Novel Anti-TM4SF1 Antibody–Drug Conjugates with Activity Against

Published OnlineFirst June 18, 2015; DOI: 10.1158/1535-7163.MCT-15-0188

Large Molecule Therapeutics Molecular Cancer Therapeutics 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, and Shou-Ching Jaminet2

Abstract

Antibody–drug conjugates (ADC) represent a promising induced complete regression of several TM4SF1-expressing therapeutic modality for managing cancer. Here, we report a tumor xenografts in nude mice, including non–smallcelllung novel humanized ADC that targets the tetraspanin-like protein cancer and pancreas, prostate, and colon cancers. As v1.10 did TM4SF1. TM4SF1 is highly expressed on the plasma mem- not react with mouse TM4SF1, it could not target the mouse branes of many human cancer cells and also on the endothelial tumor vasculature. Therefore, we generated a surrogate anti- cells lining tumor blood vessels. TM4SF1 is internalized upon mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 interaction with antibodies. We hypothesized that an ADC mpk, 2A7A-LP2 regressed several tumor xenografts without against TM4SF1 would inhibit cancer growth directly by killing noticeable toxicity. Combination therapy with v1.10-LP2 and cancer cells and indirectly by attacking the tumor vasculature. 2A7A-LP2 together was more effective than either ADC alone. We generated a humanized anti-human TM4SF1 monoclonal These data provide proof-of-concept that TM4SF1-targeting antibody, v1.10, and armed it with an auristatin cytotoxic agent ADCs have potential as anticancer agents with dual action LP2 (chemical name mc-3377). v1.10-LP2 selectively killed against tumor cells and the tumor vasculature. Such agents cultured human tumor cell lines and human endothelial cells could offer exceptional therapeutic value and warrant further that express TM4SF1. Acting as a single agent, v1.10-LP2 investigation. Mol Cancer Ther; 14(8); 1868–76. 2015 AACR.

Introduction grow beyond minimal size (1). VEGF is now recognized as the primary tumor angiogenic factor, and antibodies directed Judah Folkman envisioned that targeting the "tumor angio- against it can prevent the growth of many rapidly growing genic factor" responsible for initiating tumor angiogenesis mouse tumors (2, 3). However, in the clinic, targeting VEGF or would have clinical benefitbypreventingtheformationofthe its primary angiogenic receptor, KDR (VEGFR-2), has met with new blood vessels that tumors require if they are to survive and only limited success (4–6). Antibodies or "traps" directed against VEGF, or tyrosine kinase inhibitors that target VEGF 1Pfizer Inc., Centers for Therapeutic Innovation (CTI), Boston, Massa- receptors, are effective for a time as monotherapies in renal cell chusetts. 2The Center for Vascular Biology Research and the Depart- carcinoma and in some patients with glioblastoma multiforme. ments of Pathology, Beth Israel Deaconess Medical Center (BIDMC) When combined with chemotherapy, they delay recurrence, and Harvard Medical School, Boston, Massachusetts. 3Pfizer Inc., Global Biotherapeutic Technologies (GBT), Cambridge, Massachu- and, in some instances, prolong patient survival, but are not setts. 4Pfizer Inc., Worldwide Medicinal Chemistry, Groton, Connecti- curative. If antivascular therapy is to become more effective cut. 5Pfizer Inc., Centers for Therapeutic Innovation (CTI), San Diego, 6 and achieve its full potential, additional targets beyond the California. Pfizer Inc., Pharmacokinetics, Dynamics and Metabolism – (PDM), Pearl River, New York. 7Pfizer Inc., Drug Safety R&D, Investi- VEGF VEGFR-2 axis are required. gative Toxicology, Groton, Connecticut. 8Pfizer Inc., Biotherapeutics Transmembrane-4 L Six Family member 1 (TM4SF1) was Pharmaceutical Sciences, Andover, Massachusetts. discovered in 1986 as a tumor cell antigen recognized by the Note: Supplementary data for this article are available at Molecular Cancer mouse monoclonal antibody L6 (7, 8). TM4SF1 is an integral Therapeutics Online (http://mct.aacrjournals.org/). membrane glycoprotein structurally related to tetraspanins (8, 9). Corresponding Authors: Shou-Ching Jaminet, Department of Pathology and It is abundantly expressed on many cancer cells (7, 10), on the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, endothelial cells lining human cancer blood vessels (11), and 330 Brookline Avenue, RN-280D, Boston, MA 02215. Phone: 617-667-8156; Fax: on the endothelial cells of angiogenic blood vessels induced in 617-667-3591; E-mail: [email protected]; and Harold F. Dvorak, mice with retinopathy of prematurity (12) or by an adenovirus E-mail: [email protected] expressing VEGF-A (11). It is also weakly expressed on the doi: 10.1158/1535-7163.MCT-15-0188 endothelial cells of many normal organs and tissues (13, 14). 2015 American Association for Cancer Research. TM4SF1 regulates cell motility and intercellular adhesion in both

