antibody targeting of cell-bound muc1 sea domain kills...

Antibody targeting of cell-bound MUC1 SEA domain kills tumor cells

Edward Pichinuk ^, Itai Benhar>, Oded Jacobi^, Michael Chalik^, Lotem Weiss^,

Ravit Ziv ^, Carolyn Sympson* , Amolkumar Karwa*, Nechama I. Smorodinsky^,

Daniel B. Rubinstein#, and Daniel H. Wreschner ^+¶.

^ Department of Cell Research and Immunology Ramat Aviv, Israel, 69978,>

Department of Molecular Microbiology and Biotechnology, Tel Aviv University,

Ramat Aviv, Israel, 69978; *Biological Sciences, Covidien Pharmaceuticals, St. Louis,

MO 63042; # National Cancer Institute, National Institutes of Health, Bethesda,

Maryland, 20892 and + Biomodifying, Inc., San Diego, CA 92122

Running Title: Picomolar affinity monoclonals for cancer cell eradication

Keywords: MUC1, SEA domain, α/β junction, oncogene, cancer cell killing

¶Address correspondence to: Daniel H. Wreschner, MD., PhD. Department of Cell Research and Immunology University of Tel Aviv, Ramat Aviv, Israel, 69978

Research. on February 7, 2020. © 2012 American Association for Cancercancerres.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 April 16, 2012; DOI: 10.1158/0008-5472.CAN-12-0067

http://cancerres.aacrjournals.org/

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Abstract The cell surface glycoprotein MUC1 is a particularly appealing target for antibody targeting, being

selectively overexpressed in many types of cancers and a high proportion of cancer stem-like

cells. However the occurrence of MUC1 cleavage, which leads to the release of the extracellular

alpha-subunit into the circulation where it can sequester many anti-MUC1 antibodies, renders

the target to some degree problematic. To address this issue, we generated a set of unique

MUC1 monoclonal antibodies that target a region termed the SEA domain that remains tethered

to the cell surface after MUC1 cleavage. In breast cancer cell populations, these antibodies bound

the cancer cells with high picomolar-affinity. Starting with a partially humanized antibody,

DMB5F3, we created a recombinant chimeric antibody that bound a panel of MUC1+ cancer cells

with higher affinities relative to cetuximab® (anti-EGFR1) or tratuzumab® (anti-erbB2) control

antibodies. DMB5F3 internalization from the cell surface occurred in an efficient temperature-

dependent manner. Linkage to toxin rendered these DMB5F3 antibodies to be cytotoxic against

MUC1+ cancer cells at low picomolar concentrations. Our findings show that high affinity

antibodies to cell-bound MUC1 SEA domain exert specific cytotoxicity against cancer cells, and

they point to the SEA domain as a potential immunogen to generate MUC1 vaccines.

Introduction

MUC1 is a mucin-like glycoprotein which can generate a variety of differing isoforms (1). Of

these, the most intensely studied has been a polymorphic type I high molecular weight

transmembrane protein (MUC-TM) consisting of an extracellular domain containing 20-125

tandem repeats of 20 amino acids, followed by a transmembrane domain and a short

cytoplasmic tail (2-4). MUC1 is a heterodimer which is cleaved soon after synthesis within the

SEA module, a highly-conserved domain of 120 amino acids (4-6). Cleavage of MUC1 yields two

unequal chains: a large extracellular alpha subunit containing the tandem repeat array

specifically bound in a strong non-covalent interaction to a smaller beta subunit containing the

transmembrane and cytoplasmic domains of the molecule(4, 7).




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MUC1 is highly expressed on a range of malignancies, including breast, pancreas, ovarian,

prostate, and colon carcinomas, as well as on the malignant plasma cell of myeloma (8-13).

Because of this overexpression, MUC1 has been the subject of a great amount of attention,

primarily for its potential as a target for tumor-specific therapies. In fact, based on a number of

ranked criteria required of an optimal cancer vaccine candidate, the MUC1 protein was listed by

the National Cancer Institute Pilot Project the second best target from a list of 75 potential tumor

associated antigens(14). Moreover, within the categories of expression level, stem cell expression

and number of patients with antigen-positive cancers, the MUC1 protein received perfect

scores(14).

Most anti-MUC1 antibodies reported to date target the highly-immunogenic tandem repeat array

of the MUC1 alpha chain, [for example (15-17)]. Since the MUC1 alpha chain is not directly

tethered to the cell surface, it is also found in the peripheral circulation. There it sequesters

circulating anti-tandem repeat antibodies, limiting their ability to reach the MUC1+ tumor cells

(15, 16). In addition, deposition of immune complexes of anti-tandem alpha chain antibodies and

circulating alpha chain potentially may result in end-organ damage (16).

Targeting MUC1 epitopes bound to the cell surface would avoid antibody sequestration by

circulating alpha chain. The MUC1 SEA domain formed by the interaction of the alpha-subunit

with the extracellular portion of beta-subunit is an intricate structure which remains fixed to the

cell surface (4, 6) and significantly, results in a stable target structure. In previous initial studies

we demonstrated the proof-of-principle of generating antibodies that specifically recognize the

MUC1 SEA domain (18). The prototype DMC209 mAb described (18) has two disadvantages that

compromise its potential clinical use: It is an IgM and it has relatively low affinity for its target.

As a result of these considerations, we sought highly specific, anti-MUC1 antibodies directed

against the alpha/beta junction, that were also affinity-maturated and not of the IgM subclass.

We report here the generation of seven novel monoclonal antibodies, five Ig-gamma1 and two

IgA, that bind the MUC1 protein within its SEA domain with picomolar affinities. Our findings




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suggest that the IgG1 and IgA mAbs targeting the MUC1 SEA domain may prove beneficial in the

therapy of MUC1+ malignancies when either administered alone or in conjunction with other

anti-tumor antibodies and chemo-cytotoxic agents.

MATERIALS AND METHODS

Materials and antibodies Reagents and chemicals were obtained from Sigma (St. Louis, MO),

unless otherwise specified. Anti-MUC1 SEA-module monoclonal antibodies were generated as

described below. The anti-MUC1 tandem-repeat antibodies used here were as previously

described (19, 20).

Cell lines Human breast carcinoma lines T47D, MCF7 and ZR-75, human epidermoid carcinoma

A431 and KB cell lines, human gastric carcinoma N87 cell line and human pancreatic carcinoma

Colo357 cell line were maintained as previously described (18).

Cell Culture Cells were grown at 370C and 5% CO2, in culture media supplemented with 10% heat-

inactivated fetal calf serum, 2mM L-glutamine, 100 IU/ml penicillin and 25mg/ml streptomycin.

DA3 (mouse mammary tumor cells) and CHO-KI (Chinese Hamster Ovary cells) were grown in

Dulbecco’s modified Eagle’s medium (DMEM) or DMEM: F12 nutrient mixture(1:1), respectively.

Generation of stable DA3 mouse mammary tumor cell transfectants expressing MUC1-TM DA3

cells were co-transfected with the eukaryotic expression plasmids pCL-MUC1-TM and with

pSV2neo (which codes for neomycin resistance). Expression constructs were transfected into

cells using the calcium phosphate procedure. Stable transfectants were selected with neomycin.

Generation of bacterial recombinant MUC1-X ,SEA, SEA-4G proteins Bacterial MUC1-X ,SEA,SEA-

4G proteins were prepared as the (His)6-MUC1-Xex, (His)6-SEA and (His)6-SEA-4G fusion proteins

as previously described (21) and comprise six histidine residues at their N-terminus, followed by

the extracellular domain of the MUC1-X/SEA/SEA-4G proteins, respectively.




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Immunization of mice and generation of hybridomas Mice were initially immunized with 5

consecutive intradermal DNA immunizations spaced at 21-day intervals. The immunizing DNA

consisted of the pCL-MUC1-TM expression vector plasmid that codes for the MUC1-TM protein

(21). The extracellular domain of MUC1-X protein (recombinant bacterial MUC1-Xex, see below)

together with incomplete Freund’s adjuvant was then used to boost the mice. Bacterially

synthesized recombinant MUC1-Xex protein used for these immunizations, spontaneously self-

cleaves as previously described(4), generating the MUC1-X alpha and beta subunits that strongly,

yet non-covalently, interact with each other forming the very stable heterodimeric cleaved

MUC1-Xex protein (depicted in Figs. 1 and 4). Hybridomas were prepared by fusion of non-

secreting myeloma cells with immune splenocytes and screened by ELISA assay (see below).

ELISA for determining binding of anti-MUC1 polyclonal and monoclonal antibodies to the

extracellular domain of the MUC1-X protein ElisaImmunoAssay plates (Costar, Corning, NY) were

coated with recombinant MUC1 proteins followed by blocking. Spent culture media from the

initial hybridomas was then applied to the wells. Following incubation, samples were removed

and the wells were washed with PBS/Tween. Detection of bound antibodies was performed with

HRP-conjugated anti-mouse antibody.

Two-tiered screening for selection of anti-MUC1 monoclonal antibodies The primary screen of the

hybridomas was performed by assessing antibody binding to the extracellular domain of MUC1-X

(MUC1-Xex) as described in the ELISA assay (above). To select hybridomas secreting antibody

that recognize not only MUC1-Xex but also the complete cell surface MUC1-TM protein, those

hybridomas presenting a positive signal in the first screen were subjected to a second-tier screen.

This consisted of flow cytometric analysis using mouse cell transfectants (DA3-TM) expressing

human MUC1-TM and, in parallel, to the same parental cells (DA3-PAR) that do not express

human MUC1. This procedure ensured selection of antibody that not only bound MUC1 moieties

common to both the MUC1-X and MUC1-TM, but also recognized cell surface human MUC1-TM

as expressed by mammalian cells.

Isolation of MCF7 breast cancer side population of cancer cells A MCF7 breast cancer cell




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subpopulation that has been reported to show some characteristics of cancer stem cells (22)

were isolated as described by Finn (22). Briefly, MCF7 cells were stained with the fluorescent dye

Hoechst 33342. Due to their increased efflux of the dye a side population (SP) of MCF7 cells was

isolated by flow cytometry. The resultant cells were then confirmed as showing some

characteristics of stem cells using a battery of stem cell markers.

Preparation of ZZ-Pseudomonas exotoxin and chDMB5F3:ZZ-PE38 immunotoxin The ZZ-PE38

fusion protein is comprised of the Pseudomonas exotoxin PE38 and the ZZ-domain derived from

Protein A. The ZZ portion binds to the Fc region of IgG to form immunotoxin conjugates. Plasmid

pET22-NN-ZZ-PE38 was used for expression of soluble ZZ–Pseudomonas exotoxin A (PE38) fusion

protein secreted to the periplasm of BL-21 (DE3) Escherichia coli cells which was then purified as

previously described (23).

SDS-Polyacrylamide protein gel electrophoresis (SDS-PAGE) Proteins separated on SDS PAGE were

electro-transferred for 2 hours at 0.5Amp on to nitrocellulose filters in transfer buffer. Blots were

blocked with 5% skimmed milk followed by incubation with the primary antibody (anti-MUC1

antibodies, see Results). Bound primary antibody was detected with secondary anti-mouse

antibody conjugated to horseradish peroxidase (Chemicon International, Temecula, CA) followed

by enhanced chemiluminescence.

Cell killing assay Mouse mammary tumor cells transfected and stably expressing human MUC1-

TM (DA3-TM), the parental cells that do not express human MUC1 (DA3-PAR wild type), and

MUC1+ human cell lines (T47D and ZR-75 breast cancer cells, Colo357 pancreatic cancer cells, as

well as additional cancer cells as detailed in Results), were grown in Dulbecco’s modified Eagle’s

medium (DMEM) supplemented with 10% fetal calf serum. These cells (10,000 cells per well in

100 microliters of medium) were seeded in 96 well cell culture plates (Corning; Corning, New

York) and grown at 370C in 5%CO2 in culture media. Five hours after seeding, 50 microliters of

the chDMB5F3 was diluted by decimal dilutions starting from 1600pm and mixed with 50

microliters ZZ-PE38 toxin. The immunotoxin mixture was directly applied to the cells (final ZZ-

PE38 toxin concentration was 250ng/ml). Negative controls included adding to the target cells




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the following (instead of the chDMB5F3:ZZ-PE38 toxin immunoconjugates)- (i) concentrated

medium from parental CHO-K1 cells that is devoid of the anti-MUC1 chDMB5F3 monoclonal

antibody, (ii) ZZ-PE38 alone, (iii) chDMB5F3 monoclonal antibodies alone, devoid of the ZZ-PE38

toxin. Cell viability was assessed by measuring alkaline phosphatase activity per well.

Construction of vectors for mammalian expression in CHO (Chinese Hamster Ovary) cells of

recombinant DMB-5F3-constant region of human IgG1 [C-hIgG1] Methods used to generate these

vectors essentially followed those previously reported (23). The mammalian vectors pMAZ-IgH

and pMAZ-IgL(19) were used as backbones for the expression of the VH and VL regions of

DMB5F3 fused to human gamma1 heavy and human kappa light chains, respectively.

Surface Plasmon Resonance (SPR) binding assay Recombinant MUC1-X protein was immobilized

on a CM5 chip. As control, anti-CD20 IgG (Rituxan®) was treated in a like manner. Remaining

active groups were saturated with 1mM ethanolamine. SPR was performed using Biacore®3000

according to manufacturer's specifications (GE Healthcare, Piscataway NJ 08855). Serial dilutions

of antibody DMB5F3 (0.4-7nM) were measured for 4 minutes-association and 30 minutes -

dissociation. The chip was regenerated with 5mM NaOH. Data fitting was performed using

algorithm of the Biaevalution software.

Preparation of DMB5F3 Fab DMB5F3 IgG1 was reacted with papain to give a papain-to-antibody

ratio of 1:20 (ww) followed by incubation at 370 C for 5 hours. The reaction was stopped with

crystalline iodoacetamide at a final concentration of 0.03 M. The resultant mixture was dialyzed

against PBS pH8, passed on a protein G column, and Fab fractions collected.

Analysis of binding to MUC1/Her2/EGFR1-expressing tumor cells by flow cytometry Evaluation of

binding by flow cytometry was performed as follows: 5 x 105 cells were used in each experiment.

After trypsinization, cells were washed and monoclonal antibodies at varying dilutions were

added to the cell tubes for 1 hour at 4°C. After washing with FACS buffer, FITC-labeled goat anti-

human Fc antibody was added to the tubes for 45 min at 4 °C. Detection of bound IgG was

performed by means of flow cytometry on a FACS-Calibur (Becton Dickinson, CA), and results

analyzed with theCELLQuest program (Becton–Dickinson).




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Results

Generation of monoclonal antibodies to the cell-bound MUC1 alpha/beta junction To avoid

generating antibodies to the circulating α chain, the MUC1 immunogen should ideally be cell-

bound at all times. To comply with these criteria we used the extracellular domain of the

naturally occurring MUC1-X isoform that incorporates elements derived from both the

extracellular alpha chain and the cell bound beta chain. This provided the advantage of

incorporating the cell bound region consisting of the alpha/beta junction present in the full

naturally occurring MUC1 protein (Fig. 1), yet at the same time is devoid of the highly

immunogenic tandem-repeat array (Fig. 1, MUC1-X). The MUC1 protein used for these

immunizations is cleaved MUC1-Xex protein synthesized in bacteria. Within the bacteria, MUC1-

Xex undergoes spontaneous self-cleavage, and comprises the non-covalently interacting MUC1

alpha and beta subunits.

Using our immunization protocol (Methods), high titer anti-MUC1-X polyclonal antibody sera up

to 1:100,000 dilutions were obtained. Spleens of such mice were used for hybridoma formation

and hybridoma supernatants subjected to a two-tiered screen consisting of (a) binding to MUC1-

X protein, and (b) a more stringent second tier assessing binding to MUC1-expressing cancer cells

as assayed by flow cytometry. Hybridoma formation resulted in seven monoclonal antibodies,

five of the Ig-gamma1 isotype, designated DMB-4B4, DMB-4F4 , DMB-5F3, DMB7F3 and

DMB10F10 . Surprisingly two monoclonals of the IgA subclass were also isolated, DMB10B7 and

DMB13D11.

Cytometric analyses of monoclonal anti-junctional antibodies Flow cytometric analyses

demonstrated that all seven monoclonal antibodies bound strongly to DA3-TM cells that express

the full length MUC1. In contrast, untransfected DA3-PAR cells which do not express MUC1 were

consistently negative with all antibodies (Fig. 2). Similarly, Colo357, a MUC1+ pancreatic cancer

cell line showed unequivocal reactivity with all anti-MUC1 monocolonals (Fig. 2) as did several

breast cancer cell lines, such as T47D, MCF7 and ZR75 (data not shown). Further confirmation of

binding specificity was provided by addition of soluble exogenous MUC1-Xex protein, the




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extracellular domain of the MUC1-X protein, which abolished all reactivity (Fig. 2). These studies

underscore the fact that the DMB antibodies bind a cell-bound domain non-competitive with

moieties present in the shed alpha chain of the MUC1-TM protein. In addition to the flow

cytometric studies, initial immunohistochemical analyses were performed with one of these

monoclonal antibodies (DMB5F3) using sections of breast cancer tissue. These stainings showed

significant DMB5F3 immunoreactivity with the breast cancer cells (data not shown), results

clearly in line with the well-known overexpression of MUC1 by breast (and other)

adenocarcinoma cells, comprehensively documented in numerous studies.

Binding of anti-junctional antibodies to MCF7 side population cells that show some

characteristics of breast cancer stem cells In order to determine whether the DMB series of anti-

MUC1 junctional antibodies bind not only to differentiated MUC1+ tumor cells but also to MCF7

side population cells that show some characteristics of cancer stem cells (22), the antibodies

were reacted with the MCF-7 side population (SP). Both antibodies DMB4B4 and DMB4F4 anti-

MUC1 yielded sharp shifts in the gated cell population of both the MCF7 side population

previously shown to bear some characteristics of cancer stem cells (22) (Fig. 3, orange, middle

and right panels) and mature MCF7 cancer cells (Fig. 3, green, middle and right panels). The SP

cells bound to the anti-MUC1 antibodies were demonstrated to have multiple characteristics of

stem/progenitor cells (22). These include- [1] preferential efflux of the fluorescent DNA-binding

dye Hoechst 3342, [2] phenotypic characterization as CD44+/ CD24-/low, luminal and epithelial

markers CK15 and EpCAM, and the stem cell/progenitor marker CK19, [3] preferential formation

of mammospheres in suspension culture, and [4] gene array including preferential expression of

a variety of protein products including Wiskott-Aldrich syndrome interacting protein and insulin-

like growth factor binding protein. These findings indicate that the side population of MCF7 cells

representing cells that show some characteristics of cancer stem cells (22) can be targeted by

anti-MUC1 junctional antibodies DMB-4B4 and DMB4F4 no less effectively than MCF7 'mature'

MUC1+ tumor cells.

Despite our characterization of the SP as described above, the MCF7 SP is itself a non-

homogeneous population of cells (22) and the phenotypic identification of true stem cells




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especially in ER+ cancer cells (such as MCF7) remains a challenge (24, 25). Indeed the caveat

should be stressed that it is not clear whether this MCF7 side population represent stem cells as

appearing in ER+ cancers. Thus to what degree the anti-MUC1 junctional antibodies target true

stem cells will require a more precise characterization of SC in ER+ tumor cells.

The DMB mAbs all recognize the SEA domain yet are different from one another The DMB mAbs

were assessed by ELISA for binding to the MUC1-Xex protein, the SEA domain itself and the SEA-

4G protein, a mutant construct consisting of uncleaved α−β bound by a 4 glycine peptide (6).

These proteins were generated in bacteria, and as previously reported (4), the MUC1-Xex and

SEA domain proteins spontaneously self-cleave in bacteria, at the cleavage site FRPG|SVVV,

where | indicates cleavage, generating the interacting alpha and beta subunits (depicted in

Figure 4, bottom schema). The mutant SEA-4G protein (depicted in Figure 4, bottom schema)

comprises the amino acid sequence FRPGGGGSVVV (instead of the above wild type sequence),

resulting in a non-cleaved protein as previously described (6). Results showed that all seven

mAbs bound the three proteins (each bound to wells of an ELISA plate) at picomolar antibody

concentrations and confirmed that the primary target of these antibodies is the SEA domain.

Inspection of the binding curves of the Ig-gamma1 monoclonal antibodies to these three target

proteins demonstrated distinct binding patterns for mAbs DMB5F3, DMB4F4 and DMB7F3 to

each of these proteins (Figure 4, top panel), indicating that the precise epitope within the SEA

domain is different for each of these antibodies. Additional analyses including flow cytometry

with a battery of MUC1-expressing cancer cells (data not shown) as well as western blotting

analyses (see below), further showed that all five Ig-gamma1 monoclonal antibodies differ one

from the other in their binding characteristics.

The epitopes of anti-SEA module monoclonal antibodies DMB5F3, DMB4F4, and DMB4B4 are

largely conformational and involve elements contributed by both the alpha and the beta

subunits To define the nature of the epitopes bound by the anti-SEA antibodies, they were

assessed by western blotting with a series of SEA module constructs. Staining of SDS-PAGE gels of

the SEA module (Fig. 4, bottom panel, A) shows its cleaved constituent alpha and beta chains, as

well as protein SEA-4G, a mutant construct consisting of uncleaved alpha-beta bound by a 4




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glycine peptide(6). As expected, probing western blots with the previously described anti-beta

subunit specific mAb BOS10D2 (26) (linear epitope indicated in bottom schema, Fig. 4)

demonstrates reactivity with the beta subunit but none at the position of the alpha-subunit (lane

1). It further demonstrates strong binding to the alpha-4G-beta construct (lane 2) as well as to

small amounts of uncleaved alpha/beta protein (4)( lane 1). Antibodies DMB5F3 and DMB4F4 in

contrast react strongly with uncleaved alpha-4G-beta but are non-reactive with both the alpha

and beta subunits (Fig. 5, Panels C and D, respectively). Removal of SDS during the blotting

procedure likely allows uncleaved alpha-4G-beta protein to adopt a structure similar to that of

the SEA module, previously shown to form an unusually stable three-dimensional structure (6). In

contrast, Panel E demonstrates that antibody DMB4B4 reacts with none of the proteins, not with

alpha, not with beta subunits in isolation, nor with the alpha-4G-beta mutant isoforms,

suggesting that its binding site requires the presence of both the alpha and beta chains in an

epitope resulting from conformationally-determined cleavage.

Affinity of anti-junctional antibodies Having demonstrated the specificity of the DMB

monocolonals to a MUC1 cell-bound domain, the antibodies were examined by serial dilutions to

evaluate their affinity for intact in situ cell surface MUC1. To do so, the antibodies were reacted

with ZR-75 MUC1+ breast cancer cells in flow cytometry (Methods). ZR-75 was reacted with the

antibodies at an initial concentration of 4 microgram/ml. As seen in Figure 5 (upper panel, left),

all three antibodies, DMB-4B4, DMB-4F4, and DMB5F3 showed binding at that concentration. At

1 microgram/ml binding of DMB-4B4 and DMB-4F4 was diminished, whereas DMB5F3 clearly

showed binding (Fig. 5, middle panel). DMB5F3 continued to bind the ZR-75 cells to a

concentration as low as 62ng/ml (Fig. 5, right panel).

Binding affinity of DMB5F3 by Surface Plasmon Resonance (SPR)

As DMB5F3 showed the highest binding affinity, we concentrated further studies on this

antibody. An SPR binding assay was performed with serial dilutions of DMB5F3 ranging from 7nM

to 0.4nM as described in Methods. Over a period of 2250 seconds, the degree of dissociation is

almost imperceptible in each of the seven curves shown (Fig. 5). These studies were extended to




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a partially humanized chimeric version of DMB5F3 (chDMB5F3, see below), and demonstrated

that for chDMB5F3 an association rate (Ka 1/Ms) of 1.25 x 106 and a dissociation rate (Kd (1/s) of

7.37 x 10-6 were obtained, and an extraordinary low dissociation constant (KD) of 5.89 x 10-12M

was calculated (Panel A). To determine the binding kinetics of the Fab fragment, DMB5F3 Fab

was similarly subjected to the SPR binding assay using Biacore®3000 with serial dilutions.

Analysis of chDMB5F3 Fab (purified as in Methods) by the SPR binding assay showed an

association rate (Ka 1/Ms) of 2.37 x 106, a dissociation rate (Kd (1/s) of 1.26 x 10-3, and a

dissociation constant (KD) of 4.84 x 10-10M (Fig. 5, Panel B). Given the increased avidity

associated with a bivalent IgG, the 100-fold KD enhancement seen in the full immunoglobulin

molecule as compared to the Fab is as expected.

Generation of humanized DMB5F3 in CHO (Chinese hamster ovary) As an initial demonstration

of its clinical application, the picomolar-affinity DMB5F3 antibody was partially humanized. In

order to generate humanized DMB5F3 mAb, the H and L chains were isolated from SDS-PAGE

gels, and their N-terminal sequences (10-15a.a.) determined. Based on that amino acid sequence,

nucleotide primers were generated for use in PCR of DMB5F3 cDNA together with primers

generated from known human Ig H and L constant region sequences. Analysis of the Heavy chain

amino acid sequence revealed it to be a member of the VH3 gene family, differing in a total of 9

amino acids from germline gene VH36-60/A1/85 (not shown). Of the 9 mutations, 5 are located

in the CDR regions, while 2 are present near the usually highly-conserved N terminal of the VH

FR1 segment; one of those mutations, (from L to V) occurs at a.a. 4 from the FR1 N terminal. The

DMB5F3 VL chain is a kappa light chain, containing 5 a.a. mutations compared to its closest

germline sequence 23-48. Of the 5 mutations, 3 are present in the CDR1 and CDR3 regions.

In order to demonstrate that chimeric DMB5F3 generated by mammalian cells retains its specific

anti-MUC1 activity, recombinant chDMB5F3 was produced in CHO cells. To assess chDMB5F3

immunoreactivity, wells were coated with MUC1-Xex protein and reacted with dilutions of either

recombinant chDMB5F3 or mouse hybridoma DMB5F3 mAb (Fig. 6, panels C and D, respectively).

Both antibodies bind the MUC1-Xex protein at similar low picomolar concentrations (Fig. 6,

panel D). Flow cytometry analyses confirmed both the specificity and remarkably strong affinity




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of the recombinant chDMB5F3 antibody to the MUC1 protein. No binding was observed to

MUC1-negative parental non-transfected DA3 cells, whereas robust reactivity was seen with

mouse transfectants expressing human MUC1-TM (DA3-TM) (FIG. 6E and F, respectively). In

fact, binding was still clearly observed at a concentration of chDMB5F3 as low as 20 picoMolar.

Internalization of the DMB5F3 mAb To assess degree of internalization of the DMB5F3 mAb and

thereby its ability to transport bound drugs into the cell, MUC1-expressing cells were incubated

with DyLight649-labeled DMB5F3 either at 4oC or at 37oC, and fluorescence monitored by confocal

laser microscopy (Fig. 6G and H, respectively). At 4oC, labeled DMB5F3 was restricted to the cell

membrane, indicating binding to the cell surface only. Subsequent incubation at 37oC showed

relocalization of the labeled antibody to within the cell, reflecting highly efficient internalization

to the cell interior.

Linkage of chDMB5F3 generated by CHO to Pseudomonas ZZ-PE38 toxin and cytotoxicity of the

immunoconjugate Having seen that the DMB5F3 antibody undergoes efficient intracellular

internalization, we then assessed its ability to ferry cytotoxic moieties into the cell. The ZZ-PE38

fusion pseudomonas toxin is devoid of a cell-binding domain, and in order to effect its cell killing

activity it must be linked to an agent capable of internalizing it into the cell. Chimeric DMB5F3

(chDMB5F3) contains human Fc to which the ZZ domain (derived from Protein A) of the ZZ-PE38

toxin binds well. Parental, non-transfected cells that do not express human MUC1 are unaffected

by chDMB5F3-ZZ-PE:38 immunotoxin (Fig.6I, diamond symbols) whereas DA3 transfectants

expressing human MUC1-TM (DA3-TM) are exquisitely sensitive to chDMB5F3:ZZ-PE38 conjugate

with cytocidal activity seen at picoMolar concentrations (Fig. 6I square symbols and see inset).

These findings were extended to T47D human breast cancer cells which natively express the

MUC1 protein. The chDMB5F3:ZZ-PE38 immunotoxin conjugate was potently cytocidal to these

cells as well, with extensive cell killing observed at as low as picoMolar concentrations (Fig. 6I).

Efficacy and specificity of cell killing by chDMB5F3:ZZ-PE38 immunotoxin conjugates and

comparison of cytocidal activity with Cetuximab (Erbitux ®) and Trastuzumab (Herceptin®) As




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seen with DA3 cells transfected with human MUC1 (Fig. 6G and H), human adenocarcinoma cells

showed that following incubation at 40C labeled DMB5F3 was limited to the cell membrane,

indicating binding only to the cell surface (Fig. 7A). Subsequent incubation at 37oC demonstrated

almost complete relocalization of the labeled antibody to within the cell, reflecting highly

efficient internalization into the cell interior (Fig. 7A'). Having demonstrated internalization of

chDMB5F3 to MUC1-positive human adenocarcinoma cells, we next proceeded to assess the

cytotoxicity of immunotoxin formed with chDMB5F3, and to compare its activity with that of

Erbitux and Herceptin immunotoxins. Fixed cells demonstrated similar cell-membrane reactivity

with both chDMB5F3 and Erbitux (Fig. 7, B and B', respectively), and to Herceptin (data not

shown). Recombinant chDMB5F3:ZZ-PE38 reacted with the MUC1+ human pancreatic cancer cell

line Colo357 resulted in cell killing, and serial dilutions of the antibody revealed an IC50 of

approximately 16pM (Fig. 7C). However when chDMB5F3:ZZ-PE38 was reacted with Colo357

simultaneously with soluble recombinant extracellular domain of MUC1-X (MUC1-Xex),

competition was seen with a resultant marked reduction in cell killing (Fig.7C, orange dotted

curve). In order to demonstrate the specificity of that competition, soluble MUC1 Xex was added

to Erbitux:ZZ-PE38 at an equal concentration. MUC1 Xex failed to abrogate Erbitux immunotoxin

activity (Fig. 7C, blue dotted curve ). In a similar way, serial dilutions of chDMB5F3:ZZ-PE38

reacted with ZR75 breast cancer cells showed highly effective killing with an IC50 of 3pM

whereas addition of MUC1 Xex competitor abolished cell killing (Fig. 7D, compare continuous and

dotted orange lines). While reaction of Herceptin:ZZ-PE38 to ZR75 cells resulted in cell killing,

addition of MUC1 Xex did nothing to abrogate cell death (Fig. 7D, compare continuous and

dotted green lines). Cell lines such as A431 (epidermoid carcinoma) and N87 (gastric carcinoma)

that were found to be only very slightly positive for MUC1, as assessed by flow cytometry with

DMB5F3 mAbs, were not affected by the chDMB5F3:ZZ-PE38 immunoconjugate (data not

shown).

Taken together these results demonstrate the specificity of cell killing mediated by the

chDMB5F3:ZZ-PE38 immunotoxin, and furthermore demonstrate that in certain cancer lines,

anti-MUC1-SEA module DMB5F3 immunotoxin is the most potent of the toxin conjugates tested.




15

Discussion

Despite its potential to serve as an effective immunotherapeutic agent for a variety of high-

expressing MUC1 malignancies, the MUC1 molecule has proved to be an elusive prey. This is in

large measure due to the failure to identify and target MUC1 moieties that are permanently cell

bound (27).

Almost all anti-MUC1 antibodies reported to date are directed against the highly immunogenic

polymorphic array of 20-125 tandem repeats of a 20 amino acid sequence present in the MUC1

alpha chain [for example, (17) (28-30)]. As the alpha chain is bound non-covalently with the cell-

bound beta subunit, it is often shed from the surface of MUC1+ cells and freely circulates

peripherally. It is here that the shed alpha-subunits sequester anti-tandem repeat array

antibodies thereby precluding their ability to reach MUC1+ tumor cells (15, 16).

We previously described mechanisms whereby the cleaved junction composed of the MUC1

alpha and beta chains is formed (4). Specifically, we analyzed the potential for cleavage of an

MUC1-X isoform that contains the same intra-cellular and membrane domains as the full MUC1-

TM molecule (4). While the extracellular domain of MUC1-TM contains the highly immunogenic

tandem repeat array, the extracellular domain of MUC1-X is comprised solely of the 120 amino

acid SEA module fused to a 30 N-terminal amino acid segment of MUC1, resulting in a less

complex structure. Significantly, not only is the MUC1-X alpha/beta junctional isoform cleaved,

it is cleaved at an identical site as is the full-length MUC1-TM molecule and therefore results in

the same noncovalent interaction of the alpha and beta subunits(4).

All seven anti-MUC1 monoclonal antibodies described here are directed against the cell-bound

MUC1 SEA domain that embraces the MUC1 alpha/beta junction. Since the membrane-bound

alpha/beta junction involves an intricate structure of alpha helices and intertwined beta strands

(6), antibodies directed against the native structure would likely recognize epitopes composed of

elements contributed by both the alpha- and beta- subunits. As shown (Fig. 4), this is in fact the

case: binding by all antibodies requires intact conformation involving elements contributed by




16

both the alpha and the beta subunits. The antibodies not only conform to the expectation of

conformational binding, but they also bind the intact, native glycosylated MUC1 molecule on

MUC1+ breast cancer cells (Fig. 5) and MUC1+ pancreatic cancer cells (Fig. 2), with very high

affinity- a Kd of approximately 6 picoMolar was calculated for DMB5F3 IgG binding to MUC1

protein. Considering the large number of tandem repeat epitopes in the MUC1 alpha-subunit as

compared to the unique epitopes present in the SEA domain, the high affinity of the anti-SEA

compared to anti-alpha antibodies is all the more impressive. The DMB5F3 Kd is 3-4 logs higher

than the Kds reported for either Cetuximab(31) or Trastuzumab(32), both humanized IgGs

derived from murine hybridomas, and 3 logs higher than an anti-MUC1-tandem-repeat array

IgG1 isolated from in vitro selection of an Fab phage library.

Naked antibodies against carcinoma cell surface tumor antigens are seldom curative by

themselves and are usually administered in combination with chemotherapy. However, very

potent cytotoxic agents are often, as stand-alone therapeutics, too toxic and should be ideally

directed solely to the tumor antigen expressing cancer cell. This can be achieved via

immunoconjugates that will selectively deliver potent therapeutics to a tumor, thereby reducing

systemic toxicity.

As an initial demonstration of ultimate clinical application, the DMB5F3 anti-MUC1 was partially

humanized and produced in CHO cells as chimeric DMB5F3 (chDMB5F3), and then tested for

cytocidal activity as an immunotoxin . Expression of chDMB5F3 in CHO cells demonstrated that it

retains the same specificity and picomolar binding affinity as antibody synthesized by the original

hybridoma cells. The results demonstrate that chDMB5F3 not only binds MUC1+ malignant cells

but that as an immunoconjugate it also internalizes ZZ:P38 pseudomonas toxin resulting in very

potent cell death (Fig. 6). This is consistent with the previously reported cytocidal activity of

PE38-immunconjugates against pancreatic cancer cells and against hairy cell leukemia (33, 34). In

addition, the cytotoxic activity of chDMB5F3 immunoconjugate was compared to that of PE38

immunotoxins formed with Cetuximab (Erbitux ®) (anti-EGFR)(35) and with Trastuzumab

(Herceptin®, anti-ErbB2 or Her2)(36). In human cancer cell lines assessed for their sensitivity to

the three immunotoxin conjugates, chDMB5F3:ZZ-PE38 resulted in cytotoxicity comparable or




17

superior to the Cetuximab and Trastuzumab conjugates in pancreatic and breast cancer cells (Fig.

7).

To assess the anti-tumor activity of the chDMB5F3:ZZ-PE38 immunoconjugate within the intact

organism, we have recently initiated a study that utilizes a nude mouse model of pancreatic

tumor growth using MUC1-positive human pancreatic cancer cells. To date, our in-vivo findings

show abrogation of tumor growth corroborating the potent in-vitro cytocidal activity of

chDMB5F3:ZZ-PE38 (to be presented in a separate publication).

The anti-CD30-immunoconjugate, Brentuximab vedotin, has been recently approved for use in

CD30+ Hodgkins disease(37), where more than a third of patients with refractory Hodgkin

Lymphoma achieved complete remission and partial remissions were observed in another 40%.

This is further indication of the effective targeting capabilities of immunoconjugates and

highlights the clinical potential of such antibody-drug conjugates (ADC).

Cetuximab-induced tumor cytotoxicity has not been found to directly correlate with the degree

of tumor expression of EGFR, but to be primarily effective in the subset of EGFR+ tumors having

the KRAS mutation in colorectal adenocarcinoma and squamous cell carcinoma of the head and

neck (38, 39). Furthermore, Cetuximab has shown limited activity against other EGFR expressing

tumors such as breast cancer(40). The antitumor effect of Trastuzumab on the other hand does

in general correlate with tumor overexpression of its target protein, ErbB2 (Her2)(36). However

ErbB2 is overexpressed in only a minority subset of patients, approximately 20% of breast

cancer(36) and 22% of gastric cancers (41). Since MUC1 is overexpressed in some 70-80% of

adenocarcinomas including gastric, pancreatic, colorectal, prostate, and ovarian cancers, as well

as on the malignant plasma cell of multiple myeloma (8-13), the high degree of

chDMB5F3:immunotoxin conjugate induced cytotoxicty demonstrated here augurs well for use in

those tumors not amenable or non-responsive to therapy with other monoclonal antibodies.

Additional investigations, beyond the scope of the present report, will be required to see

whether the anti-MUC1-SEA-domain DMB monoclonal antibodies reported here possess intrinsic

anti-tumor activity within the organism when administered as naked antibodies. Such activity

shown for Cetuximab and Trastuzumab has been attributed to immunologically based and Fc




18

dependent mechanisms (42), including ADCC (antibody dependent cell mediated cytotoxicity)

and CDC (complement dependent cytotoxicity). We do not know as yet whether this will be the

case for all or any of the DMB mAbs reported here. Antibodies against the tandem repeat part of

MUC1 fail to induce complement lysis despite appreciable binding by flow cytometry. This has

been attributed to the great distance from the cell surface that complement is activated(43). In

this regard it is pertinent to note that all DMB mAbs bind MUC1-SEA-domain epitopes that are

located close by to the cell membrane making CDC a distinct possibility for DMB mAbs.

Tumor stem cells are comprised of a cell subpopulation capable of self-regeneration and are

thought to result in tumor cell repopulation following ablation of all detectable malignant disease

by anti-tumor therapies (44). The present findings indicate that the side population of MCF7 cells

previously shown to bear some characteristics of cancer stem cells (22), express MUC1+. The fact

that antibodies DMB4B4 and DMB4F4 bind this side population of MCF7 cells suggests that the

same conformationally-determined MUC1 epitopes formed by the alpha and beta chains on

differentiated tumor cells are also present on these cells.

Targeting conformationally-determined sites on the cell-bound alpha/beta MUC1 junction may

successfully overcome the difficulty encountered to date in targeting the MUC1 shed alpha chain.

Whether the ultimate optimal clinical application are the anti-junctional antibodies described

here or development of anti-alpha/beta MUC1 junction T cell vaccines (44-48)—or both—will be

answered by further studies.

Acknowledgements: This research was funded in part by grants from the Israel Cancer

Association (Project Number 20112024) and Israel Science Foundation (Project Number

1167/10). We are particularly grateful to Drs. Olivera J. Finn and Dr. Katja Engelmann from

University of Pittsburgh School of Medicine for assistance with the MCF7 side population cell

experiments.




19

References 1. Baruch, A., Hartmann, M., Zrihan-Licht, S., Greenstein, S., Burstein, M., Keydar, I.,

Weiss, M., Smorodinsky, N., and Wreschner, D. H. Preferential expression of novel MUC1 tumor antigen isoforms in human epithelial tumors and their tumor-potentiating function. Int J Cancer, 71: 741-749, 1997.

2. Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E. N., and Wilson, D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem, 265: 15286-15293, 1990.

3. Ligtenberg, M. J., Vos, H. L., Gennissen, A. M., and Hilkens, J. Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J Biol Chem, 265: 5573-5578, 1990.

4. Levitin, F., Stern, O., Weiss, M., Gil-Henn, C., Ziv, R., Prokocimer, Z., Smorodinsky, N. I., Rubinstein, D. B., and Wreschner, D. H. The MUC1 SEA module is a self-cleaving domain. J Biol Chem, 280: 33374-33386, 2005.

5. Lillehoj, E. P., Han, F., and Kim, K. C. Mutagenesis of a Gly-Ser cleavage site in MUC1 inhibits ectodomain shedding. Biochem Biophys Res Commun, 307: 743-749, 2003.

6. Macao, B., Johansson, D. G., Hansson, G. C., and Hard, T. Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin. Nat Struct Mol Biol, 13: 71-76, 2006.

7. Ligtenberg, M. J., Kruijshaar, L., Buijs, F., van Meijer, M., Litvinov, S. V., and Hilkens, J. Cell-associated episialin is a complex containing two proteins derived from a common precursor. J Biol Chem, 267: 6171-6177, 1992.

8. O'Connor, J. C., Julian, J., Lim, S. D., and Carson, D. D. MUC1 expression in human prostate cancer cell lines and primary tumors. Prostate Cancer Prostatic Dis, 8: 36-44, 2005.

9. Yonezawa, S., Byrd, J. C., Dahiya, R., Ho, J. J., Gum, J. R., Griffiths, B., Swallow, D. M., and Kim, Y. S. Differential mucin gene expression in human pancreatic and colon cancer cells. Biochem J, 276 ( Pt 3): 599-605, 1991.

10. Lapointe, J., Li, C., Higgins, J. P., van de Rijn, M., Bair, E., Montgomery, K., Ferrari, M., Egevad, L., Rayford, W., Bergerheim, U., Ekman, P., DeMarzo, A. M., Tibshirani, R., Botstein, D., Brown, P. O., Brooks, J. D., and Pollack, J. R. Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci U S A, 101: 811-816, 2004.

11. Joshi, M. D., Ahmad, R., Yin, L., Raina, D., Rajabi, H., Bubley, G., Kharbanda, S., and Kufe, D. MUC1 oncoprotein is a druggable target in human prostate cancer cells. Mol Cancer Ther, 8: 3056-3065, 2009.

12. Gold, D. V., Karanjawala, Z., Modrak, D. E., Goldenberg, D. M., and Hruban, R. H. PAM4-reactive MUC1 is a biomarker for early pancreatic adenocarcinoma. Clin Cancer Res, 13: 7380-7387, 2007.

13. Kawano, T., Ahmad, R., Nogi, H., Agata, N., Anderson, K., and Kufe, D. MUC1 oncoprotein promotes growth and survival of human multiple myeloma cells. Int J Oncol, 33: 153-159, 2008.

14. Cheever, M. A., Allison, J. P., Ferris, A. S., Finn, O. J., Hastings, B. M., Hecht, T. T., Mellman, I., Prindiville, S. A., Viner, J. L., Weiner, L. M., and Matrisian, L. M. The




20

prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res, 15: 5323-5337, 2009.

15. Prinssen, H. M., Molthoff, C. F., Verheijen, R. H., Broadhead, T. J., Kenemans, P., Roos, J. C., Davies, Q., van Hof, A. C., Frier, M., den Hollander, W., Wilhelm, A. J., Baker, T. S., Sopwith, M., Symonds, E. M., and Perkins, A. C. Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01. Cancer Immunol Immunother, 47: 39-46, 1998.

16. Gillespie, A. M., Broadhead, T. J., Chan, S. Y., Owen, J., Farnsworth, A. P., Sopwith, M., and Coleman, R. E. Phase I open study of the effects of ascending doses of the cytotoxic immunoconjugate CMB-401 (hCTMO1-calicheamicin) in patients with epithelial ovarian cancer. Ann Oncol, 11: 735-741, 2000.

17. Pegram, M. D., Borges, V. F., Ibrahim, N., Fuloria, J., Shapiro, C., Perez, S., Wang, K., Schaedli Stark, F., and Courtenay Luck, N. Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer. Breast Cancer Res, 11: R73, 2009.

18. Rubinstein, D. B., Karmely, M., Ziv, R., Benhar, I., Leitner, O., Baron, S., Katz, B. Z., and Wreschner, D. H. MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells. Cancer Res, 66: 11247-11253, 2006.

19. Mazor, Y., Keydar, I., and Benhar, I. Humanization and epitope mapping of the H23 anti-MUC1 monoclonal antibody reveals a dual epitope specificity. Mol Immunol, 42: 55-69, 2005.

20. Keydar, I., Chou, C. S., Hareuveni, M., Tsarfaty, I., Sahar, E., Selzer, G., Chaitchik, S., and Hizi, A. Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens. Proc Natl Acad Sci U S A, 86: 1362-1366, 1989.

21. Rubinstein, D. B., Karmely, M., Pichinuk, E., Ziv, R., Benhar, I., Feng, N., Smorodinsky, N. I., and Wreschner, D. H. The MUC1 oncoprotein as a functional target: immunotoxin binding to alpha/beta junction mediates cell killing. Int J Cancer, 124: 46-54, 2009.

22. Engelmann, K., Shen, H., and Finn, O. J. MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res, 68: 2419-2426, 2008.

23. Mazor, Y., Barnea, I., Keydar, I., and Benhar, I. Antibody internalization studied using a novel IgG binding toxin fusion. J Immunol Methods, 321: 41-59, 2007.

24. Ali, H. R., Dawson, S. J., Blows, F. M., Provenzano, E., Pharoah, P. D., and Caldas, C. Cancer stem cell markers in breast cancer: pathological, clinical and prognostic significance. Breast Cancer Res, 13: R118.

25. Kok, M., Koornstra, R. H., Margarido, T. C., Fles, R., