GelCode Blue staining. A single band is observed at theexpected molecular weight of approximately 60 kDa. C, Western blots ofMC-7 cell lysates probed with control, 3G5, or 3E10-3G5 show that both3G5 and 3E10-3G5 recognize and bind MDM2. D, 3E10-3G5 penetratesCOS-7 cells and localizes to the nucleus similar to 3E10 scFv alone asevidenced by anti-myc staining.
Weisbart et al.
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3E10-3G5 impairs the growth of MDM2-addictedmelanoma cellsWe next investigated the impact of 3E10-3G5 on mel-
anoma cells known to be sensitive to MDM2 inhibition(10).UACC-257melanoma cellswere incubated for 3 dayswith media containing concentrations of 3E10-3G5 rang-ing from 0 to 10 mmol/L. 3E10 and 3G5 alonewere used ascontrols and had no observable effect on the growth ormorphology of UACC-257 melanoma cells comparedwith culture medium (Fig. 2C). However, 3E10-3G5delayed the growth of the cells in a dose-responsivemanner with significant growth delay observed at a doseof 10 mmol/L (Fig. 2A). A similar effect was observed inadditional MDM2-addicted melanoma cell lines, withmarked inhibition of growth and distinct morphologicchanges observed in all of the melanoma cell lines tested(Fig. 2B and C). As expected, 3E10 and 3G5 alone had no
apparent effect on any of the melanoma cell lines (Fig.2C). Importantly, 3E10-3G5 had only a mild impact onthe growth of murine 3T3-transformed fibroblasts andhad no effect on the growth of BJ primary humanfibroblasts (Fig. 2B). Taken together these data suggestthat 3E10-3G5 successfully inhibited MDM2 in vitro andcaused a growth delay specifically in the MDM2-addicted cells.
3E10-3G5 inhibits growth of melanoma tumorsin vivo
The activity of 3E10-3G5 in vivo was tested in a humanmelanoma xenograft model. Nude mice were injectedsubcutaneously with 1 � 106 UACC-257 cells, and micewere observed or treated for 4 consecutive days withintraperitoneal injections of 1.0 mg 3E10-3G5 beginningat day 1. Four mice in the control group and 5 mice in
Figure 2. 3E10-3G5 impairs thegrowth of MDM2-addictedmelanoma cells. A, dose-responseeffect of 3E10-3G5 on the growth ofUACC-257 cells. Shown is meanresponse � SD of duplicatedeterminations. There was no effectof 3E10 or 3G5 alone. B, growth ofhuman melanoma cells (SK-MEL-103, SK-MEL-147, UACC-62,UACC-257) was inhibited at day 3 by10 mmol/L 3E10-3G5 compared withmedium alone. Results arerepresentative of 3 independentexperiments and are shown as mean� SD. 3T3 are transformed mousefibroblasts and BJ is a culture ofnormal human primary fibroblasts. C,lightmicroscopy images showing thedifferences in cell population andmorphology of melanoma cells 3days after treatment with 3E10-3G5compared with control buffer, 3E10alone, and 3G5 alone.
A Bispecific Antibody Regulates Intracellular Targets
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the experimental group developed tumors. Mice thatdeveloped tumors were then followed closely and tumorvolumes were measured. Importantly, treatment with3E10-3G5 was not associated with any clinical toxicity,as treated mice were indistinguishable from control micewith respect to their appearance and activity. However,treatment with 3E10-3G5 significantly inhibited tumorgrowth at day 20 (P ¼ 0.041) and at the termination ofthe experiment on day 22 (P¼ 0.026; Fig. 3A–C). To probethe mechanism responsible for tumor growth inhibition,we evaluated the relative levels of p53 and MDM2 inrepresentative tumors from 3 untreated mice and 3mice treated with 3E10-3G5. Treatment with 3E10-3G5increased the expression of MDM2 and p53 as shown inWestern blots of tumor lysates probed with antibodies top53 and MDM2 (Fig. 3D). Actin served as a loadingcontrol. These results are similar to changes in MDM2and p53 levels observed in cells and tumors treatedwith small-molecule inhibitors of MDM2 (10) and furthersuggest that 3E10-3G5 successfully inhibited MDM2in vivo.