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tumor (15–17) and endothelial cells (11, 18) and is clustered media, supplemented with sodium pyruvate and nonessential on the plasma membrane in intermittent microdomains amino acids for Calu-3 and SK-Mes-1, at 37 C and 5% CO2). (11, 18, 19). Because TM4SF1 is highly expressed by many Human umbilical vein endothelial cells (HUVEC, Lonza) were human cancer cells (10) and is associated with pathologic cultured according to the supplier's protocols, and used at pas- angiogenesis, we hypothesized that targeting TM4SF1 would sages 3–6. Endothelial cell purity was confirmed by flow cyto- provide a dual anticancer mechanism: killing tumor cells both metry (FACS) which yielded 100% double positive TM4SF1/ directly and also indirectly by targeting the tumor vasculature CD31 cells. 293 stably expressing human or mouse TM4SF1 (secondary mechanism). were generated by lentiviral transduction using a lentiviral ex- We reported previously that a mouse anti-human TM4SF1 pression vector from BioSettia, and packaged using ViraPower monoclonal (IgG1) antibody, 8G4, effectively destroyed the (Life Technologies). Human and mouse TM4SF1–transfected human component of the vascular network engineered in Matri- 293 cells expressed 994 and 875 TM4SF1 mRNA copies/cell, gel plugs implanted in nude mice, presumably by an antibody- respectively. Mouse anti-rabbit IgG was from Southern dependent cell-mediated cytotoxicity (ADCC) mechanism (20). Biotechnologies. 8G4 also killed human PC3 prostate cancer cells that were dependent on that network (20). Together, these data indicated Linker/payload LP2 that naked anti-TM4SF1 antibodies were able to kill TM4SF1- The structure of LP2 (chemical name mc-3377) is shown in expressing cells in both the tumor and vascular compartments Supplementary Fig. S1A. LP2 is comprised of a synthetic dolas- (20). Another murine anti-TM4SF1 monoclonal antibody, L6 tatin-10 analog that resembles in its structure momomethyl – (IgG2a kappa; refs. 7, 10), had been used to treat human tumor auristatin F (MMAF, red color in Supplementary Fig. S1A; cell lines in immunocompromised mice (10). L6, and its mouse/ ref. 27). However, a key difference of MMAF is the presence of human chimeric variant, chL6, were well tolerated and produced a N-methylated a,a-dimethyl amino acid (Aib) that replaces the objective responses in patients with several different cancers that N-methyl valine on the N-terminus of MMAF. This structural – expressed TM4SF1 (10, 13, 21 24). modification of dolastatin-10 analogs has recently been Building on our mouse experiments with 8G4, and the described by Maderna and colleagues and provides synthetic clinical experience with L6 and chL6 anti-TM4SF1 antibodies, analogs with excellent potencies and differentiated ADME prop- we hypothesized that conjugation of a cytotoxic agent to anti- erties (27). The conjugation to the antibody is accomplished fi TM4SF1 antibodies would signi cantly amplify their anticancer with the maleimidocaproyl linker (mc, blue color in Supple- activity (25). To test this hypothesis, we humanized L6, gen- mentary Fig. S1A) that is attached to the cytotoxin via an amide erating antibody v1.10; L6 and v1.10, unlike 8G4, cross-react bond. After ADC catabolism in lysosomes, ADCs generated with with cynomolgus monkey TM4SF1 (10). We then armed v1.10 this linker/payload are expected to produce Cys-capped-mc- with LP2 (chemical name mc-3377), a synthetic analog of 3377 (26). The mc linker is termed "noncleavable" because the dolastatin-10 (26); dolastatin-10 and synthetic analogs, termed release of the cytotoxic payload requires ADC catabolism in the auristatins, inhibit tubulin polymerization, and ultimately lysosome. 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 Anti-TM4SF1 antibodies and ADCs be preferentially toxic against tumor and tumor vascular endo- Mouse anti-human TM4SF1 antibodies 8G4 (20), and L6 and thelial cells with high proliferation rates, while sparing the chL6 (31), were described previously. L6 was humanized by nondividing endothelial cells of normal tissues (28–30). Complementarity Determining Regions grafting and the human- Because TM4SF1 is highly expressed by both tumor cells and ized L6 VH and VL were joined to the human IgG1 and human tumor vascular endothelium, an ideal therapeutic would target Kappa constant regions, respectively, with proprietary expression TM4SF1 on both cell types. Unfortunately, none of the mono- vectors. On the basis of structure modeling (32), we generated a fi clonal antibodies we and others have raised against human series of humanized L6 variants modi ed by the introduction of TM4SF1 cross-react with mouse TM4SF1 (7, 10, 20). Conse- back mutations from the parental mouse antibody to restore fi quently, v1.10-LP2 would be expected to exert a direct effect on binding ef ciency and antibody stability. The humanized L6 human tumor cells implanted in nude mice, but would not be variant chosen for further preclinical development, v1.10, out- expected to have an indirect effect on the xenograft's mouse performed the chL6 antibody as an ADC in a PC3 xenograft vasculature. We therefore generated a humanized anti-mouse model. Recombinant humanized v1.10 antibody was produced TM4SF1 antibody, 2A7A, and conjugated it to LP2 to test the at different scales in CHO or 293 cells. Preparation and charac- therapeutic potential of targeting the mouse tumor vasculature. terization of the rabbit/human chimeric anti-mouse/rat TM4SF1 We now report that both v1.10 and 2A7A antibody–drug antibody, 2A7A, are presented in Supplementary Materials and conjugates (ADC) are highly effective as single agents against Methods. tumor xenografts that express TM4SF1 and are still more effectivewhencombinedsoastotargetbothhumantumor Generation of anti-TM4SF1 ADCs cells and the mouse tumor vasculature. ADCs were 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- Materials and Methods mide and L-Cys were added in sequence to cap the unreacted Cell lines and reagents thiols and quench any unreacted linker-payload. After overnight Tumor cell lines were purchased from ATCC and maintained dialysis in PBS, pH 7.4, the antibodies were purified by size according to vendor recommended conditions (RPMI/10% FBS exclusion chromatography. Protein concentrations were

