Four-In-One Antibodies Have Superior Cancer Inhibitory Activity Against

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Published OnlineFirst November 4, 2014; DOI: 10.1158/0008-5472.CAN-14-1670 Cancer Therapeutics, Targets, and Chemical Biology Research

Four-in-One Antibodies Have Superior Cancer Inhibitory Activity against EGFR, HER2, HER3, and VEGF through Disruption of HER/MET Crosstalk Shi Hu1,2,3,4, Wenyan Fu5, Weihao Xu6, Yang Yang3,7, Melissa Cruz8,9, Sandra D. Berezov9, Daniel Jorissen9, Hiroaki Takeda8, and Wangdong Zhu3,4

Abstract

The anti-HER receptor antibodies cetuximab, trastuzumab, in vivo but unexpectedly also disrupted HER-MET crosstalk. and pertuzumab are used widely in clinic to treat metastatic When compared with two-in-one antibodies and a series of cancer. However, activation of the extensive crosstalk among bispecific antibodies in multiple tumor models, FL518 and theHERreceptorsaswellasotherRTKs,particularlyHER-MET CRTB6weremorebroadlyefficacious. We further showed that crosstalk, has emerged as a likely source of drug resistance. In tetra-specific antibodies were far more effective than bispecific this study, we developed two new types of tetra-specificanti- antibodies in inhibiting the growth of anti–HER-resistant can- bodies that recognize EGFR, HER2, HER3, and VEGF. These cer cells, which exhibited elevated levels of MET activation both tetra-specific antibodies, termed FL518 (four-in-one antibody) in vitro and in vivo. Overall, our results establish a new principle and CRTB6 (tetra-specific, tetravalent antibody), not only to achieve combined HER inhibition and limit drug resistance inhibited signaling mediated by these receptors in vitro and using a single antibody. Cancer Res; 75(1); 1–12. 2014 AACR.

Introduction erlotinib and gefitinib. Another family member, HER3, has recently emerged as a novel drug target because its upregulation is an Deregulation of the human epidermal growth factor receptor adverse prognostic factor in many tumor types and is associated (HER) family plays a significant role in tumorigenesis and pro- with worse survival (4–6). Although HER inhibitors have been gression (1). The basic and clinical research into two of the family widely evaluated in clinic, these promising anticancer drugs are members, epidermal growth factor receptor (EGFR/HER1) and effective only for a limited time, due to the resistance developed HER2, has led the way in transforming therapeutic biomolecules by tumor cells (7). Signaling shift has been proved for when such from bench to bedside, including the anti-EGFR antibodies cetux- intensely proliferative cancer cells faced a strong signaling inhib- imab and panitumumab, the anti-HER2 antibodies trastuzumab itor. For instance, HER3 has also been implicated in the devel- and pertuzumab (2, 3), and the EGFR tyrosine kinase inhibitors opment of resistance to anti-EGFR or anti-HER2 therapy (8–10). A shift from HER1 homodimer to HER1/HER3 heterodimer signaling in response to sustained treatment with EGFR inhibitors leads 1 Translational Medicine Research Institute and International Joint to reactivation of the PI3K/AKT pathway and bypasses the ther- Cancer Institute, The Second Military Medical University, Shanghai, P. R. China. 2Department of Clinical Medicine, The Second Military apeutic action of these compounds. Upregulation of HER3 seems Medical University, Shanghai, P. R. China. 3Department of Medical to be driven by negative feedback from AKT (11). Amplification of Imaging, Xi'an PLA 451 Hospital, Xi'an, Shaanxi, P. R. China. 4Depart- the tyrosine kinase receptor MET induces HER3 phosphorylation ment of Interventional Oncology, Xi'an PLA 451 Hospital, Xi'an, Shaanxi, P. R. China. 5Cancer Center, PLA General Hospital, PLA Post- and PI3K activation, representing another mechanism by which graduate School of Medicine, Beijing, P. R. China. 6Department of HER3 confers resistance to EGFR-targeting therapies (11, 12). Cardiology, PLA General Hospital, PLA Postgraduate School of Med- Data increasingly indicate the significant status of combined 7 icine, Beijing, P. R. China. School of Basic Medicine, Xi'an Jiao Tong inhibition strategies for directing key signaling networks of the University, Xi'an, Shaanxi, P. R. China. 8Nagoya City University Grad- uate School of Medical Sciences, Mizuho-cho, Mizuho-ku, Nagoya, HER family, including cetuximab plus trastuzumab (13), cetux- Japan. 9School of Medicine and Pharmacology, The University of imab plus bevacizumab (14), and trastuzumab plus pertuzumab Western Australia, Crawley, Australia. (2). Recently, generation of bispecific antibodies that have the Note: Supplementary data for this article are available at Cancer Research capacity to bind two different antigens simultaneously has Online (http://cancerres.aacrjournals.org/). become a new trend of engineering antibodies (15), and a number S. Hu, W. Fu, and W. Xu contributed equally to this article. of recombinant strategies have been developed to synthesize fi Corresponding Authors: Shi Hu, The Second Military Medical University, 800 bispeci c antibodies (8, 16, 17). Indeed, in 2009, catumaxomab Xiangyin Road, Shanghai 200433, P. R. China. Phone: 008601066912256; Fax: (Removab, Neovii Biotech) was approved as the first bispecific 008601066912254; E-mail: [email protected]; and Wangdong Zhu, Depart- antibody drug for the treatment of malignant ascites (18). For ment of Interventional Oncology, Xi'an PLA 451 Hospital, Xi'an, Shaanxi, P. R. anti–HER-targeted therapy, previous studies have shown that China. Phone: 86-29-85201198; Fax: 86-29-85211145; E-mail: bispecific antibodies engineered from anti-EGFR or -HER2 may [email protected] give promising results (19, 20). Moreover, two-in-one antibodies, doi: 10.1158/0008-5472.CAN-14-1670 which created a surface that specifically binds two target antigens 2014 American Association for Cancer Research. with high affinity using a single antibody binding site (21, 22),

