novel anti-tm4sf1 antibody drug conjugates with activity ......anthony barry8, leigh zawel1, anthony...

Large Molecule Therapeutics

Novel Anti-TM4SF1 Antibody–Drug Conjugateswith Activity against Tumor Cells and TumorVasculatureAlberto Visintin1, Kelly Knowlton1, Edyta Tyminski1, Chi-Iou Lin2, Xiang Zheng1,Kimberly Marquette3, Sadhana Jain3, Lioudmila Tchistiakova3, Dan Li2,Christopher J. O'Donnell4, Andreas Maderna4, Xianjun Cao5, Robert Dunn5,William B. Snyder5, Anson K. Abraham1, Mauricio Leal6, Shoba Shetty7,Anthony Barry8, Leigh Zawel1, Anthony J. Coyle1, Harold F. Dvorak2, andShou-Ching Jaminet2

Abstract

Antibody–drug conjugates (ADC) represent a promisingtherapeutic modality for managing cancer. Here, we report anovel humanized ADC that targets the tetraspanin-like proteinTM4SF1. TM4SF1 is highly expressed on the plasma mem-branes of many human cancer cells and also on the endothelialcells lining tumor blood vessels. TM4SF1 is internalized uponinteraction with antibodies. We hypothesized that an ADCagainst TM4SF1 would inhibit cancer growth directly by killingcancer cells and indirectly by attacking the tumor vasculature.We generated a humanized anti-human TM4SF1 monoclonalantibody, v1.10, and armed it with an auristatin cytotoxic agentLP2 (chemical name mc-3377). v1.10-LP2 selectively killedcultured human tumor cell lines and human endothelial cellsthat express TM4SF1. Acting as a single agent, v1.10-LP2

induced complete regression of several TM4SF1-expressingtumor xenografts in nude mice, including non–small cell lungcancer and pancreas, prostate, and colon cancers. As v1.10 didnot react with mouse TM4SF1, it could not target the mousetumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3mpk, 2A7A-LP2 regressed several tumor xenografts withoutnoticeable toxicity. Combination therapy with v1.10-LP2 and2A7A-LP2 together was more effective than either ADC alone.These data provide proof-of-concept that TM4SF1-targetingADCs have potential as anticancer agents with dual actionagainst tumor cells and the tumor vasculature. Such agentscould offer exceptional therapeutic value and warrant furtherinvestigation. Mol Cancer Ther; 14(8); 1868–76. �2015 AACR.

IntroductionJudah Folkman envisioned that targeting the "tumor angio-

genic factor" responsible for initiating tumor angiogenesiswould have clinical benefit by preventing the formation of thenew blood vessels that tumors require if they are to survive and

grow beyond minimal size (1). VEGF is now recognized as theprimary tumor angiogenic factor, and antibodies directedagainst it can prevent the growth of many rapidly growingmouse tumors (2, 3). However, in the clinic, targeting VEGF orits primary angiogenic receptor, KDR (VEGFR-2), has met withonly limited success (4–6). Antibodies or "traps" directedagainst VEGF, or tyrosine kinase inhibitors that target VEGFreceptors, are effective for a time as monotherapies in renal cellcarcinoma and in some patients with glioblastoma multiforme.When combined with chemotherapy, they delay recurrence,and, in some instances, prolong patient survival, but are notcurative. If antivascular therapy is to become more effectiveand achieve its full potential, additional targets beyond theVEGF–VEGFR-2 axis are required.

Transmembrane-4 L Six Family member 1 (TM4SF1) wasdiscovered in 1986 as a tumor cell antigen recognized by themouse monoclonal antibody L6 (7, 8). TM4SF1 is an integralmembrane glycoprotein structurally related to tetraspanins (8, 9).It is abundantly expressed on many cancer cells (7, 10), onendothelial cells lining human cancer blood vessels (11), andon the endothelial cells of angiogenic blood vessels induced inmice with retinopathy of prematurity (12) or by an adenovirusexpressing VEGF-A (11). It is also weakly expressed on theendothelial cells of many normal organs and tissues (13, 14).TM4SF1 regulates cell motility and intercellular adhesion in both

1Pfizer Inc., Centers for Therapeutic Innovation (CTI), Boston, Massa-chusetts. 2The Center for Vascular Biology Research and the Depart-ments of Pathology, Beth Israel Deaconess Medical Center (BIDMC)and Harvard Medical School, Boston, Massachusetts. 3Pfizer Inc.,Global Biotherapeutic Technologies (GBT), Cambridge, Massachu-setts. 4Pfizer Inc.,Worldwide Medicinal Chemistry, Groton, Connecti-cut. 5Pfizer Inc., Centers for Therapeutic Innovation (CTI), San Diego,California. 6Pfizer Inc., Pharmacokinetics, Dynamics and Metabolism(PDM), Pearl River, New York. 7Pfizer Inc., Drug Safety R&D, Investi-gative Toxicology, Groton, Connecticut. 8Pfizer Inc., BiotherapeuticsPharmaceutical Sciences, Andover, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Shou-Ching Jaminet, Department of Pathology andthe Center for Vascular Biology Research, Beth Israel DeaconessMedical Center,330 Brookline Avenue, RN-280D, Boston, MA 02215. Phone: 617-667-8156; Fax:617-667-3591; E-mail: [email protected]; and Harold F. Dvorak,E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0188

�2015 American Association for Cancer Research.