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determined by UV spectrophotometry. Drug:antibody ratio tional profiling (MGTP) to quantify mRNA copies per cell by (DAR) was determined as described in Supplementary Fig. S1B. normalization to 18S-rRNA, assuming that, on average, cells express approximately 106 copies of 18S-rRNA (35, 36). Cytotoxicity assays Target cells were plated at densities of 500–2,000 cells/100 mL FACS analysis culture medium per well in 96-well plates. After overnight incu- A total of 1 105 cells were reacted with the indicated bation at 37C, 100 mL of culture media containing serial dilu- antibody or ADCs in 100 mL of PBS/2%FBS for 60 minutes on tions of ADCs were added. Ninety-six hours later, 50 mLof ice and washed two times. In some instances, antibodies were CellTiter-Glo (Promega) were added to each well and the plates conjugated to a fluorophore; otherwise, an Alexa 647-labeled were read for luminescence. Data are expressed as % viability secondary anti-human Fc (Life Technologies) was added and compared with that of control untreated cells. cells were incubated for an additional 60 minutes on ice. After washing in PBS/2% FBS, fluorescence was quantified by FACS Xenografts (BD LSR Fortessa; 10,000 events collected/point). fi Xenografts were performed at Crown Bioscience Inc., P zer To determine the relative number of TM4SF1 copies on Oncology, and BIDMC, as indicated. Six- to eight-week-old the cell surface, we stained cells with saturating amounts female athymic nude mice (strain 490, homozygous, Charles (30–100 mg/mL) of Alexa488 (Life Technologies) conjugated River) were used in the American sites, whereas BALB/c nude anti-TM4SF1 antibodies (chL6 or 2A7A) or isotype control mice (HFK Bioscience) were used at Crown Bioscience. Mice were antibodies. The degree of labeling (DOL) for each antibody inoculated subcutaneously with 1 to 5 million tumor cells to was determined using a Nanodrop spectrophotometer [moles produce 200–300 mm3-sized tumors within 15 days. Tumor- dye/mole protein ¼ absorbance494/(71,000 protein concen- bearing mice were injected intraperitoneally (Crown Bioscience tration (mol/L)]. Calibration curves were generated using the fi Inc. and BIDMC) or intravenously (P zer Oncology) with ADCs Quantum Alexa488 MESF kit (Bangs Laboratories, Inc.). The as indicated. A nontargeting monoclonal antibody (8.8, human geometric mean fluorescence values obtained from the test IgG1) conjugated to LP2 was used as a control at 10 mpk (mg/kg). samples were then divided by the DOL and the background Tumor volumes were calculated as described (34). Animals were (isotype control–stained cells) subtracted. TM4SF1 receptor housed and handled according to institution-approved animal numbers were calculated from a calibration curve. care protocols. Mice were terminated with CO2 inhalation followed by cervical dislocation. Results Gene expression analysis Expression of TM4SF1 in human cancers TM4SF1 gene expression levels were estimated from the Cancer We used immunohistochemistry to confirm literature reports Cell Line Encyclopedia (CCLE, http://www.broadinstitute.org/ (10) that TM4SF1 protein was strongly expressed on the tumor ccle/home). RNA expression was quantified with the Affymetrix and vascular cells of cancers resected from patients. Collaborating microarray platform. Normalization of gene expression was done with Indivumed, we found strong tumor cell staining in 10 of by applying the Robust Multi-array Average algorithm. For vali- 13 liver, 16 of 20 non–small cell lung (NSCLC), 4 of 20 breast, and dation of the microarray data, we employed multi-gene transcrip- 3 of 20 colon cancer patient samples (Supplementary Fig. S2), in

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.

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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 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 endothelial cells that express high endogenous levels of TM4SF1, that is, approximately 100 copies of TM4SF1 mRNA/cell (11, 20) and 4–5 105 surface protein molecules/cell. As shown in Fig. 1A, v1.10 bound to cultured HUVECs and human lung microvascular endothelial cells (HLEC) with an apparent Kd of 2 to 5 nmol/L (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 4 (Supplementary Fig. S1B). The resulting anti-human TM4SF1 ADC (v1.10-LP2) bound to HUVECs and HLECs in Figure 2. a manner similar to v1.10 (Fig. 1A). Neither v1.10 nor v1.10- TM4SF1 expression in different human tumor cell lines and their sensitivity LP2 interacted with cells that did not express human TM4SF1, 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. for example, 293 cells or 293 cells that were stably transfected TM4SF1 mRNA expression data were extracted from the CCLE (www. with mouse TM4SF1 (Fig. 1B). broadinstitute.org/ccle/home; ref. 47). mRNA levels (y-ordinate) are – To achieve cytotoxicity, the antibody toxin conjugate must expressed in a RNA-normalized linear fluorescence intensity scale. IC50 be internalized and trafficked to lysosomes, where the anti- values (x-ordinate) were calculated from full 10-point dose response body is catabolized and the toxic payload is released. HUVEC curves (Supplementary Fig. S4) using GraphPad software. Except for NCI- internalized substantial amounts of v1.10 by 30 minutes and H358 (indicated by black dashed circle), sensitivity to v1.10-LP2 correlated well with TM4SF1 RNA expression levels. Six NSCLC tumor cell lines there was essentially complete colocalization of antibody (horizontal arrows) that express different levels of TM4SF1 were selected with LAMP1-positive vesicles (late lysosomes) by 4 hours for xenograft studies (Fig. 3A). B and C, quantification of per cell mRNA (Supplementary Fig. S3). copy numbers and surface protein levels, respectively, by MGTP and FACS v1.10-LP2 ADC was highly effective in killing HUVECs and analysis on five NSCLC and on the breast cancer cell line MCF-7. Mean fluorescence values were obtained using saturating amounts of Alexa488- HLECs (Fig. 1C). For both cell lines, the IC50 of v1.10-LP2 killing was in the low nanomolar range (1.7 nmol/L for HUVECs and labeled chimeric L6, and were converted to receptor numbers using calibration beads (Bangs Laboratories Inc.). Data represent the mean of 2.2 nmol/L for HLECs), while a control nontargeting human three independent determinations. IgG1 antibody, 8.8-LP2, did not kill endothelial cell at concentrations below 100 nmol/L.

Selection of TM4SF1-expressing tumor cell lines Expression of TM4SF1 in human tumor xenografts Tumor cell lines expressing high levels of TM4SF1 were iden- Seven different tumor xenografts (Supplementary Fig. S5A) tiﬁed with an in silico survey of public and proprietary gene were grown as xenografts, either at Crown Bioscience Inc. in expression databases (CCLE, Oncomine, Gene Logic, and OTP) Balb/c nude mice or at BIDMC in athymic 490 nude mice. SK- and from prior publications (7, 8, 16, 37). As a group, NSCLC MES-1, A549, SW620, MiaPaCa2, and PC3 tumors expressed tumor cell lines expressed the highest levels of TM4SF1 among more than 50 TM4SF1 mRNA copies/106 18S copies as deter- solid tumors (Fig. 2A). However, many other cancer cell lines also mined by MGTP (35, 36), whereas NCI-H460 and Calu6 ranked high, including those originating in ovary, liver, pancreas, tumors expressed fewer than 50 copies. Tumor xenografts that prostate, colon, and breast (data not shown). TM4SF1 expression were grown in athymic 490 nude mice at BIDMC expressed levels gave similar rank order whether measured as mRNA copy substantially higher levels of mouse TM4SF1, as well as the numbers (Fig. 2B) or as cell surface protein (Fig. 2C). speciﬁc endothelial cell markers CD144 and VEGFR-2, than tumorsgrowninBalb/cnudemiceatCrownBioscienceInc. Sensitivity of various cancer cell lines to v1.10-LP2 (Supplementary Fig. S5B); TM4SF1 mRNA assays of both As with endothelial cells, the apparent Kd of v1.10 binding to Crown Bioscience and BIDMC xenograft tumors were per- several tumor cell lines was in the single digit nanomolar range. formed at BIDMC. These data suggest that tumor xenografts Cell lines with low TM4SF1 RNA expression levels (e.g., Calu-6 grownindifferentnudemousestrains generate different levels expresses only 4.2 104 surface copies of TM4SF1/cell), were of vascular density. largely insensitive to v1.10-LP2. In general, in vitro sensitivity to v1.10-LP2 correlated with TM4SF1 RNA expression levels, with Effect of v1.10-LP2 ADCs on NSCLCs and other tumor IC50 values in the low nanomolar range for most of the cell lines xenografts tested (Fig. 2A and Supplementary Fig. S4). The exception was To assess the therapeutic potential of anti-TM4SF1 ADCs NCI-H358, for which we have no explanation. in vivo, select NSCLC tumor cell lines (horizontal arrows

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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 approximately 300 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 control ADC 8.8-LP2 (10 mpk; fuchsia). ADCs were administered q4d4 (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, Pﬁzer Oncology (PO) implanted tumors in Matrigel and injected antibodies intravenously.

in Fig. 2A) were implanted subcutaneously in nude mice. concentration range of 0.5 to 16 mg ADC/mL (Supplementary When tumors had grown to approximately 200 mm3 Fig. S6A). (300 mm3 for A549 and SK-MES-1 cells), mice received intra- peritoneal injections of 100 mL of PBS, or PBS containing 3 2A7A, a humanized rabbit anti-mouse TM4SF1 monoclonal or 10 mpk of v1.10-LP2, or 10 mpk of control 8.8-LP2. antibody Treatments were administered at 4-day intervals for four cycles v1.10 does not recognize mouse TM4SF1. This is not sur- (q4d4). prising in that the amino acid sequences of human and mouse v1.10-LP2 induced complete tumor regression, defined as TM4SF1 extracellular loop 2 differ significantly (20), and nei- nonpalpable tumors, in nearly all mice bearing NSCLC xeno- ther we nor others have been able to develop an antibody that grafts that expressed high levels of TM4SF1 (Fig. 3A). Calu-6 reacts with both human and mouse TM4SF1. Therefore, to cells, which express low levels of TM4SF1 and were insensitive target the mouse TM4SF1 expressed on endothelial cell lining to v1.10-LP2 in culture, were also poorly sensitive to v1.10-LP2 the tumor xenograft vasculature, we developed a humanized in vivo. rabbit surrogate monoclonal antibody, 2A7A. 2A7A recognized Efficacy of v1.10-LP2 was also evaluated in human cancer mouse TM4SF1, but did not react with human TM4SF1. As xenografts originating in colon (SW620), prostate (PC3), and shown in Fig. 4A, 2A7A bound to 293 cells that had been stably pancreas (MiaPaca-2, Capan-1, and Panc-1). Similar results were transduced with mouse TM4SF1 (293mTM4SF1)andtothe obtained whether drugs were administered intravenously or immortalized mouse microvascular endothelial cell line, MS1. intraperitoneally, and whether tumors were, or were not, However, 2A7A did not bind to untransfected parental 293 cells implanted in Matrigel (Fig. 3B). Complete regressions were or to cells expressing human TM4SF1 such as HUVEC (Fig. 4B). achieved in the majority of the tumors tested at 3 mpk, and, at Consistent with its binding pattern, 2A7A-LP2, was highly mTM4SF1 10 mpk, in all of the models except for SW620 at the Pfizer cytotoxic to MS1 cells and 293 with IC50 values of Oncology site where tumors did not regress but remained growth 0.015 and 0.003 nmol/L, respectively (Fig. 4C). The number static for approximately 50 days. of mouse TM4SF1 molecules on the surface of 293mTM4SF1 or v1.10-LP2 was well tolerated at all three study sites. Mice did MS1 cells was calculated to be 5.2 105 and 5.7 105 copies/ not exhibit signs of toxicity such as changes in grooming habits or cell, respectively, that is, levels comparable with those of weight loss at either 3 or 10 mpk. On the basis of the A549, human TM4SF1 expressed by HUVEC (4 105 copies/cell) MiaPaCa2 and PC3 regression profiles and the pharmacokinetics and HLEC (5 105 copies/cell). However, the potency of the of v1.10-LP2 in nude mice, we calculated a serum effective 2A7A-LP2 ADC toward cultured rodent endothelial cells was

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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 endothelial cell, but not with untransfected parental 293 cells. B, MS1 cells (top) stained with 2A7A- Alexa488, whereas HUVEC (bottom) did not. C, cytotoxicity assays. 293 cells were not susceptible to either 2A7A-LP2 or 8.8-LP2 (top). 2A7A-LP2 efﬁciently killed 293mTM4SF1 (middle), and MS1 cells (bottom), 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.

about two orders of magnitude higher than that of v1.10-LP2 also to a lesser extent to the vasculature of nearby normal against human HUVEC and HLEC, possibly due to the 10-fold tissues, but did not bind tumor cells which expressed human higher binding afﬁnity of 2A7A for mouse TM4SF1 (0.5 nmol/L) TM4SF1 (Fig. 5A). In contrast, v1.10-LP2, similarly injected versus that of v1.10 for human TM4SF1 (5 nmol/L). intraperitoneally into mice bearing PC3 tumors, localized almost entirely to the tumor cells which express human Distribution and antitumor activity of 2A7A-LP2 TM4SF1 and was not detected in tumor or adjacent normal 2A7A-LP2 injected intraperitoneally into mice bearing PC3 tissue blood vessels that express mouse TM4SF1 (Fig. 5B). A xenografts bound strongly to the mouse tumor vasculature, and nontargeting isotype-matched control antibody, 8.8-LP2, did

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). Forty-eight hours 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, q4d4, vertical dotted lines) were started when tumors had reached a size of approximately 300 mm3. Experiments were performed at BIDMC, 5 mice per group.

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not localize to the tumor or to the host vasculature (not A shown). Calu-6 2,400 Using the same q4d4 protocol used for v1.10-LP2 (Fig. 3), 8.8-LP2 (10 mpk) v1.10-LP2 (3 mpk) 2A7A-LP2 at 3 mpk induced partial (PC3) or complete (A549, 1,800

± SEM 2A7A-LP2 (3 mpk) MiaPaCa2, and SW620) regressions (Fig. 5C). MiaPaCa2 regres- 3 Combination (3+3) sion persisted for almost 50 days, whereas A549 and SW620 1,200 tumors recurred at around 8 days after the fourth injection of 600 2A7A-LP2, and PC3 tumors began to recur even before the fourth injection. Treatment with 2A7A-LP2, like that with the Volume mm Volume 0 control ADC 8.8-LP2, was well tolerated at the 3 mpk dose level 010203040 with mice exhibiting no signs of systemic toxicity such as Days changes in grooming behavior or weight loss (Supplementary Fig. S6B). However, at higher doses of 5 to 10 mpk, significant B weight loss was observed immediately after the fourth injection NCI-H460 2,400 (data not shown). 8.8-LP2 (10 mpk) v1.10-LP2 (3 mpk) 1,800 2A7A-LP2 (3 mpk) ± SEM Simultaneous targeting of TM4SF1 expressed on tumor cells 3 Combination (3+3) 1,200 and on tumor vasculature The foregoing studies with v1.10-LP2 and 2A7A-LP2 estab- 600 lished that targeting TM4SF1 expressed by either tumor cells or 0 by the tumor vasculature could regress tumor xenografts. Because mm Volume 010203040 the two approaches are nonredundant, we hypothesized that the combined effects of targeting tumor and vasculature would Days be additive. To test this hypothesis, we studied Calu-6 and NCI- Figure 6. H460 tumors whose cells express low levels of TM4SF1 in Combination treatment of refractory tumor xenografts with ADC directed culture (Fig. 2B and C). Also, whereas cultured NCI-H460 cells against human and mouse TM4SF1. Clau-6 (A) and NCI-H460 (B) did respond to v1.10-LP2, Calu-6 did not (Supplemen- 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 or tary Fig. S4A). Furthermore, Calu-6 xenografts were resistant to 2A7A-LP2, alone or in combination, q4d4 (vertical dotted lines). Mean v1.10-LP2 as a single agent (Fig. 3A). tumor volumes SEM, 10 mice per group. As shown in Fig. 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- cultured human endothelial cells and for many cancer cells H460 model was resistant to v1.10-LP2 in vivo, but responded (Figs. 1A and 2), and, when reacted with plasma membrane to 2A7A-LP2, delaying tumor progression by approximately 15 TM4SF1, was internalized (Supplementary Fig. S3), a property days (Fig. 6B). Combination therapy delayed tumor growth for an essential for ADC efficacy. When conjugated to a proprietary additional 10 days. 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 Discussion induced complete regressions in five of six different NSCLC TM4SF1 is an important housekeeping gene that is required tumor xenografts (Fig. 3A) and of tumor xenografts of pancreas, for the polarization and migration of cultured endothelial cell prostate, and colon origin (Fig. 3B). In general, sensitivity to (11, 18). It is also highly expressed by many different human v1.10-LP2 correlated well with TM4SF1 expression at both the cancer cells (7, 10) and has important roles in cancer initiation, RNA and protein levels and as measured both in vitro and migration, and invasion (38). Earlier studies with L6, an anti- in vivo. Naked v1.10 antibody (3 mpk, q4d4) had slight antibody targeting human TM4SF1, exhibited low toxicity and gave PC3 xenograft tumor activity, presumably due to ADCC, and promising results in mouse tumor xenografts and in a small limited tumor growth during the four injection cycle; however, number of cancer patients, presumably via mechanisms involv- PC3 tumors did not regress and grew rapidly after treatment ing CDC and ADCC (21, 39). Together these findings suggested ceased (data not shown). Thus, the antitumor activity of v1.10- that therapy could be enhanced by targeting TM4SF1 with an LP2 is largely implemented by ADC. ADC approach. ADCs have entered the clinic and are currently ADCs targeting TM4SF1 offer the opportunity to attack both used to target tumors expressing many different antigens, cancer cells and cancer-associated vascular endothelium. A con- including CD33 (40), Her2 (41), and CD30 (42); more than cern, of course, is that such ADCs will also damage the vascular 100 additional ADCs directed against different tumor cell endothelium of normal organs. Four cycles of 2A7A-LP2 at 3 mpk targets populate the preclinical and clinical pipelines (43– were well tolerated, despite antibody binding to normal vascu- 46). An ADC against a target expressed by both tumor cells lature (Fig. 5A, yellow arrows), with no changes in body weight and the tumor vasculature would be expected to offer excep- (Supplementary Fig. S6B), animal activity, or grooming behavior. tional therapeutic benefit. Treatments could be safely increased to 10 cycles of 3 mpk 2A7A- Here, we report the development of v1.10, a humanized LP2 at 2-day intervals (data not shown). Thus, 2A7A-LP2 has a version of the previously described anti-TM4SF1 mouse mono- clear predilection for damaging xenograft tumor endothelial cells clonal antibody L6. v1.10 has low nanomolar affinity for versus normal endothelial cell. However, at higher doses (5–10