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have aroused abundant interest in the field. Moreover, the in vivo Coimmunoprecipitation assays properties, such as pharmacokinetics, stability, and effector func- Coimmunoprecipitation assay was used to evaluate the tion, of the IgG-based bispecific antibodies are expected to be HER/MET heterodimerization as previously described (28, 30). similar to those of standard monoclonal antibodies, which are Cells were lysed in 1 mL RPMI lysis buffer [1% v/v DevelopTriton crucial for clinical use. Thanks to the design of two-in-one anti- X-100, 1% w/v CHAPS, 10 mmol/L HEPES (pH 7.2)], in RPMI bodies, we were able to create IgG-based tetra-specific antibodies medium containing 0.2 mmol/L PMSF, 10 mg/mL leupeptin, 10 using current antibody engineering methodologies. U/mL aprotinin, and 1 mmol/L Na3VO4. Heterodimers were immunoprecipitated from 500 mL of lysate using indicated anti- Materials and Methods bodies covalently coupled to agarose (Pierce ultralink) for 1 hour at 36.5C. Recombinant human EGF and HRG (R&D Systems) Cell lines, antibodies, and animals were added at a final concentration of 0.5 nmol/L, respectively. All cancer cell lines were obtained from the American Type Complexes were washed twice in lysis buffer and resuspended in Culture Collection (ATCC), except HCC827 and gastric cancer cell SDS sample buffer and boiled. Samples were separated on a 4% to line MNK45, which were purchased from the German Collection 12% polyacrylamide gel (Novex) and electro-blotted onto nitro- of Microorganisms and Cell Cultures. All the cell lines were cellulose membranes. Blots were blocked in 10% BSA/TBST and authenticated twice by morphologic and isoenzyme analyses probed with indicated antibodies. Western blot and protein during the study period. Cell lines were routinely checked for immunoprecipitation analyses were performed after the CST contamination by mycoplasma using Hoechst staining and con- protocol. sistently found to be negative. Cells were cultured in DMEM medium supplemented with 10% FCS in 5% CO at 37.8Cin 2 Cell proliferation assay a humidified incubator. Cetuximab was purchased from Merck. H1666 cells (4,000 cells/well) and H1993 cells (3,000 cells/ Trastuzumab and bevacizumab were purchased from Roche Ltd. well) were plated in 96-well plates. The next day, cells were treated Six-week-old female BALB/c nude mice and eight- to 12-week-old with indicated concentrations of antibody in 1% serum-contain- C.B-17 SCID mice were obtained from the Shanghai Experimental ing medium. Depending on the cell line, ligand was added Animal Center of the Chinese Academy of Sciences (Shanghai, simultaneously. After 3 to 4 days, AlamarBlue (Invtiogen) was China). All animals were treated in accordance with guidelines of added to the wells and fluorescence was read using a 96-well the Committee on Animals of the Chinese Academy of Sciences. fluorometer with excitation at 530 nm and emission of 590 nm. The results are shown as relative fluorescence units (RFU). For Designed antibodies MTS assay, cells were treated with 10 mg/mL recombinant anti- Four-in-oneCrossMabFL518 weregenerated fromMEHD7945A bodies. Recombinant human EGF, HRG, and VEGF (R&D Sys- (23) and bH1-44 (24) as previously described (25). Four-in-one tems) were added to a final concentration of 0.5 nmol/L, respec- dual-variable domain antibodies (DVD-Ig) methodology (26) was tively. After an additional 4- to 7-day incubation, cell proliferation utilized to generate DVD Ig CRTB6, together with the "knobs into was determined by Cell Titer 96 AQueous One Solution Cell holes" (KiH) methodology and the CrossMab methodology. Bi- Proliferation Assay Kit (Promega). All assays were performed specific crossmabs CT9, CB12, RT6, and RB4 were generated from independently three times. cetuximab/trastuzumab, cetuximab/bevacizumab, RG7116 (anti- HER3)/trastuzumab, and RG7116/bevacizumab. In vivo therapy study C.B-17 SCID mice were inoculated with 5 106 BxPC3 fi Construction, expression, and protein puri cation cells suspended in HBSS. For MDA-MB-231, BT474, MCF-7, – Residues 1 619 of the extracellular domain of EGFR (EGFR- HCC1954, or ReHCC1954 in vivo xenograft studies, 3 106 – – ECD), residues 1 513 of the HER3-ECD, residues 1 646 of BT474 or MCF-7 cells, 4 106 HCC1954, or ReHCC1954 cells – HER2-ECD, and residues 8 109 of human VEGF were prepared were inoculated into the mammary fat pads of female BALB/c as described previously (27, 28) except that we employed the nude mice implanted with 0.72-mg 60-day release 17b-estra- þ pcDNA3.1( )-expressing vector (Invitrogen) and the FreeStyle diol pellets (Innovative Research of America; ref. 31). For 293 expression system (Invitrogen). Four-in-one antibodies HCC827 or ReHCC827 xenograft studies, 5 106 HCC827 fi fi and bispeci c crossmabs were cloned, expressed, and puri ed or ReHCC827 cells were inoculated into BALB/c nude mice. following reported procedures (29). Mice were randomly split into groups of 10 mice each as tumor volumes reached an average of about 150 mm3.Multipledose Immunoblotting studies comprised 4 weeks of treatment, with the first dose (day Cells were incubated with the indicated antibodies in serum- of randomization) being a 2 loading dose. Tumors were free medium for 1 hour at 36.5 C, then treated with EGF (0.5 measured with calipers at least once a week for the duration nmol/L), HRG (0.5 nmol/L), VEGF (0.5 nmol), or not for 15 of the study. Tumors were measured with digital calipers, and minutes. After washing, the cells were lysed and the lysates tumor volumes were calculated by the formula: volume ¼ were subjected to SDS-PAGE and immunoblotted with length (width)2/2. antibodies against EGFR, phospho-EGFR-Tyr1068, phospho- HER2-Tyr1221/1222, phospho-HER3-Tyr1289, phospho-MET- Statistical analysis Tyr1234/5, AKT, phospho-AKT-Ser473, phospho-STAT3-Ser705, Statistical analysis was performed by the Student unpaired t test p44/42 MAPK, phospho-p44/42 ERK1/2 Thr202/Tyr204, EGFR, to identify significant differences unless otherwise indicated, with HER2, and MET (all from Cell Signaling Technology), and HER3 P value of less than 0.05 considered significant difference. Addi- (Santa Cruz). All assays were performed independently three tional methods can be found in Supplementary Materials and times. Methods.