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tumor (15–17) and endothelial cells (11, 18) and is clusteredon the plasma membrane in intermittent microdomains(11, 18, 19). Because TM4SF1 is highly expressed by manyhuman cancer cells (10) and is associated with pathologicangiogenesis, we hypothesized that targeting TM4SF1 wouldprovide a dual anticancer mechanism: killing tumor cells bothdirectly and also indirectly by targeting the tumor vasculature(secondary mechanism).

We reported previously that a mouse anti-human TM4SF1monoclonal (IgG1) antibody, 8G4, effectively destroyed thehuman component of the vascular network engineered in Matri-gel plugs implanted in nude mice, presumably by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism (20).8G4 also killed human PC3 prostate cancer cells that weredependent on that network (20). Together, these data indicatedthat naked anti-TM4SF1 antibodies were able to kill TM4SF1-expressing cells in both the tumor and vascular compartments(20). Another murine anti-TM4SF1 monoclonal antibody, L6(IgG2a–kappa; refs. 7, 10), had been used to treat human tumorcell lines in immunocompromised mice (10). L6, and its mouse/human chimeric variant, chL6, were well tolerated and producedobjective responses in patients with several different cancers thatexpressed TM4SF1 (10, 13, 21–24).

Building on our mouse experiments with 8G4, and theclinical experience with L6 and chL6 anti-TM4SF1 antibodies,we hypothesized that conjugation of a cytotoxic agent to anti-TM4SF1 antibodies would significantly amplify their anticanceractivity (25). To test this hypothesis, we humanized L6, gen-erating antibody v1.10; L6 and v1.10, unlike 8G4, cross-reactwith cynomolgus monkey TM4SF1 (10). We then armed v1.10with LP2 (chemical name mc-3377), a synthetic analog ofdolastatin-10 (26); dolastatin-10 and synthetic analogs, termedauristatins, inhibit tubulin polymerization, and ultimatelyinduce G2–M cell-cycle arrest and cell death at low picomolarintracellular concentrations (27). Tubulin inhibitors have beenextensively investigated as vascular targeting agents and seem tobe preferentially toxic against tumor and tumor vascular endo-thelial cells with high proliferation rates, while sparing thenondividing endothelial cells of normal tissues (28–30).

Because TM4SF1 is highly expressed by both tumor cells andtumor vascular endothelium, an ideal therapeutic would targetTM4SF1 on both cell types. Unfortunately, none of the mono-clonal antibodies we and others have raised against humanTM4SF1 cross-react with mouse TM4SF1 (7, 10, 20). Conse-quently, v1.10-LP2 would be expected to exert a direct effect onhuman tumor cells implanted in nude mice, but would not beexpected to have an indirect effect on the xenograft's mousevasculature. We therefore generated a humanized anti-mouseTM4SF1 antibody, 2A7A, and conjugated it to LP2 to test thetherapeutic potential of targeting the mouse tumor vasculature.We now report that both v1.10 and 2A7A antibody–drugconjugates (ADC) are highly effective as single agents againsttumor xenografts that express TM4SF1 and are still moreeffective when combined so as to target both human tumorcells and the mouse tumor vasculature.

Materials and MethodsCell lines and reagents

Tumor cell lines were purchased from ATCC and maintainedaccording to vendor recommended conditions (RPMI/10% FBS

media, supplemented with sodium pyruvate and nonessentialamino acids for Calu-3 and SK-Mes-1, at 37�C and 5% CO2).Human umbilical vein endothelial cells (HUVEC, Lonza) werecultured according to the supplier's protocols, and used at pas-sages 3–6. Endothelial cell purity was confirmed by flow cyto-metry (FACS) which yielded 100% double positive TM4SF1/CD31 cells. 293 stably expressing human or mouse TM4SF1were generated by lentiviral transduction using a lentiviral ex-pression vector from BioSettia, and packaged using ViraPower(Life Technologies). Human and mouse TM4SF1–transfected293 cells expressed 994 and 875 TM4SF1 mRNA copies/cell,respectively. Mouse anti-rabbit IgG was from SouthernBiotechnologies.

Linker/payload LP2The structure of LP2 (chemical name mc-3377) is shown in

Supplementary Fig. S1A. LP2 is comprised of a synthetic dolas-tatin-10 analog that resembles in its structure momomethylauristatin F (MMAF, red color in Supplementary Fig. S1A;ref. 27). However, a key difference of MMAF is the presence ofa N-methylated a,a-dimethyl amino acid (Aib) that replaces theN-methyl valine on the N-terminus of MMAF. This structuralmodification of dolastatin-10 analogs has recently beendescribed by Maderna and colleagues and provides syntheticanalogs with excellent potencies and differentiated ADME prop-erties (27). The conjugation to the antibody is accomplishedwith the maleimidocaproyl linker (mc, blue color in Supple-mentary Fig. S1A) that is attached to the cytotoxin via an amidebond. After ADC catabolism in lysosomes, ADCs generated withthis linker/payload are expected to produce Cys-capped-mc-3377 (26). The mc linker is termed "noncleavable" because therelease of the cytotoxic payload requires ADC catabolism in thelysosome.

Anti-TM4SF1 antibodies and ADCsMouse anti-human TM4SF1 antibodies 8G4 (20), and L6 and

chL6 (31), were described previously. L6 was humanized byComplementarity Determining Regions grafting and the human-ized L6 VH and VL were joined to the human IgG1 and humanKappa constant regions, respectively, with proprietary expressionvectors. On the basis of structure modeling (32), we generated aseries of humanized L6 variants modified by the introduction ofback mutations from the parental mouse antibody to restorebinding efficiency and antibody stability. The humanized L6variant chosen for further preclinical development, v1.10, out-performed the chL6 antibody as an ADC in a PC3 xenograftmodel. Recombinant humanized v1.10 antibody was producedat different scales in CHO or 293 cells. Preparation and charac-terization of the rabbit/human chimeric anti-mouse/rat TM4SF1antibody, 2A7A, are presented in Supplementary Materials andMethods.

Generation of anti-TM4SF1 ADCsADCswere prepared by partial reduction of the antibodies with

tris(2-carboxyethyl)phosphine followed by coupling to maleimi-docapronic-auristatin (mc3377; ref. 33). Excess of N-ethylmalei-mide and L-Cys were added in sequence to cap the unreactedthiols and quench any unreacted linker-payload. After overnightdialysis in PBS, pH 7.4, the antibodies were purified by sizeexclusion chromatography. Protein concentrations were

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determined by UV spectrophotometry. Drug:antibody ratio(DAR) was determined as described in Supplementary Fig. S1B.

Cytotoxicity assaysTarget cells were plated at densities of 500–2,000 cells/100 mL

culture medium per well in 96-well plates. After overnight incu-bation at 37�C, 100 mL of culture media containing serial dilu-tions of ADCs were added. Ninety-six hours later, 50 mL ofCellTiter-Glo (Promega) were added to each well and the plateswere read for luminescence. Data are expressed as % viabilitycompared with that of control untreated cells.

XenograftsXenografts were performed at Crown Bioscience Inc., Pfizer

Oncology, and BIDMC, as indicated. Six- to eight-week-oldfemale athymic nude mice (strain 490, homozygous, CharlesRiver) were used in the American sites, whereas BALB/c nudemice (HFK Bioscience) were used at Crown Bioscience. Mice wereinoculated subcutaneously with 1 to 5 million tumor cells toproduce 200–300 mm3-sized tumors within 15 days. Tumor-bearing mice were injected intraperitoneally (Crown BioscienceInc. and BIDMC) or intravenously (Pfizer Oncology) with ADCsas indicated. A nontargeting monoclonal antibody (8.8, humanIgG1) conjugated to LP2was used as a control at 10mpk (mg/kg).Tumor volumes were calculated as described (34). Animals werehoused and handled according to institution-approved animalcare protocols. Mice were terminated with CO2 inhalation fol-lowed by cervical dislocation.

Gene expression analysisTM4SF1 gene expression levels were estimated from the Cancer

Cell Line Encyclopedia (CCLE, http://www.broadinstitute.org/ccle/home). RNA expression was quantified with the Affymetrixmicroarray platform. Normalization of gene expression was doneby applying the Robust Multi-array Average algorithm. For vali-dation of themicroarray data, we employedmulti-gene transcrip-

tional profiling (MGTP) to quantify mRNA copies per cell bynormalization to 18S-rRNA, assuming that, on average, cellsexpress approximately 106 copies of 18S-rRNA (35, 36).

FACS analysisA total of 1 � 105 cells were reacted with the indicated

antibody or ADCs in 100 mL of PBS/2%FBS for 60 minutes onice and washed two times. In some instances, antibodies wereconjugated to a fluorophore; otherwise, an Alexa 647-labeledsecondary anti-human Fc (Life Technologies) was added andcells were incubated for an additional 60 minutes on ice. Afterwashing in PBS/2% FBS, fluorescence was quantified by FACS(BD LSR Fortessa; 10,000 events collected/point).

To determine the relative number of TM4SF1 copies onthe cell surface, we stained cells with saturating amounts(30–100 mg/mL) of Alexa488 (Life Technologies) conjugatedanti-TM4SF1 antibodies (chL6 or 2A7A) or isotype controlantibodies. The degree of labeling (DOL) for each antibodywas determined using a Nanodrop spectrophotometer [molesdye/mole protein ¼ absorbance494/(71,000 � protein concen-tration (mol/L)]. Calibration curves were generated using theQuantum Alexa488 MESF kit (Bangs Laboratories, Inc.). Thegeometric mean fluorescence values obtained from the testsamples were then divided by the DOL and the background(isotype control–stained cells) subtracted. TM4SF1 receptornumbers were calculated from a calibration curve.

ResultsExpression of TM4SF1 in human cancers

We used immunohistochemistry to confirm literature reports(10) that TM4SF1 protein was strongly expressed on the tumorand vascular cells of cancers resected from patients. Collaboratingwith Indivumed, we found strong tumor cell staining in 10 of13 liver, 16 of 20non–small cell lung (NSCLC), 4 of 20breast, and3 of 20 colon cancer patient samples (Supplementary Fig. S2), in

Figure 1.v1.10 andv1.10-LP2 (mc-3377) reactivitywith, and cytotoxicity against, culturedhuman endothelial cells. A, FACSanalysis of reactivity of v1.10 and v1.10-LP2 on HUVEC and HLEC. Geometricmean fluorescence (MFI) values areplotted versus antibody concentration.Apparent Kd values (EC50) weregenerated usingGraph Pad software. B,FACS analysis demonstrates that v1.10interacts strongly with 293 cells stablytransduced with human TM4SF1(293hTM4SF1, clear profile), but not with293 cells (gray profile) or with 293stably transduced with mouse TM4SF1(293mTM4SF1, black profile). C,cytotoxicity of HUVEC and HLECtreated with varying amounts of v1.10-LP2 and a control ADC, 8.8-LP2. Cellviability was assessed at 96 hours byluminometry.

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all cases the tumor vasculature and the vasculature of adjacentnormal tissues also stained.

Binding properties and cytotoxic effects of v1.10 and v1.10-LP2 on cultured endothelial cells

8G4 and L6 interact with different epitopes on externalloop 2 of human TM4SF1. Because L6 reacts with cynomolgusTM4SF1 (7, 10), whereas 8G4 does not (20), we selected L6for further studies and prepared v1.10, a humanized clone ofL6. We analyzed v1.10's reactivity with cultured humanendothelial cells that express high endogenous levels ofTM4SF1, that is, approximately 100 copies of TM4SF1mRNA/cell (11, 20) and 4–5 � 105 surface protein mole-cules/cell. As shown in Fig. 1A, v1.10 bound to culturedHUVECs and human lung microvascular endothelial cells(HLEC) with an apparent Kd of 2 to 5 nmol/L (the antibodyconcentration that gave 50% of the maximal binding by FACSanalysis). v1.10 was then conjugated to an auristatin-likepayload LP2 (chemical name mc-3377) to an average DARof 4 (Supplementary Fig. S1B). The resulting anti-humanTM4SF1 ADC (v1.10-LP2) bound to HUVECs and HLECs ina manner similar to v1.10 (Fig. 1A). Neither v1.10 nor v1.10-LP2 interacted with cells that did not express human TM4SF1,for example, 293 cells or 293 cells that were stably transfectedwith mouse TM4SF1 (Fig. 1B).

To achieve cytotoxicity, the antibody–toxin conjugate mustbe internalized and trafficked to lysosomes, where the anti-body is catabolized and the toxic payload is released. HUVECinternalized substantial amounts of v1.10 by 30 minutes andthere was essentially complete colocalization of antibodywith LAMP1-positive vesicles (late lysosomes) by 4 hours(Supplementary Fig. S3).

v1.10-LP2 ADC was highly effective in killing HUVECs andHLECs (Fig. 1C). For both cell lines, the IC50 of v1.10-LP2 killingwas in the low nanomolar range (1.7 nmol/L for HUVECs and2.2 nmol/L for HLECs), while a control nontargeting humanIgG1 antibody, 8.8-LP2, did not kill endothelial cell at concen-trations below 100 nmol/L.

Selection of TM4SF1-expressing tumor cell linesTumor cell lines expressing high levels of TM4SF1 were iden-

tified with an in silico survey of public and proprietary geneexpression databases (CCLE, Oncomine, Gene Logic, and OTP)and from prior publications (7, 8, 16, 37). As a group, NSCLCtumor cell lines expressed the highest levels of TM4SF1 amongsolid tumors (Fig. 2A). However, many other cancer cell lines alsoranked high, including those originating in ovary, liver, pancreas,prostate, colon, and breast (data not shown). TM4SF1 expressionlevels gave similar rank order whether measured as mRNA copynumbers (Fig. 2B) or as cell surface protein (Fig. 2C).

Sensitivity of various cancer cell lines to v1.10-LP2As with endothelial cells, the apparent Kd of v1.10 binding to

several tumor cell lines was in the single digit nanomolar range.Cell lines with low TM4SF1 RNA expression levels (e.g., Calu-6expresses only �4.2 � 104 surface copies of TM4SF1/cell), werelargely insensitive to v1.10-LP2. In general, in vitro sensitivity tov1.10-LP2 correlated with TM4SF1 RNA expression levels, withIC50 values in the low nanomolar range for most of the cell linestested (Fig. 2A and Supplementary Fig. S4). The exception wasNCI-H358, for which we have no explanation.

Expression of TM4SF1 in human tumor xenograftsSeven different tumor xenografts (Supplementary Fig. S5A)

were grown as xenografts, either at Crown Bioscience Inc. inBalb/c nude mice or at BIDMC in athymic 490 nude mice. SK-MES-1, A549, SW620, MiaPaCa2, and PC3 tumors expressedmore than 50 TM4SF1 mRNA copies/106 18S copies as deter-mined by MGTP (35, 36), whereas NCI-H460 and Calu6tumors expressed fewer than 50 copies. Tumor xenografts thatwere grown in athymic 490 nude mice at BIDMC expressedsubstantially higher levels of mouse TM4SF1, as well as thespecific endothelial cell markers CD144 and VEGFR-2, thantumors grown in Balb/c nude mice at Crown Bioscience Inc.(Supplementary Fig. S5B); TM4SF1 mRNA assays of bothCrown Bioscience and BIDMC xenograft tumors were per-formed at BIDMC. These data suggest that tumor xenograftsgrown in different nude mouse strains generate different levelsof vascular density.

