Novel Anti-TM4SF1 Antibody Drug Conjugates with Activity ... · 7 Pfizer Inc., Drug Safety R&D, Investigative Toxicology, Eastern Point Road, Groton, CT 06340 8 Pfizer Inc., Biotherapeutics

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Novel Anti-TM4SF1 Antibody Drug Conjugates with Activity against Tumor Cells and Tumor Vasculature

Alberto 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*, Shou-Ching Jaminet2*

1 Pfizer Inc., Centers for Therapeutic Innovation (CTI), 3 Blackfan circle, Boston, MA 02115 2 The Center for Vascular Biology Research and the Departments of Pathology, Beth Israel Deaconess Medical Center (BIDMC) and Harvard Medical School, RN280D, 330 Brookline Ave, Boston, MA 02215 3 Pfizer Inc., Global Biotherapeutic Technologies (GBT), 700 Main Street, Cambridge, MA 02139 4 Pfizer Inc., Worldwide Medicinal Chemistry, Eastern Point Road, Groton, CT 06340 5 Pfizer Inc., Centers for Therapeutic Innovation (CTI), 10700 Science Center Drive, San Diego, CA 92121 6 Pfizer Inc., Pharmacokinetics, Dynamics and Metabolism (PDM), 401 N. Middletown Road, Pearl River, NY 10965 7 Pfizer Inc., Drug Safety R&D, Investigative Toxicology, Eastern Point Road, Groton, CT 06340 8 Pfizer Inc., Biotherapeutics Pharmaceutical Sciences, One Burtt Road, Andover, MA 01810

*Corresponding authors: Shou-Ching S. Jaminet and Harold F. Dvorak, Department of Pathology and the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, RN-280D, Boston, MA02215, USA. Telephone and E-mail: [email protected] (617-667-8156); [email protected] (617-667-8529); Fax: 617-667-3591.

Conflict of interest: All of the authors except HFD, SCJ and CIL were Pfizer’s employees during the completion of the project. HFD, CSJ, DL and CIL declare no conflict of interest.

Running title: anti-TM4SF1 ADCs

Keywords: TM4SF1, Antibody Drug Conjugate (ADC), Vascular Targeting, Anti-angiogenesis, Tumor Targeting, Xenografts

Financial Information: This study was funded by Pfizer CTI through an award to co-PIs H.F. Dvorak and S.C. Jaminet at Beth Israel Deaconess Medical Center.

on April 25, 2020. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 18, 2015; DOI: 10.1158/1535-7163.MCT-15-0188

http://mct.aacrjournals.org/

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Abstract

Antibody Drug Conjugates (ADCs) represent a promising therapeutic modality for managing cancer.

Here we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is

highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells

lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized

that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly

by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal

antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-

LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1.

Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor

xenografts in nude mice, including NSCLC and pancreas, prostate and colon cancers. Since v1.10 did not

react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a

surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed

several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-

LP2 together was more effective than either ADC alone. These data provide proof of concept that

TM4SF1-targeting ADCs have potential as anti-cancer agents with dual action against tumor cells and

the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further

investigation.




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Introduction

Judah Folkman envisioned that targeting the “tumor angiogenic factor” responsible for initiating tumor

angiogenesis would have clinical benefit by preventing the formation of the new blood vessels that tumors

require if they are to survive and grow beyond minimal size (1). Vascular endothelial growth factor (VEGF) is

now recognized as the primary tumor angiogenic factor, and antibodies directed against it can prevent the

growth of many rapidly growing mouse tumors (2, 3). However, in the clinic, targeting VEGF or its primary

angiogenic receptor, KDR (VEGFR-2), has met with only limited success (4-6). Antibodies or “traps” directed

against VEGF, or tyrosine kinase inhibitors that target VEGF receptors, are effective for a time as

monotherapies in renal cell carcinoma and in some patients with glioblastoma multiforme. When combined

with chemotherapy, they delay recurrence, and, in some instances, prolong patient survival, but are not curative.

If anti-vascular therapy is to become more effective and achieve its full potential, additional targets beyond the

VEGF-VEGFR-2 axis are required.

Transmembrane-4 L Six Family member 1 (TM4SF1) was discovered in 1986 as a tumor cell antigen

recognized by the mouse monoclonal antibody L6 (7, 8). TM4SF1 is an integral membrane glycoprotein

structurally related to tetraspanins (8, 9). It is abundantly expressed on many cancer cells (7, 10), on endothelial

cells (EC) lining human cancer blood vessels (11), and on the EC of angiogenic blood vessels induced in mice

with retinopathy of prematurity (12) or by an adenovirus expressing VEGF-A (11). It is also weakly expressed

on the EC of many normal organs and tissues (13, 14). TM4SF1 regulates cell motility and intercellular

adhesion in both tumor (15-17) and endothelial cells (11, 18) and is clustered on the plasma membrane in

intermittent microdomains (11, 18, 19). Because TM4SF1 is highly expressed by many human cancer cells (10)

and is associated with pathological angiogenesis, we hypothesized that targeting TM4SF1 would provide a dual

anti-cancer mechanism: killing tumor cells both directly and also indirectly by targeting the tumor vasculature

(secondary mechanism).




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We reported previously that a mouse anti-human TM4SF1 monoclonal (IgG1) antibody, 8G4,

effectively destroyed the human component of the vascular network engineered in Matrigel plugs implanted in

nude mice, presumably by an ADCC mechanism (20). 8G4 also killed human PC3 prostate cancer cells that

were dependent on that network (20). Together these data indicated that 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) (7, 10), had been used to treat human tumor cell lines in

immuno-compromised mice (10). L6, and its mouse/human chimeric variant, chL6, were well tolerated and

produced objective responses in patients with several different cancers that expressed TM4SF1 (10, 13, 21-24).

Building on our mouse experiments with 8G4, and the clinical experience with L6 and chL6 anti-

TM4SF1 antibodies, we hypothesized that conjugation of a cytotoxic agent to anti-TM4SF1 antibodies would

significantly amplify their anti-cancer activity (25). To test this hypothesis, we humanized L6, generating

antibody v1.10; L6 and v1.10, unlike 8G4, cross-react with cynomolgus monkey TM4SF1 (10). We then armed

v1.10 with LP2 (chemical name mc-3377), a synthetic analog of dolastatin-10 (26); dolastatin-10 and synthetic

analogs, termed auristatins, inhibit tubulin polymerization and ultimately induce G2–M cell-cycle arrest and cell

death at low picomolar intracellular concentrations (27). Tubulin inhibitors have been extensively investigated

as vascular targeting agents and seem to be preferentially toxic against tumor and tumor vascular endothelial

cell (EC) with high proliferation rates, while sparing the non-dividing EC of normal tissues (28-30).

Because TM4SF1 is highly expressed by both tumor cells and tumor vascular endothelium, an ideal

therapeutic would target TM4SF1 on both cell types. Unfortunately, none of the monoclonal antibodies we and

others have raised against human TM4SF1 cross-react with mouse TM4SF1 (7, 10, 20). Consequently, v1.10-

LP2 would be expected to exert a direct effect on human tumor cells implanted in nude mice, but would not be

expected to have an indirect effect on the xenograft’s mouse vasculature. We therefore generated a humanized




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anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2 to test the therapeutic potential of targeting the

mouse tumor vasculature. We now report that both v1.10 and 2A7A ADCs are highly effective as single agents

against tumor xenografts that express TM4SF1 and are still more effective when combined so as to target both

human tumor cells and the mouse tumor vasculature.

Materials and Methods

Cell lines and reagents

Tumor cell lines were purchased from ATCC (Manassas, VA) and maintained according to vendor

recommended conditions (RPMI/10% FBS media, supplemented with sodium pyruvate and non-essential amino

acids for Calu-3 and SK-Mes-1, at 37ºC and 5% CO2). Human umbilical vein EC (HUVEC, Lonza,

Walkersville, MD) were cultured according to the supplier’s protocols, and used at passages 3-6. EC purity was

confirmed by flow cytometry (FACS) which yielded 100% double positive TM4SF1/CD31 cells. 293 stably

expressing human or mouse TM4SF1 were generated by lentiviral transduction using a lentiviral expression

vector from BioSettia (San Diego, CA), and packaged using ViraPower (Life Technologies, Grand Island, NY).

Human and mouse TM4SF1 transfected 293 cells expressed 994 and 875 TM4SF1 mRNA copies/cell,

respectively. Mouse anti-rabbit IgG was from Southern Biotechnologies (Birmingham, AL).

Linker/payload LP2

The structure of LP2 (chemical name mc-3377) is shown in Supplementary Fig. S1,A. LP2 is comprised of a

synthetic dolastatin-10 analog that resembles in its structure momomethyl auristatin F (MMAF, red color in

Supplementary Fig. S1,A) (27). However, a key difference of MMAF is the presence of a N-methylated α,α-

dimethyl amino acid (Aib) that replaces the N-methyl valine on the N-terminus of MMAF. This structural

modification of dolastatin-10 analogs has recently been described by Maderna et al. and provides synthetic

analogs with excellent potencies and differentiated ADME properties (27). The conjugation to the antibody is




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accomplished with the maleimidocaproyl linker (mc, blue color in Supplementary Fig. S1,A) that is attached to

the cytotoxin via an amide bond. After ADC catabolism in lysosomes, ADC’s generated with this

linker/payload are expected to produce Cys-capped-mc-3377 (26). The mc linker is termed “non-cleavable”

because the release of the cytotoxic payload requires ADC catabolism in the lysosome.

Anti-TM4SF1 antibodies and ADC

Mouse anti-human TM4SF1 antibodies 8G4 (20), and L6 and chL6 (31), were described previously. L6 was

humanized by Complementarity Determining Regions (CDR) grafting and the humanized L6 VH and VL were

joined to the human IgG1 and human Kappa constant regions, respectively, with proprietary expression vectors.

Based on structure modeling (32) we generated a series of humanized L6 variants modified by the introduction

of back-mutations from the parental mouse antibody to restore binding efficiency and antibody stability. The

humanized L6 variant chosen for further preclinical development, v1.10, outperformed the chL6 antibody as an

ADC in a PC3 xenograft model. Recombinant humanized v1.10 antibody was produced at different scales in

CHO or 293 cells. Preparation and characterization of the rabbit/human chimeric anti-mouse/rat TM4SF1

antibody, 2A7A, are presented in Supplementary Methods.

Generation of anti-TM4SF1 antibody-drug conjugates (ADCs)

ADCs were prepared by partial reduction of the antibodies with tris(2-carboxyethyl)phosphine followed by

coupling to maleimidocapronic-auristatin (mc3377) (33). Excess of N-ethylmaleimide and L-Cys were added in

sequence to cap the unreacted thiols and quench any unreacted linker-payload. After overnight dialysis in PBS,

pH 7.4, the antibodies were purified by size exclusion chromatography. Protein concentrations were determined

by UV spectrophotometry. Drug:Antibody Ratio (DAR) was determined as described in Supplementary Fig.

S1,B.

Cytotoxicity assays




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Target cells were plated at densities of 500-2,000 cells/100 μl culture medium per well in 96 well plates. After

overnight incubation at 37ºC, 100 ul of culture media containing serial dilutions of ADC were added. 96h later,

50 µl of CellTiter-Glo (Promega, Madison, WI) were added to each well and the plates were read for

luminescence. Data are expressed as % viability compared with that of control untreated cells.

Xenografts

Xenografts were performed at Crown Bioscience Inc. (CB, Beijing, China); at Pfizer Oncology (PO, Pearl

River, NY); and at BIDMC (BI), as indicated. Six to eight weeks old female athymic nude mice (strain 490,

homozygous, Charles River, Wilmington) were used in the American sites, whereas BALB/c nude mice (HFK

Bioscience, Beijing) were used at Crown Bioscience. Mice were inoculated subcutaneously with 1-5 million

tumor cells to produce 200-300 mm3-sized tumors within 15 days. Tumor-bearing mice were injected

intraperitoneally (CB, BI) or intravenously (PO) with ADCs as indicated. A non-targeting monoclonal antibody

(8.8, human IgG1) conjugated to LP2 was used as a control at 10 mpk (mg/kg). Tumor volumes were calculated

as described (34). Animals were housed and handled according to institution-approved animal care protocols.

Mice were terminated with CO2 inhalation followed by cervical dislocation.

Gene expression analysis

TM4SF1 gene expression levels were estimated from the Cancer Cell Line Encyclopedia (CCLE,

http://www.broadinstitute.org/ccle/home). RNA expression was quantified with the Affymetrix microarray

platform. Normalization of gene expression was done by applying the Robust Multi-array Average (RMA)

algorithm. For validation of the microarray data, we employed multi-gene transcriptional profiling (MGTP) to

quantify mRNA copies per cell by normalization to 18S-rRNA, assuming that, on average, cells express ~106

copies of 18S-rRNA (35, 36).

FACS analysis




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1x105 cells were reacted with the indicated antibody or ADC in 100 µl of PBS/2%FBS for 60 minutes on ice

and washed 2x. In some instances, antibodies were conjugated to a fluorophore; otherwise, an Alexa 647-

labeled secondary anti-human Fc (Life Technologies) was added and cells were incubated for an additional 60

minutes on ice. After washing 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 on the cell surface, we stained cells with saturating

amounts (30-100 µg/ml) of Alexa488 (Life Technologies)-conjugated anti-TM4SF1 antibodies (chL6 or 2A7A)

or isotype control antibodies. The degree of labeling (DOL) for each antibody was determined using a

Nanodrop spectrophotometer [moles dye/mole protein = absorbance494/(71,000 x protein concentration (M)].

Calibration curves were generated using the Quantum Alexa488 MESF kit (Bangs Laboratories, Inc., Fishers,

IN). The geometric mean fluorescence values obtained from the test samples were then divided by the DOL and

the background (isotype control stained cells) subtracted. TM4SF1 receptor numbers were calculated from a

calibration curve.

Results

Expression of TM4SF1 in human cancers

We used immunohistochemistry to confirm literature reports (10) that TM4SF1 protein was strongly expressed

on the tumor and vascular cells of cancers resected from patients. Collaborating with Indivumed (Hamburg,

Germany), we found strong tumor cell staining in 10 of 13 liver, 16 of 20 non-small cell lung (NSCLC), 4 of 20

breast, and 3 of 20 colon cancer patient samples (Supplementary Fig. S2); in all cases the tumor vasculature and

the vasculature of adjacent normal tissues also stained.

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




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8G4 and L6 interact with different epitopes on external loop 2 of human TM4SF1. Because L6 reacts with

cynomolgus TM4SF1 (7, 10), whereas 8G4 does not (20), we selected L6 for further studies and prepared

v1.10, a humanized clone of L6. We analyzed v1.10’s reactivity with cultured human EC that express high

endogenous levels of TM4SF1, i.e., ~100 copies of TM4SF1 mRNA/cell (11, 20) and 4 - 5x105 surface protein

molecules/cell. As shown in Fig. 1A, v1.10 bound to cultured human umbilical vein EC (HUVEC) and human

lung microvascular EC (HLEC) with an apparent Kd of 2-5 nM (the antibody concentration that gave 50% of

the maximal binding by FACS analysis). v1.10 was then conjugated to an auristatin-like payload LP2 (chemical

name mc-3377) to an average DAR of four (Supplementary Fig. S1,B). The resulting anti-human TM4SF1

ADC (v1.10-LP2) bound to HUVEC and HLEC in a manner similar to v1.10 (Fig. 1A). Neither v1.10 nor

v1.10-LP2 interacted with cells that did not express human TM4SF1, e.g., 293 cells or 293 cells that were stably

transfected with mouse TM4SF1 (Fig 1B).

To achieve cytotoxicity, the antibody-toxin conjugate must be internalized and trafficked to lysosomes,

where the antibody is catabolized and the toxic payload is released. HUVEC internalized substantial amounts of

v1.10 by 30 min and there was essentially complete co-localization of antibody with LAMP1 positive vesicles

(late lysosomes) by 4 hours (Supplementary Fig. S3).

v1.10-LP2 ADC was highly effective in killing HUVEC and HLEC (Fig. 1C). For both cell lines, the

IC50 of v1.10-LP2 killing was in the low nanomolar range (1.7 nM for HUVEC and 2.2 nM for HLEC), while a

control non-targeting human IgG1 antibody, 8.8-LP2, did not kill EC at concentrations below 100 nM.

Selection of TM4SF1-expressing tumor cell lines

Tumor cell lines expressing high levels of TM4SF1 were identified with an in silico survey of public and

proprietary gene expression databases (CCLE, Oncomine, Gene Logic and OTP) and from prior publications (7,

8, 16, 37). As a group, NSCLC tumor cell lines expressed the highest levels of TM4SF1 among solid tumors

(Fig 2A). However, many other cancer cell lines also ranked high, including those originating in ovary, liver,




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pancreas, prostate, colon and breast (data not shown). TM4SF1 expression levels gave similar rank order

whether measured as mRNA copy numbers (Fig. 2B) or as cell surface protein (Fig. 2C).

Sensitivity of various cancer cell lines to v1.10-LP2

As with EC, 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-6 expresses only ~4.2x104 surface copies

of TM4SF1/cell), were largely insensitive to v1.10-LP2. In general, in vitro sensitivity to v1.10-LP2 correlated

with TM4SF1 RNA expression levels, with IC50 values in the low nanomolar range for most of the cell lines

tested (Fig. 2A and Supplementary Fig. S4). The exception was NCI-H358, for which we have no explanation.

Expression of TM4SF1 in human tumor xenografts

Seven different tumor xenografts (Supplementary Fig. S5,A) were grown as xenografts, either at Crown Bio

(CB) in Balb/c nude mice or at BIDMC (BI) in athymic 490 nude mice. SK-MES-1, A549, SW620, MiaPaCa2

and PC3 tumors expressed more than 50 TM4SF1 mRNA copies/106 18S copies as determined by MGTP (35,

36), whereas NCI-H460 and Calu6 tumors expressed fewer than 50 copies. Tumor xenografts that were grown

in athymic 490 nude mice at BI expressed substantially higher levels of mouse TM4SF1, as well as the specific

EC markers CD144 and VEGFR-2, than tumors grown in Balb/c nude mice at CB (Supplementary Fig. S5,B).

These data suggest that tumor xenografts grown in different nude mouse strains generate different levels of

vascular density.

Effect of v1.10-LP2 ADC on NSCLC and other tumor xenografts

In order to assess the therapeutic potential of anti-TM4SF1 ADCs in vivo, select NSCLC tumor cell

lines (horizontal arrows in Fig. 2A) were implanted subcutaneously in nude mice. When tumors had grown to

~200 mm3 (300 mm3 for A549 and SK-MES-1 cells), mice received intraperitoneal injections of 100 µl of PBS,




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or PBS containing 3 or 10 mpk of v1.10-LP2, or 10 mpk of control 8.8-LP2. Treatments were administered at

four-day intervals for four cycles (q4dx4).

v1.10-LP2 induced complete tumor regression, defined as non-palpable tumors, in nearly all mice

bearing NSCLC xenografts that expressed high levels of TM4SF1 (Fig. 3A). Calu-6 cells, which express low

levels of TM4SF1 and were insensitive to v1.10-LP2 in culture, were also poorly sensitive to v1.10-LP2 in vivo.

Efficacy of v1.10-LP2 was also evaluated in human cancer xenografts originating in colon (SW620),

prostate (PC3) and pancreas (MiaPaca-2, Capan-1, and Panc-1). Similar results were obtained whether drugs

were administered intravenously or intraperitoneally, and whether tumors were, or were not, implanted in

Matrigel (Fig. 3B). Complete regressions were achieved in the majority of the tumors tested at 3 mpk, and, at 10

mpk, in all of the models except for SW620 at the PO site where tumors did not regress but remained growth

static for ~50 days.

v1.10-LP2 was well-tolerated at all three study sites. Mice did not exhibit signs of toxicity such as

changes in grooming habits or weight loss at either 3 or 10 mpk. Based on the A549, MiaPaCa2 and PC3

regression profiles and the pharmacokinetics (PK) of v1.10-LP2 in nude mice, we calculated a serum effective

concentration range of 0.5 to 16 µg ADC/ml (Supplementary Fig. S6,A).

2A7A, a humanized rabbit anti-mouse TM4SF1 monoclonal antibody

v1.10 does not recognize mouse TM4SF1. This is not surprising in that the amino acid sequences of human and

mouse TM4SF1 extracellular loop 2 differ significantly (20), and neither we nor others have been able to

develop an antibody that reacts with both human and mouse TM4SF1. Therefore, to target the mouse TM4SF1

expressed on EC lining the tumor xenograft vasculature, we developed a humanized rabbit surrogate

monoclonal antibody, 2A7A. 2A7A recognized mouse TM4SF1, but did not react with human TM4SF1. As

shown in Fig. 4A, 2A7A bound to 293 cells that had been stably transduced with mouse TM4SF1 (293mTM4SF1)




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and to the immortalized mouse microvascular EC line, MS1. However, 2A7A did not bind to untransfected

parental 293 cells or to cells expressing human TM4SF1 such as HUVEC (Fig. 4B). Consistent with its binding

pattern, 2A7A-LP2 was highly cytotoxic to MS1 cells and 293mTM4SF1 with IC50 values of 0.015 and 0.003 nM,

respectively (Fig. 4C). The number of mouse TM4SF1 molecules on the surface of 293mTM4SF1 or MS1 cells

was calculated to be 5.2x105 and 5.7x105 copies/cell, respectively, i.e., levels comparable to those of human

TM4SF1 expressed by HUVEC (~4x105 copies/cell) and HLEC (~5x105 copies/cell). However, the potency of

the 2A7A-LP2 ADC toward cultured rodent EC was about two orders of magnitude higher than that of v1.10-

LP2 against human HUVEC and HLEC, possibly due to the 10-fold higher binding affinity of 2A7A for mouse

TM4SF1 (0.5 nM) versus that of v1.10 for human TM4SF1 (~5 nM).

Distribution and anti-tumor activity of 2A7A-LP2

2A7A-LP2 injected intraperitoneally into mice bearing PC3 xenografts bound strongly strongly to the mouse

tumor vasculature, and also to a lesser extent to the vasculature of nearby normal tissues, but did not bind tumor

cells which expressed human TM4SF1 (Fig. 5A). By contrast, v1.10-LP2, similarly injected intraperitoneally

into mice bearing PC3 tumors, localized almost entirely to the tumor cells which express human TM4SF1 and

was not detected in tumor or adjacent normal tissue blood vessels that express mouse TM4SF1 (Fig. 5B). A

non-targeting isotype-matched control antibody, 8.8-LP2, did not localize to the tumor or to the host vasculature

(not shown).

Using the same q4dx4 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 regression persisted for almost 50

days, whereas A549 and SW620 tumors recurred at around 8 days after the 4thinjection of 2A7A-LP2, and PC3

tumors began to recur even before the 4th injection. Treatment with 2A7A-LP2, like that with the control ADC

8.8-LP2, was well tolerated at the 3 mpk dose level with mice exhibiting no signs of systemic toxicity such as




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changes in grooming behavior or weight loss (Supplementary Fig. S6,B). However, at higher doses of 5-10

mpk, significant weight loss was observed immediately after the 4th injection (data not shown).

Simultaneous targeting of TM4SF1 expressed on tumor cells and on tumor vasculature

The foregoing studies with v1.10-LP2 and 2A7A-LP2 established that targeting TM4SF1 expressed by either

tumor cells or by the tumor vasculature could regress tumor xenografts. Because the two approaches are non-

redundant, we hypothesized that the combined effects of targeting tumor and vasculature would be additive. To

test this hypothesis, we studied Calu-6 and NCI-H460 tumors whose cells express low levels of TM4SF1 in

culture (Fig. 2B, C). Also, whereas cultured NCI-H460 cells did respond to v1.10-LP2, Calu-6 did not

(Supplementary Fig. S4,A). Further, Calu-6 xenografts were resistant to v1.10-LP2 as a single agent (Fig. 3A).

As shown in Figure 6A, 3 mpk v1.10-LP2 or 2A7A-LP2 alone only minimally impacted the growth of

Calu-6 xenografts. However, when combined, these ADCs added an additional 15 days to progression free

survival. Despite in vitro sensitivity, the NCI-H460 model was resistant to v1.10-LP2 in vivo, but responded to

2A7A-LP2, delaying tumor progression by ~15 days (Fig. 6B). Combination therapy delayed tumor growth for

an additional 10 days.

Discussion

TM4SF1 is an important housekeeping gene that is required for the polarization and migration of cultured EC

(11, 18). It is also highly expressed by many different human cancer cells (7, 10) and has important roles in

cancer initiation, migration and invasion (38). Earlier studies with L6, an antibody targeting human TM4SF1,

exhibited low toxicity and gave promising results in mouse tumor xenografts and in a small number of cancer

patients, presumably via mechanisms involving CDC and ADCC (21, 39). Together these findings suggested

that therapy could be enhanced by targeting TM4SF1 with an ADC approach. ADCs have entered the clinic and

are currently used to target tumors expressing many different antigens, including CD33 (40), Her2 (41), CD30




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(42); more than 100 additional ADCs directed against different tumor cell targets populate the preclinical and

clinical pipelines (43-46). An ADC against a target expressed by both tumor cells and the tumor vasculature

would be expected to offer exceptional therapeutic benefit.

Here we report the development of v1.10, a humanized version of the previously described anti-

TM4SF1 mouse monoclonal antibody L6. v1.10 has low nanomolar affinity for cultured human EC and for

many cancer cells (Figs. 1A, 2), and, when reacted with plasma membrane TM4SF1, was internalized

(Supplementary Figs. S3), a property essential for ADC efficacy. When conjugated to a proprietary tubulin

inhibitor, LP2 (chemical name mc-3377), the resulting ADC was highly cytotoxic against both cultured

endothelial (Fig. 1C) and tumor cells (Supplementary Fig. S4). v1.10-LP2 induced complete regressions in five

of six different NSCLC tumor xenografts (Fig. 3A) and of tumor xenografts of pancreas, prostate and colon

origin (Fig. 3B). In general, sensitivity to v1.10-LP2 correlated well with TM4SF1 expression at both the RNA

and protein levels and as measured both in vitro and in vivo. Naked v1.10 antibody (3 mpk, q4dx4) had slight

anti-PC3 xenograft tumor activity, presumably due to ADCC, and limited tumor growth during the four

injection cycle; however, PC3 tumors did not regress and grew rapidly after treatment ceased (data not shown).

Thus, the anti-tumor activity of v1.10-LP2 is largely implemented by ADC.

ADCs targeting TM4SF1 offer the opportunity to attack both cancer cells and cancer-associated vascular

endothelium. A concern, of course, is that such ADCs will also damage the vascular endothelium of normal

organs. Four cycles of 2A7A-LP2 at 3 mpk were well tolerated, despite antibody binding to normal vasculature

(Fig. 5A, yellow arrows), with no changes in body weight (Supplementary Fig. S6,B), 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 a clear predilection for damaging xenograft tumor EC versus normal

EC. However, at higher doses (5-10 mpk, q4dx4), 2A7A-LP2 caused substantial weight loss and even lethality.




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Consistent with the findings of Hellstrom with L6 (10), v1.10-ADC was well-tolerated in cynomolgus

monkeys at doses up to 12 mg/kg, although the pharmacological relevance of cynomolgus monkey to humans

has not been fully established for this target. The clinical experience with L6 and chL6 (and their radio-

conjugated counterparts) is also consistent with our mouse data. Although L6 did not carry a payload, it was

competent to trigger CDC and ADCC, accumulated in EC-rich tissues such as lungs, and exhibited little toxicity

in patients (21, 39). A number of tubulin inhibitors, including vinca alkaloids, auristatins, taxanes, tubulysin and

combretastatins, have been extensively researched and developed as anti-angiogenic drugs, and the resistance of

normal endothelium to such agents has been well documented in the clinic (28-30).

Several important considerations need to be resolved before our mouse findings can be extended to

human cancer patients. First, the binding affinity of 2A7A antibody to mouse TM4SF1 (0.5 nM) was 10-fold

higher than the apparent Kd of v1.10 antibody to human TM4SF1 (~5 nM); this might translate into better

activity in different mouse strains, but could also increase toxicity compared with v1.10-LP2. 2A7A-LP2 is 50-

100 fold more potent in killing immortalized mouse EC than is v1.10-LP2 in killing cultured human EC or

tumor cells, possibly because of better target binding properties. It is therefore unclear how well 2A7A-LP2

approximates the activity of v1.10-LP2 against human vasculature. Second, while the overall distribution of

TM4SF1 in mouse organs is qualitatively similar to that in humans, we have not yet determined whether the

TM4SF1 expression levels are quantifiably comparable. Third, the in vivo models used here involve rapidly

growing tumor xenografts implanted subcutaneously in inbred mice. Whether the vasculature of these tumors

appropriately phenocopies the tumor vasculature of established human cancers remains to be determined.

Finally, 2A7A-LP2 showed an extremely rapid distribution phase in mice, falling two logs in serum

concentration in less than an hour after injection (Supplementary Fig. S6,A). On the one hand, a rapid fall was

not unexpected in that anti-TM4SF1 antibodies bind to the EC of normal organs throughout the body. However,

the reported distribution phase of the chL6 antibody in human cancer patients was ~5 days (37). Hence, 2A7A-




16

LP2 may be a poor predictor of therapeutic index or pharmacokinetics in humans. Nevertheless, at very low

plasma levels, 2A7A-LP2 effectively targeted human xenografts (Fig. 5) and achieved 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) and the other (2A7A-LP2) acting against mouse tumor vascular EC TM4SF1 (indirect

mechanism), are effective as single agents in treating mouse tumor xenografts (Figs. 3, 5), and that, when

combined (Fig. 6), are still more effective. The response is likely determined by a number of variables,

including TM4SF1 surface expression levels on tumor and vascular endothelial cells. Overall tumor vascularity

may also be a factor as we observed differences in vascular markers in tumors grown in different strains of nude

mice (Fig. S5,B). Unfortunately, v1.10-LP2, does not react with murine TM4SF1, and so we do not at present

have a single ADC that is reactive with both human tumor and mouse vascular TM4SF1 for use in preclinical

studies. Nonetheless, our data validate the potential of TM4SF1 as an attractive human cancer target and

provide proof of concept that targeting TM4SF1 with an ADC can be exploited therapeutically to treat human

cancer xenografts. v1.10-LP2 may be the first in a new class of drugs with the bifunctional capacity to target a

molecule, TM4SF1, that is expressed both by tumor cells and by the tumor vasculature.

Acknowledgements

We thank the following Pfizer scientists and staff: Eric Bennett and Rita Agostinelli for humanization of L6 and

production of recombinant antibodies; Dikran Aivazian, Dana Castro, and Rachel Roach for generation of

2A7A; Peter Pop-Damkov and Samantha Crocker for bioanalytical and PK studies; Lauren Gauthier for studies

with non-human primates; Matthew Doroski, Hud Risley, Alexander Porte and Zecheng Chen for the synthesis

of mc-3377 and Evangelia Hatzis and Darcy Paige for program management. Maoping Gao




17

(CrownBiosciences) and Stephanie Shi coordinated outsourced animal studies. We also thank Yu Liu and Jane

Seo-Jung Sa for xenograft studies at BIDMC.




18

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23

Figure Legends

Figure 1. v1.10 and v1.10-LP2 (mc-3377) reactivity with, and cytotoxicity against, cultured human

endothelial cells. A, FACS analysis of reactivity of v1.10 and v1.10-LP2 on HUVEC and HLEC. Geometric

mean fluorescence (MFI) values are plotted versus antibody concentration. Apparent Kd values (EC50) were

generated using Graph Pad software. B, FACS analysis demonstrates that v1.10 interacts strongly with 293 cells

stably transduced with human TM4SF1 (293hTM4SF1, clear profile), but not with 293 cells (gray profile) or with

293 stably transduced with mouse TM4SF1 (293mTM4SF1, black profile). C, Cytotoxicity of HUVEC and HLEC

treated with varying amounts of v1.10-LP2 and a control ADC, 8.8-LP2. Cell viability was assessed at 96 hours

by luminometry.

Figure 2. TM4SF1 expression in different human tumor cell lines and their sensitivity to 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 Cancer Cell Line

Encyclopedia (www.broadinstitute.org/ccle/home) (47). mRNA levels (y-ordinate) are expressed in a RNA-

normalized linear fluorescence intensity scale. IC50 values (x-ordinate) were calculated from full 10-point dose

response curves (Supplementary Fig. S4) using Graph pad software. Except for NCI-H358 (indicated by black

dashed circle), sensitivity to v1.10-LP2 correlated well with TM4SF1 RNA expression levels. Six NSCLC

tumor cell lines (horizontal arrows) that express different levels of TM4SF1 were selected for xenograft studies

(Fig. 3A). B,C, Quantification of per cell mRNA copy numbers and surface protein levels, respectively, by

MGTP and FACS analysis on five NSCLC and on the breast cancer cell line MCF-7. Mean fluorescence values

were obtained using saturating amounts of Alexa488-labeled chimeric L6, and were converted to receptor

numbers using calibration beads (Bangs Laboratories Inc.). Data represent the mean of three independent

determinations.




24

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

~200-300 mm3 (Calu3, SK-MES-1, EBC-1, NCI-H1299, Calu-6) or ~300 mm3 (A549). Mice were then treated

intraperitoneally with 100 μl PBS (blue), v1.10-LP2 [3 mpk (green) or 10 mpk (red)], or a matching control

ADC 8.8-LP2 (10 mpk; fuchsia). ADCs were administered q4dx4 (vertical dotted lines). v1.10-LP2 produced

sustained complete regressions in all models at 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 per group 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 injection schemes

shown in (A), the Pfizer Oncology (PO) implanted tumors in Matrigel and injected antibodies intravenously.

Figure 4. 2A7A and 2A7A- LP2 (mc-3377) reactivity with and cytotoxicity against different cultured cells.

A, FACS analysis demonstrates that 2A7A-Alexa488 conjugate reacts strongly with 293 cells that stably

express mouse TM4SF1 (293mTM4SF1) and with MS1 immortalized mouse EC, but not with untransfected

parental 293 cells. B, MS1 cells (upper panel) stained with 2A7A-Alexa488, whereas HUVEC (lower panel) did

not. C, Cytotoxicity assays. 293 cells were not susceptible to either 2A7A-LP2 or 8.8-LP2 (upper panel). 2A7A-

LP2 efficiently killed 293mTM4SF1 (middle panel) and MS1 cells (lower panel), whereas control 8.8-LP2 was not

toxic to any of the cells at the concentrations used. Percent cell viability was determined at 96 hours using the

CellTiter-Glo kit. Results are from two independent experiments which gave similar results.




25

Figure 5. Distribution of 2A7A-LP2 (mc-3377) and v1.10-LP2 (mc-3377) in PC3 tumor xenografts and the

effect of 2A7A-LP2 on tumor xenografts. PC3 tumor cells were implanted in athymic nude mice and allowed

to grow to 300 mm3. Mice were then injected intraperitoneally with 3 mpk 2A7A-LP2 (A) or v1.10-LP2 (B).

48h later, tumors were harvested after intravenous injections of FITC-dextran to visualize blood vessels. Fresh

frozen sections were stained with Alexa594 conjugated anti-human IgG antibodies to visualize 2A7A-LP2 (A)

or v1.10-LP2 (B). Dashed white lines indicate tumor-host interface. 2A7A-LP2 was localized to tumor (white

arrows) and host (yellow arrows) blood vessels, largely obscuring green FITC-dextran staining (A). In contrast,

v1.10-LP2 was localized to tumor cells (B) and green FITC-dextran stained vessels are clearly seen. C, Effects

of 2A7A-LP2 (red lines) or control 8.8-LP2 (black lines) on A549, MiaPaCa2, PC3, and SW620 human tumor

xenografts. Treatments (3 mpk, q4dx4, vertical dotted lines) were started when tumors had reached a size of

~300 mm3. Experiments were performed at BIDMC, 5 mice per group.

Figure 6. Combination treatment of refractory tumor xenografts with ADC directed against human and

mouse TM4SF1. Clau-6 (A) and NCI-H460 (B) xenograft tumors were grown in Balb/c nude mice (Crown

Bio) to ~200-300 mm3 and were treated with v1.10-LP2 or 2A7A-LP2, alone or in combination, q4dx4 (vertical

dotted lines). Mean tumor volumes ± SEM, 10 mice per group.






















Published OnlineFirst June 18, 2015.Mol Cancer Ther Alberto Visintin, Kelly Knowlton, Edyta Tyminski, et al. against Tumor Cells and Tumor VasculatureNovel Anti-TM4SF1 Antibody Drug Conjugates with Activity

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