1874 Mol Cancer Ther; 14(8) August 2015 Molecular Cancer Therapeutics

Anti-TM4SF1 ADCs

mpk, q4d4), 2A7A-LP2 caused substantial weight loss and even (Supplementary Fig. S5B). Unfortunately, v1.10-LP2 does not lethality. react with murine TM4SF1, and so we do not at present have a Consistent with the findings of Hellstrom with L6 (10), single ADC that is reactive with both human tumor and mouse v1.10-ADC was well-tolerated in cynomolgus monkeys at doses vascular TM4SF1 for use in preclinical studies. Nonetheless, up to 12 mg/kg, although the pharmacologic relevance of our data validate the potential of TM4SF1 as an attractive cynomolgus monkey to humans has not been fully established human cancer target and provide proof-of-concept that target- for this target. The clinical experience with L6 and chL6 (and ing TM4SF1 with an ADC can be exploited therapeutically their radio-conjugated counterparts) is also consistent with our to treat human cancer xenografts. v1.10-LP2 may be the first mouse data. Although L6 did not carry a payload, it was in a new class of drugs with the bifunctional capacity to target competent to trigger CDC and ADCC, accumulated in endo- a molecule, TM4SF1, that is expressed both by tumor cells thelial cell–rich tissues such as lungs, and exhibited little and by the tumor vasculature. toxicity in patients (21, 39). A number of tubulin inhibitors, including vinca alkaloids, auristatins, taxanes, tubulysin, and fl combretastatins, have been extensively researched and devel- Disclosure of Potential Con icts of Interest fl oped as antiangiogenic drugs, and the resistance of normal No potential con icts of interest were disclosed. endothelium to such agents has been well documented in the – clinic (28 30). Authors' Contributions Several important considerations need to be resolved before Conception and design: A. Visintin, K. Knowlton, K. Marquette, L. Tchistiakova, our mouse findings can be extended to human cancer patients. C.J. O'Donnell, W. Snyder, L. Zawel, H.F. Dvorak, S.-C. Jaminet First, the binding affinity of 2A7A antibody to mouse TM4SF1 Development of methodology: A. Visintin, E. Tyminski, X. Zheng, S. Jain, L. Tchistiakova, A. Maderna, X. Cao, R. Dunn, W. Snyder, S.-C. Jaminet (0.5 nmol/L) was 10-fold higher than the apparent Kd of v1.10 antibody to human TM4SF1 (5 nmol/L); this might translate Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Knowlton, E. Tyminski, C.-I. Lin, X. Zheng, S. Jain, into better activity in different mouse strains, but could also D. Li, X. Cao, A. Barry, H.F. Dvorak, S.-C. Jaminet increase toxicity compared with v1.10-LP2. 2A7A-LP2 is 50- to Analysis and interpretation of data (e.g., statistical analysis, biostatistics, 100-fold more potent in killing immortalized mouse endo- computational analysis): A. Visintin, K. Knowlton, E. Tyminski, X. Zheng, thelial cells than is v1.10-LP2 in killing cultured human K. Marquette, S. Jain, L. Tchistiakova, D. Li, A.K. Abraham, M. Leal, A. Barry, endothelial cells or tumor cells, possibly because of better H.F. Dvorak, S.-C. Jaminet target binding properties. It is therefore unclear how well Writing, 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, 2A7A-LP2 approximates the activity of v1.10-LP2 against R. Dunn, W. Snyder, A.K. Abraham, M. Leal, S. Shetty, A. Barry, H.F. Dvorak, human vasculature. Second, while the overall distribution of S.-C. Jaminet TM4SF1 in mouse organs is qualitatively similar to that in Administrative, technical, or material support (i.e., reporting or organizing humans, we have not yet determined whether the TM4SF1 data, constructing databases): D. Li, M. Leal, H.F. Dvorak, S.-C. Jaminet expression levels are quantifiably comparable. Third, the in Study supervision: A. Visintin, A.J. Coyle, H.F. Dvorak, S.-C. Jaminet fi vivo models used here involve rapidly growing tumor xeno- Other (project leader for this program on P zer's side; supervised and overviewed all the activities pertaining to the program done at Pfizer; also grafts implanted subcutaneously in inbred mice. Whether the wrote the manuscript together with Dr. Dvorak): A. Visintin vasculature of these tumors appropriately phenocopies the Other (served as a team member in studying this target; involved in tumor vasculature of established human cancers remains to be scientific data interpretation, some study design, and review/revision of determined. Finally, 2A7A-LP2 showed an extremely rapid manuscript): S. Shetty distribution phase in mice, falling two logs in serum concentration in less than an hour after injection (Supplementary Fig. Acknowledgments S6A). On the one hand, a rapid fall was not unexpected in that The authors thank the following Pfizer scientists and staff: Eric Bennett and anti-TM4SF1 antibodies bind to the endothelial cells of nor- Rita Agostinelli for humanization of L6 and production of recombinant anti- mal organs throughout the body. However, the reported bodies, Dikran Aivazian, Dana Castro, and Rachel Roach for generation of distribution phase of the chL6 antibody in human cancer 2A7A, Peter Pop-Damkov and Samantha Crocker for bioanalytical and Phar- patients was approximately 5 days (37). Hence, 2A7A-LP2 macokinetic studies; Lauren Gauthier for studies with non-human primates, may be a poor predictor of therapeutic index or pharmaco- Matthew Doroski, Hud Risley, Alexander Porte, and Zecheng Chen for the kinetics in humans. Nevertheless, at very low plasma levels, synthesis of mc-3377, and Evangelia Hatzis and Darcy Paige for program management. Maoping Gao (CrownBiosciences) and Stephanie Shi coordinat- 2A7A-LP2 effectively targeted human xenografts (Fig. 5) and ed outsourced animal studies. The authors also thank Yu Liu and Jane Seo-Jung achieved an even better outcome when combined with v1.10- Sa for xenograft studies at BIDMC. LP2 (Fig. 6). In sum, we have shown that two ADCs, one (v1.10-LP2) acting against human tumor TM4SF1 (direct mechanism) and Grant Support the other (2A7A-LP2) acting against mouse tumor vascular This study was funded by Pfizer CTI through an award to co-principal endothelial cell TM4SF1 (indirect mechanism), are effective as investigators (H.F. Dvorak and S.C. Jaminet) at Beth Israel Deaconess Medical Center. single agents in treating mouse tumor xenografts (Figs. 3 The costs of publication of this article were defrayed in part by the and 5), and that, when combined (Fig. 6), are still more payment of page charges. This article must therefore be hereby marked effective. The response is likely determined by a number of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate variables, including TM4SF1 surface expression levels on this fact. tumor and vascular endothelial cells. Overall tumor vascularity may also be a factor as we observed differences in vascular Received March 4, 2015; revised June 5, 2015; accepted June 7, 2015; markers in tumors grown in different strains of nude mice published OnlineFirst June 18, 2015.

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1876 Mol Cancer Ther; 14(8) August 2015 Molecular Cancer Therapeutics

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

Alberto Visintin, Kelly Knowlton, Edyta Tyminski, et al.

Mol Cancer Ther 2015;14:1868-1876. Published OnlineFirst June 18, 2015.

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