DiscussionWe have shown that treatment with a 3E10-3G5
bispecific antibody impairs the growth of melanomacells in vitro and in vivo. This growth delay is likely theresult of increased levels of activated p53 that havebeen freed from inhibition by MDM2 by the action ofthe 3G5 antibody fragment. In accordance with thishypothesis, elevated levels of p53 were observed intumors in mice treated with the bispecific antibody. Wealso noted that these tumors exhibited increased levelsof MDM2, which is consistent with results obtained by
others with MDM2 inhibitors, such as Nutlin-3, and islikely the result of increased levels of p53 drivingadditional production of MDM2 (10). Because MDM2has numerous p53-independent effects (10), it is pos-sible that the impact of 3E10-3G5 on the melanoma cellsand tumors is the result of an effect on diverse meta-bolic pathways in addition to its impact on p53 func-tion. Although we administered micromolar amountsof 3E10-3G5 to mice, only nanomolar amounts areinternalized intracellularly consistent with antigen-binding specific effects.
We previously constructed and showed efficacy of acell-penetrating bispecific antibody composed of 3E10scFv and the scFv fragment of mAb PAb421, an antibodythat binds and restores the function of some p53 mutants(6). In the present study, we have extended our cell-penetrating bispecific antibody technology by showingthe effectiveness of this approach in modulating MDM2activity in vivo. Because p53 activity can be inhibited bymutation and/or overexpression of MDM2, combinationtherapy with 3E10-3G5 and 3E10-PAb421 may proveparticularly useful in selected tumor cells.
Our studies establish proof-of-principle for the use ofthe cell-penetrating antibody 3E10 as a transport vehicleto deliver therapeutic antibody fragments directed tointracellular and intranuclear targets. The exquisite anti-gen-binding specificity of antibodies delivered into intra-cellular compartments will likely result in improved ther-apeutic indices by avoiding off-target binding responsiblefor toxic side effects of small-molecule inhibitors. Inaddition, cell-penetrating bispecific antibodies can bedesigned that bind intracellular epitopes, such as tran-scription factors and DNA repair proteins, that cannot
Figure 3. 3E10-3G5 inhibits humanmelanoma xenograft growth invivo. Nude mice were injectedsubcutaneously with 1 � 106
UACC-257 cells on day 1 and thenobserved in control group (A) ortreated by intraperitonealadministration of 1.0mg 3E10-3G5on days 1 to 4 (B). C, mean tumorvolume � SEM after injection ofcells into control and treated mice.D, tumors in mice treated with3E10-3G5 exhibited increasedlevels of p53 and MDM2 as shownby Western blotting for p53 andMDM2 in tumors from3control and3 3E10-3G5–treated mice.
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presently be targeted with small-molecule inhibitors andare currently considered undruggable. The use of cell-penetrating bispecific antibodies in targeted moleculartherapy will significantly broaden the spectrum of acces-sible intracellular targets and may have a profoundimpact in cancer therapy.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: R.H. Weisbart, R.N. NishimuraDevelopment of methodology: R.H. Weisbart, C. Cloninger, R.N.NishimuraAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.H.Weisbart, J.F. Gera, G. Chan, J.E. Hansen, E.Li, C. Cloninger, R.N. NishimuraAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): R.H. Weisbart, R.N. NishimuraWriting, review, and/or revision of the manuscript: R.H. Weisbart, J.F.Gera, J.E. Hansen, R.N. Nishimura
Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G. Chan, E. Li, C. CloningerStudy supervision: R.H. Weisbart, R.N. Nishimura
Conducted in vivo experiment: C. CloningerContributed reagents for experiment and characterized antibody-binding site: A.J. Levine
AcknowledgmentsThe authors thank Maria Soengas, Melanoma Laboratory, Spanish
National Cancer Research Centre for contributing melanoma cell lines.
Grant SupportThis work was supported by a Merit Review Grant from the Veterans
Affairs awarded to R.H. Weisbart.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 May 9, 2012; revised July 18, 2012; accepted July 26, 2012;published OnlineFirst August 3, 2012.
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A Bispecific Antibody Regulates Intracellular Targets
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