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Figure 1. Design and characterization of four-in-one antibodies. A, schematic representation of MEHD7954A that binds to EGFR and HER3 and bH1-44 that binds to HER2 and VEGF. Models were generated from PDB entry 3P11, 3P0Y, 1YY9, 1M6B, 3BE1, and 3BDY. B, schematic representation of the four-in-one antibody FL518. C, schematic representation of tetra-speciﬁc, tetravalent DVD-Ig CRTB6. D, FL518 binding to immobilized EGFR-ECD, HER3-ECD, HER2-ECD, or VEGF in the presence of the indicated soluble competitor. 1¼0.01 mg/mL; 10¼0.1 mg/mL; 100¼1 mg/mL; 1,000¼10 mg/mL. Results are expressed as FL518 concentration versus optical density (OD). E, four-in-one antibodies binding to immobilized indicated protein in the presence of the indicated biotin-competitor antibodies. Detection was carried out with alkaline phosphatase-conjugated avidin. Results are expressed as antibody concentration versus optical density. F–I, immunoblots examining the ability of 10 mg/mL of control IgG, MEHD7954A, bH1-44, cetuximab, RG7116, trastuzumab, bevacizumab, FL518, or CRTB6 to inhibit the EGF-stimulated phosphorylation of EGFR and ERK1/2 in Caco-2 cells (F), the HRG-stimulated phosphorylation of HER3, AKT, and ERK1/2 in MCF-7 cells (G), the ligand-independent phosphorylation of HER2 and AKT in BT474 cells (H), and the ligand-independent phosphorylation of HER2 and AKT in BT474 cells (I).

Results directed against EGFR and HER3 and bH1-44 (22), which is described as binding VEGF and HER2 (Fig. 1A). Two meth- Design and characterization of a four-in-one antibody in an odologies were used in the generation of the four-in-one IgG-like format antibody (denoted as FL518). Heterodimerization of the two We ﬁrst sought to generate a four-in-one antibody using heavy chains was overcome by the KiH methodology (32, 33), two two-in-one antibodies, including MEHD7945A (21) and the discrimination between the two light-chain/heavy-

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Figure 2. Four-in-one antibodies have superior antitumor activity in vitro and in vivo. A, schematic representation of bispeciﬁc CrossMabs. B and C, H1666 cells (B) or H1993 cells (C) were treated with increasing concentrations of the indicated antibodies in the presence of EGF (0.5 nmol/L), HRG (0.5 nmol/L), and VEGF (0.5 nmol/L). The concentrations shown on the x axis reﬂect the concentration of a single antibody or the combination of antibodies. (Continued on the following page.)

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chain interactions was achieved using the CrossMab method- cate the favorable stability properties of the four-in-one ology (Fig. 1B; ref. 34). antibodies. Because one Fab of FL518 can bind either EGFR or HER3 and the other Fab can bind either HER2 or VEGF, a certain concen- Four-in-one antibodies potently inhibit receptor tration of FL518 antibodies can directly bind to EGFR, HER3, phosphorylation of EGFR, HER2, HER3, and VEGFR2 HER2, and VEGF on the surface of the cell membrane during the To evaluate their inhibitory efficacy on EGFR and downstream same period. However, this kind of four-in-one antibody has its phosphorylation of ERK1/2, we pretreated high EGFR-expressing own limits in that one antibody molecule can only binds two of Caco-2 cells (38) with parental antibodies, FL518, or CRTB6 the targets. Thus, we employ the DVD-Ig methodology (35), the before EGF stimulation and determined that FL518 and CRTB6 KiH methodology, and the CrossMab methodology to make a inhibited phosphorylation of EGFR and ERK1/2, respectively (Fig. tetra-specific, tetravalent antibody termed as CRTB6 (Fig. 1C). For 1F), with an effect similar to the MEHD7945A and the mono- this antibody design, one IgG-like antibody molecular now can specific antibody cetuximab. No significant inhibition was bind to four targets. We use the V region of cetuximab and observed for bH1-44, trastuzumab, or bevacizumab. Moreover, RG7116 (36) in the Fab arms for EGFR and HER3, for their ability four-in-one antibodies had a superior inhibitory effect on the to block the EGF- and HRG-dependent homo- or hetero-dimer- phosphorylation of ERK1/2. Next, we chose MCF-7 cells for which ization and signaling and the V region of trastuzumab, which has HRG treatment potently activates the HER2/HER3 pathway in our shown its unique effect on ligand-independent EGFR/HER2 or assay. Treatment with both FL518 and CRTB6 before HRG stim- HER3/HER2 signaling (37). In addition, anti-VEGF antibody ulation potently inhibited the phosphorylation of HER3 and bevacizumab was added to make this tetra-specific, tetravalent markedly decreased the phosphorylation of AKT and ERK1/2 antibody more comparable with FL518. Because the dimerization (Fig. 1G). Treatment with MEHD7945A or RG7116 achieved and communication among HER family members are the key similar results in the phosphorylation of HER3 but was less events of EGFR, HER2, and HER3, there is reason to suspect that effective in phosphorylating AKT and ERK1/2. this tetra-specific, tetravalent antibody may be more efficacious Moreover, we assessed ligand-independent HER2 signaling as than FL518. well as VEGF-stimulated phosphorylation of VEGFR2 in BT474 FL518 binding to immobilized HER3-ECD or immobilized cells. Treatment with FL518 or CRTB6 potently inhibited the auto- HER2-ECD was observed to reduce in a dose-dependent man- phosphorylation of HER2 (Fig. 1H) and VEGF-stimulated phos- ner with increasing amounts of EGFR-ECD or VEGF, respec- phorylation of VEGFR2 (Fig. 1I) and decreased the phosphory- tively. Conversely, FL518 was competed away from immobi- lation of AKT. MEHD7945A modestly decreased the phosphor- lized EGFR-ECD or immobilized VEGF by soluble HER3-ECD ylation of HER2 but had no effect on the phosphorylation of or HER2-ECD protein, respectively. As expected, given their VEGFR2, whereas the bH1-44 had a comparable effect on the relative binding constants, higher concentrations of soluble phosphorylation of VEGFR2 and a small effect on that of HER2. EGFR-ECD or VEGF were needed to compete with the binding These antibodies were less effective in the phosphorylation of AKT of FL518 to immobilized HER3-ECD or HER2-ECD. These data than the four-in-one antibodies. were consistent with the parental antibodies MEHD7945A and bH1-44 (Fig. 1D). In contrast, no such data can be detected for Four-in-one antibodies have superior antitumor activity in vitro DVD-Ig CRTB6 (Supplementary Fig. S1). Competitive binding and in vivo assays employing antibodies were also performed and showed Having established FL518 and CRTB6 as potent inhibitors of that the epitopes of these four-in-one antibodies overlap with EGFR, HER3, HER2, and VEGF signaling, we sought to compare the epitopes of MEHD7945A and bH1-44 on EGFR, HER3, them with bispecific antibodies in cell proliferation assays with the HER2, and VEGF (Fig. 1E). stimulation of EGF, HRG, and VEGF. Thus, in contrast to the The affinities of the four-in-one antibodies and the parental parental antibodies MEHD7945A and bH1-44, we generated antibodies for EGFR, HER3, HER2, and VEGF were determined another four bispecific antibodies (CT9, CB12, RT6, and RB4) by surface plasmon resonance (SPR). As suggested by molec- using the KiH methodology and the CrossMab methodology ular modeling, the affinities of the four-in-one antibodies to from cetuximab/trastuzumab, cetuximab/bevacizumab, RG7116/ their respective ligands did not differ significantly from those trastuzumab, and RG7116/bevacizumab (Fig. 2A). Characteriza- of the parental antibodies and simultaneous binding to both tion of these bispecific antibodies shows similar or better inhibi- ligands could be shown by SPR assay (Supplementary Table tion profiles than their parental antibodies in our in vitro and in vivo S1). In addition, as summarized in Supplementary Table S2, assays (Supplementary Fig. S2). We chose the human non–small the main PK parameters of FL518 and CRTB6 in mice were very cell lung cancer (NSCLC) cell line H1666 (11) that expresses EGFR, close to those of MEHD7945A, bH1-44, and trastuzumab. The EGFR ligands, HER2, and HER3 in our cell proliferation assay. data presented in Supplementary Table S2 also show that CB12 and bH1-44 did not drastically inhibit cell growth, but a FL518 and CRTB6 had PK properties analogous to those of dose-dependent decrease in proliferation was observed with RB6, a conventional IgG molecule. Taken together, these data indi- CT9, and RB4. MEHD7945A also exhibited significant inhibition

(Continued.) Cell proliferation relative to an untreated control was measured after 7 or 6 days using AlamarBlue staining. D, MTS assay examining the effects of 10 mg/mL of the indicated antibodies on breast or pancreatic cancer cell proliferation in the absence or presence of EGF, HRG, and VEGF (0.5 nmol/L each). Results are shown as the percentage of control cell proliferation. Error bars, SD; , P < 0.5; , P < 0.01, compared with control (CTRL) group. All assays were performed independently three times. E, BxPC3, MDA-MB-231, and MCF-7 tumor-bearing mice were treated weekly using control IgG (10 mg/kg), CT9 (10 mg/kg), CB12 (10 mg/kg), RT6 (10 mg/kg), RB4 (10 mg/kg), MEHD7945A (10 mg/kg), bH1-44 (10 mg/kg), MEHD7945A plus bH1-44 (5 mg/each), FL518 (10 mg/kg), or CRTB6 (10 mg/kg). The ﬁrst dose was given as a 2 loading dose. Arrows, treatments; data are presented as the mean tumor volume SEM. Percent tumor growth inhibition was evaluated at day 26.