Effect of v1.10-LP2 ADCs on NSCLCs and other tumorxenografts

To assess the therapeutic potential of anti-TM4SF1 ADCsin vivo, select NSCLC tumor cell lines (horizontal arrows

Figure 2.TM4SF1 expression in different human tumor cell lines and their sensitivityto v1.10-LP2 (mc-3377). A, plot of TM4SF1 mRNA expression by 12 non-small cell lung cancer (NSCLC) cell lines against sensitivity to v1.10-LP2.TM4SF1 mRNA expression data were extracted from the CCLE (www.broadinstitute.org/ccle/home; ref. 47). mRNA levels (y-ordinate) areexpressed in a RNA-normalized linear fluorescence intensity scale. IC50

values (x-ordinate) were calculated from full 10-point dose responsecurves (Supplementary Fig. S4) using GraphPad software. Except for NCI-H358 (indicated by black dashed circle), sensitivity to v1.10-LP2 correlatedwell with TM4SF1 RNA expression levels. Six NSCLC tumor cell lines(horizontal arrows) that express different levels of TM4SF1 were selectedfor xenograft studies (Fig. 3A). B and C, quantification of per cell mRNAcopy numbers and surface protein levels, respectively, by MGTP and FACSanalysis on five NSCLC and on the breast cancer cell line MCF-7. Meanfluorescence values were obtained using saturating amounts of Alexa488-labeled chimeric L6, and were converted to receptor numbers usingcalibration beads (Bangs Laboratories Inc.). Data represent the mean ofthree independent determinations.

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in Fig. 2A) were implanted subcutaneously in nude mice.When tumors had grown to approximately 200 mm3

(300 mm3 for A549 and SK-MES-1 cells), mice received intra-peritoneal injections of 100 mL of PBS, or PBS containing 3or 10 mpk of v1.10-LP2, or 10 mpk of control 8.8-LP2.Treatments were administered at 4-day intervals for four cycles(q4d�4).

v1.10-LP2 induced complete tumor regression, defined asnonpalpable tumors, in nearly all mice bearing NSCLC xeno-grafts that expressed high levels of TM4SF1 (Fig. 3A). Calu-6cells, which express low levels of TM4SF1 and were insensitiveto v1.10-LP2 in culture, were also poorly sensitive to v1.10-LP2in vivo.

Efficacy of v1.10-LP2 was also evaluated in human cancerxenografts originating in colon (SW620), prostate (PC3), andpancreas (MiaPaca-2, Capan-1, and Panc-1). Similar results wereobtained whether drugs were administered intravenously orintraperitoneally, and whether tumors were, or were not,implanted in Matrigel (Fig. 3B). Complete regressions wereachieved in the majority of the tumors tested at 3 mpk, and, at10 mpk, in all of the models except for SW620 at the PfizerOncology site where tumors did not regress but remained growthstatic for approximately 50 days.

v1.10-LP2 was well tolerated at all three study sites. Mice didnot exhibit signs of toxicity such as changes in grooming habits orweight loss at either 3 or 10 mpk. On the basis of the A549,MiaPaCa2 and PC3 regression profiles and the pharmacokineticsof v1.10-LP2 in nude mice, we calculated a serum effective

concentration range of 0.5 to 16 mg ADC/mL (SupplementaryFig. S6A).

2A7A, a humanized rabbit anti-mouse TM4SF1 monoclonalantibody

v1.10 does not recognize mouse TM4SF1. This is not sur-prising in that the amino acid sequences of human and mouseTM4SF1 extracellular loop 2 differ significantly (20), and nei-ther we nor others have been able to develop an antibody thatreacts with both human and mouse TM4SF1. Therefore, totarget the mouse TM4SF1 expressed on endothelial cell liningthe tumor xenograft vasculature, we developed a humanizedrabbit surrogate monoclonal antibody, 2A7A. 2A7A recognizedmouse TM4SF1, but did not react with human TM4SF1. Asshown in Fig. 4A, 2A7A bound to 293 cells that had been stablytransduced with mouse TM4SF1 (293mTM4SF1) and to theimmortalized mouse microvascular endothelial cell line, MS1.However, 2A7A did not bind to untransfected parental 293 cellsor to cells expressing human TM4SF1 such as HUVEC (Fig. 4B).Consistent with its binding pattern, 2A7A-LP2, was highlycytotoxic to MS1 cells and 293mTM4SF1 with IC50 values of0.015 and 0.003 nmol/L, respectively (Fig. 4C). The numberof mouse TM4SF1 molecules on the surface of 293mTM4SF1 orMS1 cells was calculated to be 5.2 � 105 and 5.7 � 105 copies/cell, respectively, that is, levels comparable with those ofhuman TM4SF1 expressed by HUVEC (�4 � 105 copies/cell)and HLEC (�5 � 105 copies/cell). However, the potency of the2A7A-LP2 ADC toward cultured rodent endothelial cells was

Figure 3.v1.10-LP2 (mc-3377)-induced regression of human tumor xenografts. A, different NSCLC tumor cell lines that express varying levels of TM4SF1 (Fig. 2A)were grown subcutaneously in nude mice to a size of approximately 200 to 300 mm3 (Calu3, SK-MES-1, EBC-1, NCI-H1299, Calu-6) or approximately300 mm3 (A549). Mice were then treated intraperitoneally with 100 mL PBS (blue), v1.10-LP2 [3 mpk (green) or 10 mpk (red)], or a matching controlADC 8.8-LP2 (10 mpk; fuchsia). ADCs were administered q4d�4 (vertical dotted lines). v1.10-LP2 produced sustained complete regressions in all modelsat both 3 and 10 mpk doses, except for the low TM4SF1 expressing Calu-6 model, where treatment even at 10 mpk only delayed tumor growth.Experiments were performed either at BIDMC (BI, 4 or 5 mice per group in 490 athymic nude mice) or at Crown Biosciences Inc. (CB, 10 mice pergroup in Balb/c nude mice). Insets indicate the number of animals achieving complete regression at 3 mpk. B, tumor xenograft models of pancreatic(MiaPaCa2, Panc-1), prostate (PC3), and colon (SW620) origin. In addition to tumors prepared with the same tumor growth and antibody injectionschemes shown in A, Pfizer Oncology (PO) implanted tumors in Matrigel and injected antibodies intravenously.

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about two orders of magnitude higher than that of v1.10-LP2against human HUVEC and HLEC, possibly due to the 10-foldhigher binding affinity of 2A7A for mouse TM4SF1 (0.5 nmol/L)versus that of v1.10 for human TM4SF1 (�5 nmol/L).

Distribution and antitumor activity of 2A7A-LP22A7A-LP2 injected intraperitoneally into mice bearing PC3

xenografts bound strongly to the mouse tumor vasculature, and

also to a lesser extent to the vasculature of nearby normaltissues, but did not bind tumor cells which expressed humanTM4SF1 (Fig. 5A). In contrast, v1.10-LP2, similarly injectedintraperitoneally into mice bearing PC3 tumors, localizedalmost entirely to the tumor cells which express humanTM4SF1 and was not detected in tumor or adjacent normaltissue blood vessels that express mouse TM4SF1 (Fig. 5B). Anontargeting isotype-matched control antibody, 8.8-LP2, did

Figure 4.2A7A and 2A7A-LP2 (mc-3377)reactivity with and cytotoxicityagainst different cultured cells. A,FACS analysis demonstrates that2A7A-Alexa488 conjugate reactsstrongly with 293 cells that stablyexpress mouse TM4SF1 (293mTM4SF1)and with MS1 immortalized mouseendothelial cell, but not withuntransfected parental 293 cells. B,MS1 cells (top) stained with 2A7A-Alexa488, whereas HUVEC (bottom)did not. C, cytotoxicity assays. 293cells were not susceptible to either2A7A-LP2 or 8.8-LP2 (top). 2A7A-LP2efficiently killed 293mTM4SF1 (middle),and MS1 cells (bottom), whereascontrol 8.8-LP2was not toxic to any ofthe cells at the concentrations used.Percent cell viability was determinedat 96 hours using the CellTiter-Glo kit.Results are from two independentexperiments which gave similarresults.

Figure 5.Distribution of 2A7A-LP2 (mc-3377) and v1.10-LP2 (mc-3377) in PC3 tumor xenografts and theeffect of 2A7A-LP2 on tumor xenografts. PC3tumor cells were implanted in athymic nudemiceand allowed to grow to 300mm3. Micewere theninjected intraperitoneally with 3 mpk 2A7A-LP2(A) or v1.10-LP2 (B). Forty-eight hours later,tumors were harvested after intravenousinjections of FITC-dextran to visualize bloodvessels. Fresh frozen sections were stained withAlexa594 conjugated anti-human IgG antibodiesto visualize 2A7A-LP2 (A) or v1.10-LP2 (B).Dashed white lines indicate tumor–hostinterface. 2A7A-LP2 was localized to tumor(white arrows) and host (yellow arrows) bloodvessels, largely obscuring green FITC-dextranstaining (A). In contrast, v1.10-LP2 was localizedto tumor cells (B) and green FITC-dextranstained vessels are clearly seen. C, effects of2A7A-LP2 (red lines) or control 8.8-LP2 (blacklines) on A549, MiaPaCa2, PC3, and SW620human tumor xenografts. Treatments (3 mpk,q4d�4, vertical dotted lines) were started whentumors had reached a size of approximately 300mm3. Experiments were performed at BIDMC, 5mice per group.

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not localize to the tumor or to the host vasculature (notshown).

Using the same q4d�4 protocol used for v1.10-LP2 (Fig. 3),2A7A-LP2 at 3 mpk induced partial (PC3) or complete (A549,MiaPaCa2, and SW620) regressions (Fig. 5C). MiaPaCa2 regres-sion persisted for almost 50 days, whereas A549 and SW620tumors recurred at around 8 days after the fourth injection of2A7A-LP2, and PC3 tumors began to recur even before thefourth injection. Treatment with 2A7A-LP2, like that with thecontrol ADC 8.8-LP2, was well tolerated at the 3 mpk dose levelwith mice exhibiting no signs of systemic toxicity such aschanges in grooming behavior or weight loss (SupplementaryFig. S6B). However, at higher doses of 5 to 10 mpk, significantweight loss was observed immediately after the fourth injection(data not shown).

Simultaneous targeting of TM4SF1 expressed on tumor cellsand on tumor vasculature

The foregoing studies with v1.10-LP2 and 2A7A-LP2 estab-lished that targeting TM4SF1 expressed by either tumor cells orby the tumor vasculature could regress tumor xenografts. Becausethe two approaches are nonredundant, we hypothesized thatthe combined effects of targeting tumor and vasculature wouldbe additive. To test this hypothesis, we studied Calu-6 and NCI-H460 tumors whose cells express low levels of TM4SF1 inculture (Fig. 2B and C). Also, whereas cultured NCI-H460 cellsdid respond to v1.10-LP2, Calu-6 did not (Supplemen-tary Fig. S4A). Furthermore, Calu-6 xenografts were resistant tov1.10-LP2 as a single agent (Fig. 3A).

As shown in Fig. 6A, 3 mpk v1.10-LP2 or 2A7A-LP2 alone onlyminimally impacted the growth of Calu-6 xenografts. However,when combined, these ADCs added an additional 15 days toprogression-free survival. Despite in vitro sensitivity, the NCI-H460 model was resistant to v1.10-LP2 in vivo, but respondedto 2A7A-LP2, delaying tumor progression by approximately 15days (Fig. 6B). Combination therapy delayed tumor growth for anadditional 10 days.

DiscussionTM4SF1 is an important housekeeping gene that is required

for the polarization and migration of cultured endothelial cell(11, 18). It is also highly expressed by many different humancancer cells (7, 10) and has important roles in cancer initiation,migration, and invasion (38). Earlier studies with L6, an anti-body targeting human TM4SF1, exhibited low toxicity and gavepromising results in mouse tumor xenografts and in a smallnumber of cancer patients, presumably via mechanisms involv-ing CDC and ADCC (21, 39). Together these findings suggestedthat therapy could be enhanced by targeting TM4SF1 with anADC approach. ADCs have entered the clinic and are currentlyused to target tumors expressing many different antigens,including CD33 (40), Her2 (41), and CD30 (42); more than100 additional ADCs directed against different tumor celltargets populate the preclinical and clinical pipelines (43–46). An ADC against a target expressed by both tumor cellsand the tumor vasculature would be expected to offer excep-tional therapeutic benefit.

Here, we report the development of v1.10, a humanizedversion of the previously described anti-TM4SF1 mouse mono-clonal antibody L6. v1.10 has low nanomolar affinity for

cultured human endothelial cells and for many cancer cells(Figs. 1A and 2), and, when reacted with plasma membraneTM4SF1, was internalized (Supplementary Fig. S3), a propertyessential for ADC efficacy. When conjugated to a proprietarytubulin inhibitor, LP2 (chemical name mc-3377), the resultingADC was highly cytotoxic against both cultured endothelial(Fig. 1C) and tumor cells (Supplementary Fig. S4). v1.10-LP2induced complete regressions in five of six different NSCLCtumor xenografts (Fig. 3A) and of tumor xenografts of pancreas,prostate, and colon origin (Fig. 3B). In general, sensitivity tov1.10-LP2 correlated well with TM4SF1 expression at both theRNA and protein levels and as measured both in vitro andin vivo. Naked v1.10 antibody (3 mpk, q4d�4) had slight anti-PC3 xenograft tumor activity, presumably due to ADCC, andlimited tumor growth during the four injection cycle; however,PC3 tumors did not regress and grew rapidly after treatmentceased (data not shown). Thus, the antitumor activity of v1.10-LP2 is largely implemented by ADC.

ADCs targeting TM4SF1 offer the opportunity to attack bothcancer cells and cancer-associated vascular endothelium. A con-cern, of course, is that such ADCs will also damage the vascularendothelium of normal organs. Four cycles of 2A7A-LP2 at 3mpkwere well tolerated, despite antibody binding to normal vascu-lature (Fig. 5A, yellow arrows), with no changes in body weight(Supplementary Fig. S6B), animal activity, or grooming behavior.Treatments could be safely increased to 10 cycles of 3 mpk 2A7A-LP2 at 2-day intervals (data not shown). Thus, 2A7A-LP2 has aclear predilection for damaging xenograft tumor endothelial cellsversus normal endothelial cell. However, at higher doses (5–10

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Figure 6.Combination treatment of refractory tumor xenografts with ADC directedagainst human and mouse TM4SF1. Clau-6 (A) and NCI-H460 (B)xenograft tumors were grown in Balb/c nude mice (Crown Biosciences,Inc.) to approximately 200 to 300 mm3 and were treated with v1.10-LP2 or2A7A-LP2, alone or in combination, q4d�4 (vertical dotted lines). Meantumor volumes � SEM, 10 mice per group.

Visintin et al.





mpk, q4d�4), 2A7A-LP2 caused substantial weight loss and evenlethality.

Consistent with the findings of Hellstrom with L6 (10),v1.10-ADC was well-tolerated in cynomolgus monkeys at dosesup to 12 mg/kg, although the pharmacologic relevance ofcynomolgus monkey to humans has not been fully establishedfor this target. The clinical experience with L6 and chL6 (andtheir radio-conjugated counterparts) is also consistent with ourmouse data. Although L6 did not carry a payload, it wascompetent to trigger CDC and ADCC, accumulated in endo-thelial cell–rich tissues such as lungs, and exhibited littletoxicity in patients (21, 39). A number of tubulin inhibitors,including vinca alkaloids, auristatins, taxanes, tubulysin, andcombretastatins, have been extensively researched and devel-oped as antiangiogenic drugs, and the resistance of normalendothelium to such agents has been well documented in theclinic (28–30).

Several important considerations need to be resolved beforeour mouse findings can be extended to human cancer patients.First, the binding affinity of 2A7A antibody to mouse TM4SF1(0.5 nmol/L) was 10-fold higher than the apparent Kd of v1.10antibody to human TM4SF1 (�5 nmol/L); this might translateinto better activity in different mouse strains, but could alsoincrease toxicity compared with v1.10-LP2. 2A7A-LP2 is 50- to100-fold more potent in killing immortalized mouse endo-thelial cells than is v1.10-LP2 in killing cultured humanendothelial cells or tumor cells, possibly because of bettertarget binding properties. It is therefore unclear how well2A7A-LP2 approximates the activity of v1.10-LP2 againsthuman vasculature. Second, while the overall distribution ofTM4SF1 in mouse organs is qualitatively similar to that inhumans, we have not yet determined whether the TM4SF1expression levels are quantifiably comparable. Third, the invivo models used here involve rapidly growing tumor xeno-grafts implanted subcutaneously in inbred mice. Whether thevasculature of these tumors appropriately phenocopies thetumor vasculature of established human cancers remains to bedetermined. Finally, 2A7A-LP2 showed an extremely rapiddistribution phase in mice, falling two logs in serum concen-tration in less than an hour after injection (Supplementary Fig.S6A). On the one hand, a rapid fall was not unexpected in thatanti-TM4SF1 antibodies bind to the endothelial cells of nor-mal organs throughout the body. However, the reporteddistribution phase of the chL6 antibody in human cancerpatients was approximately 5 days (37). Hence, 2A7A-LP2may be a poor predictor of therapeutic index or pharmaco-kinetics in humans. Nevertheless, at very low plasma levels,2A7A-LP2 effectively targeted human xenografts (Fig. 5) andachieved an even better outcome when combined with v1.10-LP2 (Fig. 6).

In sum, we have shown that two ADCs, one (v1.10-LP2)acting against human tumor TM4SF1 (direct mechanism) andthe other (2A7A-LP2) acting against mouse tumor vascularendothelial cell TM4SF1 (indirect mechanism), are effective assingle agents in treating mouse tumor xenografts (Figs. 3and 5), and that, when combined (Fig. 6), are still moreeffective. The response is likely determined by a number ofvariables, including TM4SF1 surface expression levels ontumor and vascular endothelial cells. Overall tumor vascularitymay also be a factor as we observed differences in vascularmarkers in tumors grown in different strains of nude mice

(Supplementary Fig. S5B). Unfortunately, v1.10-LP2 does notreact with murine TM4SF1, and so we do not at present have asingle ADC that is reactive with both human tumor and mousevascular TM4SF1 for use in preclinical studies. Nonetheless,our data validate the potential of TM4SF1 as an attractivehuman cancer target and provide proof-of-concept that target-ing TM4SF1 with an ADC can be exploited therapeuticallyto treat human cancer xenografts. v1.10-LP2 may be the firstin a new class of drugs with the bifunctional capacity to targeta molecule, TM4SF1, that is expressed both by tumor cellsand by the tumor vasculature.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception anddesign:A. Visintin, K. Knowlton, K.Marquette, L. Tchistiakova,C.J. O'Donnell, W. Snyder, L. Zawel, H.F. Dvorak, S.-C. JaminetDevelopment of methodology: A. Visintin, E. Tyminski, X. Zheng, S. Jain,L. Tchistiakova, A. Maderna, X. Cao, R. Dunn, W. Snyder, S.-C. JaminetAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Knowlton, E. Tyminski, C.-I. Lin, X. Zheng, S. Jain,D. Li, X. Cao, A. Barry, H.F. Dvorak, S.-C. JaminetAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Visintin, K. Knowlton, E. Tyminski, X. Zheng,K. Marquette, S. Jain, L. Tchistiakova, D. Li, A.K. Abraham, M. Leal, A. Barry,H.F. Dvorak, S.-C. JaminetWriting, review, and/or revision of the manuscript: A. Visintin, K. Knowlton,E. Tyminski, X. Zheng, K. Marquette, S. Jain, L. Tchistiakova, C.J. O'Donnell,R. Dunn, W. Snyder, A.K. Abraham, M. Leal, S. Shetty, A. Barry, H.F. Dvorak,S.-C. JaminetAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Li, M. Leal, H.F. Dvorak, S.-C. JaminetStudy supervision: A. Visintin, A.J. Coyle, H.F. Dvorak, S.-C. JaminetOther (project leader for this program on Pfizer's side; supervised andoverviewed all the activities pertaining to the program done at Pfizer; alsowrote the manuscript together with Dr. Dvorak): A. VisintinOther (served as a team member in studying this target; involved inscientific data interpretation, some study design, and review/revision ofmanuscript): S. Shetty

AcknowledgmentsThe authors thank the following Pfizer scientists and staff: Eric Bennett and

Rita Agostinelli for humanization of L6 and production of recombinant anti-bodies, Dikran Aivazian, Dana Castro, and Rachel Roach for generation of2A7A, Peter Pop-Damkov and Samantha Crocker for bioanalytical and Phar-macokinetic studies; Lauren Gauthier for studies with non-human primates,Matthew Doroski, Hud Risley, Alexander Porte, and Zecheng Chen for thesynthesis of mc-3377, and Evangelia Hatzis and Darcy Paige for programmanagement. Maoping Gao (CrownBiosciences) and Stephanie Shi coordinat-ed outsourced animal studies. The authors also thank Yu Liu and Jane Seo-JungSa for xenograft studies at BIDMC.

Grant SupportThis study was funded by Pfizer CTI through an award to co-principal

investigators (H.F. Dvorak and S.C. Jaminet) at Beth Israel Deaconess MedicalCenter.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 4, 2015; revised June 5, 2015; accepted June 7, 2015;published OnlineFirst June 18, 2015.

Anti-TM4SF1 ADCs





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2015;14:1868-1876. Published OnlineFirst June 18, 2015.Mol Cancer Ther Alberto Visintin, Kelly Knowlton, Edyta Tyminski, et al. Tumor Cells and Tumor Vasculature

Drug Conjugates with Activity against−Novel Anti-TM4SF1 Antibody

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