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in the cell proliferation assay. However, the greatest inhibition of (MEHD7945A plus bH1-44, TGI 66%). Moreover, the tumor cell growth was obtained with the four-in-one antibodies (Fig. 2B). growth of MCF-7 xenografts (Fig. 2E, right) was inhibited by Intriguingly, four-in-one antibodies potently inhibited cell prolif- bispecific antibodies MEHD7945A (TGI 39%), bH1-44 (TGI eration at lower antibody concentrations than the combination of 38%), CT9 (TGI 19%), CB12 (TGI 33%), RT6 (TGI 29%), and MEHD7945A and bH1-44, and the tetravalent DVD-Ig CRTB6 was RB4 (TGI 22%). Although the combined blockade in cell signal- more effective than FL518. The greater potency may be due to the ing by a combination of bispecific antibodies resulted in a gain in four-in-one antibodies' unique ability to simultaneously engage the inhibitory effect [MEHD7945A plus BH1-44 (TGI 50%)], more receptors on the surface of cells. The inhibition mechanism FL518 was more effective in inhibiting tumor growth (TGI underlying the tetravalent antibody is hard to predict because it 73%, P < 0.001), and the tetravalent antibody CRTB6 had the depends not only on the affinity for the antigens but also on the best inhibition rate (TGI 100%, P < 0.00001) of tumor xenografts local antigen concentration. Similar inhibition results can be ob- in nude mice. served for H1993 cells, which also exhibited a high level of expression of EGFR, HER2, and HER3 (Fig. 2C; ref. 25). The combi- Only four-in-one antibodies have the ability to disrupt nation of MEHD7945A and bH1-44 caused significant inhibition HER/MET crosstalk of cell proliferation; however, four-in-one antibodies had a better The above results showed that simultaneously targeting inhibition profile. several proliferation and survival signals will provide better Next, we performed similar proliferation assays for breast clinical outcomes; however, two observations drew our atten- cancer MDA-MB-231 (high EGFR expression; ref. 36), BT474 tion.First,moresignificant inhibition of AKT and ERK1/2 (high HER2 expression), and MCF-7 (low HER2 expression) signaling was attained using a single agent of four-in-one cells as well as for BxPC3 cells (21), a pancreatic cancer cell line antibodies than with any other antibody applied in the same driven by HER3 activation (Fig. 2D). In MDA-MB-231 cells, concentration. Second, in the in vitro and in vivo cancer cell bispecific antibodies did not exhibit significant inhibition, but proliferation assays, four-in-one antibodies, especially DVD-Ig the combination of MEHD7945 and bH1-44 had a consider- CRTB6, had a far greater inhibition rate than other antibodies, able toxic effect. However, four-in-one antibodies, particularly even the combination of parental antibodies. These data show the DVD-Ig CRTB6, showed a unique inhibitory effect on these that simultaneously targeting EGFR, HER2, HER3, and VEGF cells. In BT474 cells, MCF-7 cells and BxPC cells, anti-HER3 may involve a mechanism of inhibition other than only antibodies RT6, RB4, and MEHD7945A had better performance inhibition of the phosphorylation of EGFR, HER2, HER3, and than anti-EGFR/HER2 antibodies or anti-VEGF antibodies, VEGFR2. Interestingly, in MKN45 cells (39) and HCC827 (40) whereas FL518 inhibited proliferation more potently than cells stimulated with EGF and HRG, although phosphorylation bispecific antibodies and the combination of MEHD7945A of EGFR, HER2, HER3 could be inhibited by MEHD7945A or and bH1-44. CRTB6 exerted the greatest inhibitory effect in bH1-44, downstream ERK1/2 or STAT3 signaling was not all the cell lines. Taken together, these data suggested that four- interrupted (Fig. 3A), even at an extremely high dose (Fig. in-one antibodies exhibit increased antiproliferative activity 3B). Moreover, phosphorylation of receptors was even signif- compared with bispecific antibodies in vitro. icantly inhibited by the combination of these two types of To test four-in-one antibody activity in vivo, BxPC3 cells were antibodies. Downstream ERK1/2 was only inhibited with an injected subcutaneously into nude mice, and established tumors IC50 value of 18.6 mg/mL, and STAT3 signaling was not were treated once a week with four-in-one antibodies or bispecific interrupted in MKN45 cells (Fig. 3C). Particularly, phosphor- antibodies. For BxPC3 cells, RT6 inhibited tumor growth by 55% ylation of MET, the oncogene that is often coexpressed in compared with the vehicle control (P < 0.001; Fig. 2E, left), and tumors with EGF family receptors, was only inhibited when a RB4 suppressed tumor growth by 66% (P < 0.05), whereas CT9, significant high dose of a combination of MEHD7945A and CB12, and RB4 had only marginal effects. bH1-44 had a small bH1-44 was administered (IC50: 13.5 mg/mL). However, the effect and MEHD7945A significantly inhibited tumor growth by inhibition of MET was attained by the four-in-one antibodies 72%, whereas the four-in-one antibody FL518 inhibited tumor Fl518 and CRTB6 with an IC50 value of 0.37 mg/mL and 0.14 growth by nearly 92% compared with the vehicle control (P < mg/mL, respectively. Downstream phosphorylation of STAT3 0.0001), which was even more effective than the combination of occurred with an IC50 value of 0.17 and phosphorylation of P < parental antibodies ( 0.001, MEHD7945A plus bH1-44, TGI ERK1/2occurredwithanIC50 value of 0.28 mg/mL (Fig. 3D 77%). Notably, tumor growth was inhibited nearly 100% with and E). Accordingly, only four-in-one antibodies have the CRTB6 treatment compared with the vehicle control (P < 0.0001), unique ability to disrupt the MET/EGFR or MET/HER3 hetero- and the established tumors were ultimately eradicated in tumor- dimers in MKN45 cells (Fig. 3F). We concluded that blocking bearing mice. MET/HER crosstalk is a novel mechanism underlying the To further investigate and compare the potency of four-in-one superior inhibition ability of four-in-one antibodies. antibodies with other antibodies in additional xenograft models, breast cancer MDA-MB-231 and MCF-7 cells were injected sub- A MET/HER signaling shift is crucial for the resistance to cutaneously into female BALB/c nude mice. In our assay, the HER-directed therapeutic antibodies tumor growth of MDA-MB-231 xenografts (Fig. 2E, middle) was Because anti-HER antibodies cetuximab, trastuzumab, and inhibited by MEHD7945A (TGI 57%), BH1-44 (TGI 28%), CT9 pertuzumab are three widely used clinical antibody drugs, we (TGI 12%), CB12 (TGI 5%), RT6 (TGI 43%), and RB4 (TGI 34%), first generated cetuximab-resistant HCC827 cells (ReHCC827) whereas the combined blockade in cell signaling caused by FL518 using previously described methods (40). Next, we modeled the and CRTB6 was most effective in inhibiting tumor growth development of acquired resistance treating the trastuzumab- (TGI 83% and TGI 97%, respectively). The combination of insensitive breast cancer cell line HCC1954 cells in vivo by expos- bispecific antibodies did not significantly enhance inhibition ing xenografts to increasing concentrations of trastuzumab and

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Figure 3. Only four-in-one antibodies have the ability to disrupt HER/MET crosstalk. A, immunoblots examining the ability of 10 mg/mL of the indicated antibodies to inhibit the HER ligand (0.5 nmol/L EGF and 0.5 nmol/L HRG)–stimulated phosphorylation of EGFR, HER2, HER3, MET, AKT, ERK1/2, and STAT3 in MKN45 and HCC827 cells. B, immunoblots examining the ability of 50 mg/mL of the indicated antibodies to inhibit the HER ligand– stimulated phosphorylation of ERK1/2 and STAT3 in MKN45 cells. C–E, MKN45 cells treated with the indicated concentrations of MEHD7945A plus bH1-44, FL518, or CRTB6 were stimulated with HER ligands for 10 minutes. Cell lysates were immunoblotted to detect pMET, pERK1/2, pSTAT3, and total b-actin. IC50 is the concentration of antibodies that inhibits 50% of phosphorylation of indicated proteins. F, immunoblots examining the ability of 10 mg/mL of the indicated antibodies disrupt the MET/EGFR or MET/HER3 heterodimers in MKN45 cells.

pertuzumab to generate dual-resistance cells ReHCC1954. After- that enhanced MET/EGFR and MET/HER3 crosstalk and signal- ward, we validated the resistant phenotype in vivo and in vitro (Fig. ing may be acquired antibody resistance mechanisms. 4A and B). Importantly, although the cancer cell line HCC1954 can be inhibited by trastuzumab plus pertuzumab (Fig. 4B), Four-in-one antibodies, but not bispecific antibodies, cancer cells developed resistance to this current-evaluated treat- overcome resistance to HER-directed therapeutic antibodies ment strategy in clinic in our assay. Next, we tested phosphotyr- in vitro and in vivo osine signaling in parental and resistant cell lines and found that Our experiments have established that for cetuximab admin- the amounts of MET, phosphorylation of MET, phosphorylation istration–induced acquired resistance ReHCC827 cells, EGFR of HER3, phosphorylation of ERK1/2, and phosphorylation of signaling has shifted to EGFR/MET signaling, whereas for STAT3 were significantly enhanced in the resistant cells compared ReHCC1954 cells, HER2/HER3 signaling has shifted to HER3/ with the parental cells (Fig. 4C). Consistent with this, the resistant MET signaling. Because four-in-one antibodies have been shown cells showed a dramatic increase in EGFR/MET and HER3/MET to block MET signaling, inhibiting MET/EGFR or MET/HER3 heterodimers (Fig. 4C). In addition, the resistant cells expressed crosstalk, next we tested whether they could overcome acquired higher levels of EGF-like ligands (EGF, HRG, BTC, and HGF) than resistance to the HER direct therapy antibodies. Similar to the the parental cells (Fig. 4D). These data indicate that overexpres- previous result, only four-in-one antibodies potently inhibited sion of MET and EGF-like ligands may be associated with resistant EGF-induced MET/EGFR heterodimerization/signaling and HRG- phenotypes. dependent MET/HER3 heterodimerization/signaling as well as Next, we examined the effect of siRNA knockdown of MET on downstream phosphorylation of ERK1/2, STAT3, and AKT in the the resistant cell lines. Transfection of MET siRNA dramatically resistant cells (Fig. 5A). Accordingly, the extent of in vitro growth downregulated the amount of MET in both cell lines (Fig. 4E). inhibition by four-in-one antibodies was similar to the parental Treatment with MET siRNA effectively inhibited EGF-induced and resistant cells, whereas bispecific antibodies did not show MET/EGFR signaling and cell growth (Fig. 4F) in ReHCC827 significant inhibition in the resistant cells (Fig. 5B). Four-in-one cells. Our data also showed that treatment with MET siRNA antibody treatment significantly inhibited the tumor growth in resulted in effective inhibition of HRG-mediated HER3 signaling both established parental and resistant cell tumors in all tumor- and cell growth (Fig. 4G) in ReHCC1954 cells. In addition, MET bearing mice (Fig. 5C), whereas bispecific antibody treatment siRNA treatment in both cell lines considerably resensitized failed to lead to significant tumor inhibition. We also showed that resistant cells to cetuximab or dual trastuzumab and pertuzumab the combination of bispecific antibodies was significantly less treatment (Fig. 4F and G). Together, these data further suggest effective than four-in-one antibodies in inhibiting the in vitro

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Figure 4. MET/HER signaling shift is crucial for the resistance to HER-directed therapeutic antibodies. A and B, left, MTS assay evaluating cell proliferation of the indicated parental cancer cell lines and resistant sublines upon treatment with 10 mg/mL of control IgG, 10 mg/mL of cetuximab, or trastuzumab plus pertuzumab (5 mg/mL each). Error bars, SD. Right, tumor volume of HCC827, ReHCC827, HCC1954, or ReHCC1954 tumor xenografts after treatment with 10 mg/kg of control IgG or cetuximab or tastuzumab plus pertuzumab. Data are shown as the mean SEM. C, immunoblots comparing major cell signaling changes between the indicated parental breast cancer cell lines and their corresponding resistant sublines. D, real-time quantitative PCR analysis of expression of EGF-like ligands. Data are shown as the mean SD. E, ReHCC827 and ReHCC1954 cells were transfected with 100 pmol control or MET siRNA or were not transfected. (Continued on the following page.)

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Figure 5. Four-in-one antibodies, but not bispecific antibodies, overcome HER-directed therapeutic antibodies in vitro and in vivo. A, coimmunoprecipitation assay and immunoblots evaluating the effects of 10 mg/mL of the indicated pretreatments on EGF-induced MET/EGFR heterodimerization and signaling in ReHCC827 cell lines (left) or on HRG-induced MET/EGFR heterodimerization and signaling in ReHCC1954 cell lines (right). B, MTS assay evaluating cell proliferation of the indicated parental cancer cell lines and resistant sublines upon treatment with 10 mg/mL of indicated antibodies. Error bars, SD. Left, HCC827 and ReHCC827 cells. Right, HCC1954 and ReHCC1954 cells. C, tumor volume of HCC827 and ReHCC827 tumor xenografts (left) and HCC1954 and ReHCC1954 (right) tumor xenografts after treatment with the indicated antibodies. Data are shown as the mean SEM. proliferation of resistant cells (Fig. 5B and C). Consistent with and one VEGF, one HER3 and HER2, or one HER3 and one VEGF this, the in vivo antitumor activity of the antibodies in combina- simultaneously. Moreover, we generated another four-in-one tion was much lower than that of four-in-one antibodies in the DVD-Ig–like antibody, CRTB6, which is capable of binding one resistant xenograft mouse model (Fig. 5B and C). EGFR, one HER2, one HER3, and one VEGF simultaneously. To our knowledge, this is the first study to describe this type of Discussion antibody. Interestingly, four-in-one antibodies were more active Herein, we provide evidence that a four-in-one antibody, than all the bispecific antibodies in all in vitro and in vivo models. FL518, generated from two-in-one antibodies, MEHD7945A and Unexpectedly, our cell signaling assays revealed that four-in-one bH1-44, is capable of binding one EGFR and one HER2, one EGFR antibodies not only exhibited a blocking effect on EGFR, HER2,

(Continued.) Two days after transfection, cells were lysed and subjected to SDS-PAGE and immunoblotted with indicated antibodies. F and G, left, ReHCC827 cells or ReHCC1954 cells were transfected with 100 pmol control or MET siRNA or were not transfected. Two days after transfection, the cells were incubated with 10 mg/mL of cetuximab or 10 mg/mL of trastuzumab plus pertuzumab (5 mg/mL each) in serum-free medium for 1 hour at 37C. The cells were then treated with EGF (0.5 nmol/L) or HRG (0.5 nmol/L) for 10 minutes. After washing, the cells were lysed and subjected to SDS-PAGE and immunoblotted with indicated antibodies. Right, ReHCC827 cell proliferation or ReHCC1954 cell proliferation were determined by MTS assay, and EGF or HRG was added at a ﬁnal concentration of 0.5 nmol/L.

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HER3, and VEGF signaling but also had the capacity to block HER/ ly evaluate ADCC effect between four-in-one antibodies and MET crosstalk. Combination use of the anti-EGFR/HER3 anti- bispecific antibodies. An evaluation of whether targeted antibody body MEHD7945A plus the anti-HER2/VEGF antibody bH1-44 treatment of cancer could reduce tumor burden in syngeneic has a similar effect but required an extremely high dose. Although wild-type mice (47) is ongoing. Moreover, it is also interesting in the DVD-Ig design of four-in-one antibodies, cetuximab, tras- that whether other RTK crosstalks may involve in the HER recep- tuzumab, bevacizumab, and a new anti-HER3 antibody RG7116 tors that can be disrupted by novel antibodies, especially recent were used to be comparable with FL518. However, as the efficacy evidence had shown that the RTK AXL may be a key mediator of of adding bevacizumab in anti–HER-targeted therapy has been cetuximab resistance (48), providing a rationale for the cetuximab controversial and the cardiac safety may also arise as a disadvan- usage that would raise the crosstalk between EGFR and other tage, using other options for this arm may gain more potent RTKs. However, a detailed mechanism needs further investiga- therapeutic biomacromolecules, especially those antibodies that tion. In addition, it is well known that binding of cetuximab to recently have been suggested to enhance the immune response EGFR also resulted in induction of apoptosis, whereas HER2/ of antibody drugs, such as anti-CTLA4 antibody ipilimumab or HER3 signaling inhibits cell death through the PI3K–AKT– mTOR anti-CD3 antibody. pathway. AKT includes three distinct enzymes, each of which is a Sensitivity to a specific anticancer agent evolves during admin- member of the protein kinase family that is specific for serine– istration (8, 39). A variety of compensatory mechanisms may threonine and that inhibits apoptosis. Nevertheless, trastuzumab underlie acquired resistance to EGFR- or HER2-targeted agents, alone did not efficiently induce cancer cell apoptosis, partly due to including switching dimerization partners (7), upregulation of its weak effect on the HRG-dependent HER2/HER3 activation. ligands for HER3 or EGFR (24, 26, 41), or overexpression of However, combined therapy to block HER2/HER3 signaling, such particular receptors, especially HER3, which are identified as an as trastuzumab plus pertuzumab, is currently under investigation evolved response to current anti-HER/ErbB agents needing early in clinical use. Thus, four-in-one antibodies, particularly the tetra- and direct blockade (42). In a previous study, a bispecific anti- specific, tetravalent antibody, which combined anti-HER antibo- body generated from trastuzumab and pertuzumab also sup- dies in a single antibody, would amplify the apoptosis effect. All in ported this hypothesis, overcoming acquired resistance to trastu- all, our data greatly support the hypothesis that simultaneously zumab by effectively blocking HER2/HER3 heterodimerization targeting more than one proliferation and survival signal or (19). Moreover, receptor crosstalk of MET plays a critical role in targeting a resistance mechanism will provide better clinical out- the development of resistance to EGFR family inhibitors. Ampli- comes. The unique potential of FL518 and CRTB6 to target EGFR, fication of MET occurs in patients with NSCLC who develop HER2, HER3, and VEGF to interrupt MET/HER crosstalk and delay resistance to the RTK inhibitors such as gefitinib or erlotinib direct anti-HER drug resistance warrants their consideration as (11, 43, 44). In NSCLC cells selected for resistance to gefitinib promising anticancer therapies in the clinic. or erlotinib in vitro, MET is amplified and activated and stimulates fl MET/EGFR or HER3 phosphorylation and signaling to AKT Disclosure of Potential Con icts of Interest fl (11, 25). In Met-amplified gastric cancer cell lines, EGFR and No potential con icts of interest were disclosed. HER3 are basally phosphorylated, which is abrogated in the Authors' Contributions presence of Met inhibitors (23). Stimulation with both HGF and Conception and design: S. Hu, W. Fu, W. Xu, Y. Yang, M. Cruz, S.D. Berezov, EGF promotes downstream activation of several signaling path- D. Jorissen, H. Takeda, W. Zhu ways, including AKT, ERK1/2, and STAT3 (45). Met is also Development of methodology: S. Hu, W. Fu expressed at elevated levels in HER2-amplified human breast Acquisition of data (provided animals, acquired and managed patients, cancers, and cooperative signaling between MET and HER2 might provided facilities, etc.): W. Fu, W. Xu, Y. Yang, M. Cruz Analysis and interpretation of data (e.g., statistical analysis, biostatistics, be a mechanism by which MET promotes cancer progression (46). computational analysis): S. Hu, W. Fu, M. Cruz, S.D. Berezov, D. Jorissen HER3/MET crosstalk and signaling were observed to play a crucial Writing, review, and/or revision of the manuscript: S. Hu, W. Zhu role in the resistance to direct HER2 blocking therapy, especially Administrative, technical, or material support (i.e., reporting or organizing for the cancer cell lines resistant to the dual therapy of trastuzu- data, constructing databases): H. Takeda mab plus pertuzumab, which is currently under evaluation in Study supervision: W. Zhu clinics and has promoted good outcomes (2). Because MET was proved to be a select partner of HER family members, it is not Acknowledgments surprising that use of anti-HER agents would result in the acti- The authors thank Dr. Lijun Zhou, of Navy General Hospital, for critical review of the article; Celplor LLC and ProMab Biotechnologies, Inc. for help on vation of HER/MET dimerization and signaling. In our present mammalian cell–based recombinant protein production; Alamo Laboratories report, only simultaneous targeting of EGFR, HER2, and HER3 Inc. and Hefeng Inc. for help on cell proliferation assays; Crown Bioscience Inc. proved efficient to block the crosstalk between HER/MET, and and Western Biotechnology Inc. for help on xenograft studies; and Dr. Cheng superiority of four-in-one antibodies was also observed. Although Chen, of Tianjin University, and Dr. Tai An, of Union Medical College, for the combination of two-in-one antibodies to block EGFR, HER2, helpful discussions. and HER3 can also inhibit such crosstalk, four-in-one antibodies have the unique advantage of low dose. Grant Support Direct oncogenic-signal stress through competing with natural This work was supported by the Chinese National Natural Science Founda- ligands of HERs and inducing blockade of the HER signal path- tion (grant nos. 81273311, 81172119, and 31470897). way, and antibody-dependent cellular cytotoxicity (ADCC) effect The costs of publication of this article were defrayed in part by the payment of advertisement mediated through the Fc portion of the antibody, are the two page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mechanisms that underline the antitumor effect of anti-HER antibodies. However, in our in vivo data, xenografts were using Received June 20, 2014; revised September 12, 2014; accepted October 18, immunodeficiency mice for human cancer cells, we cannot direct- 2014; published OnlineFirst November 4, 2014.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2014 American Association for Cancer Research. Cancer Correction Research Correction: Four-in-One Antibodies Have Superior Cancer Inhibitory Activity against EGFR, HER2, HER3, and VEGF through Disruption of HER/MET Crosstalk Shi Hu, Wenyan Fu, Weihao Xu, Yang Yang, Melissa Cruz, Sandra D. Berezov, Daniel Jorissen, Hiroaki Takeda, and Wangdong Zhu

In the original version of this article (1), the authors discovered that the cancer cell lines MCF-7, BT474, HCC827, and HCC1954 were contaminated with HeLa cells. All experiments in which these cell lines were used (Figs. 1–5) have been repeated with new stocks of cell lines obtained from the ATCC. The ﬁndings and conclusions remain the same. The errors have been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.

References 1. Hu S, Fu W, Xu W, Yang Y, Cruz M, Berezov SD, et al. Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk. Cancer Res 2015;75:159–70.

Published online July 1, 2019. Cancer Res 2019;79:3525 doi: 10.1158/0008-5472.CAN-19-1515 Ó2019 American Association for Cancer Research.

www.aacrjournals.org 3525 Published OnlineFirst November 4, 2014; DOI: 10.1158/0008-5472.CAN-14-1670

Four-in-One Antibodies Have Superior Cancer Inhibitory Activity against EGFR, HER2, HER3, and VEGF through Disruption of HER/MET Crosstalk

Shi Hu, Wenyan Fu, Weihao Xu, et al.

Cancer Res Published OnlineFirst November 4, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-1670

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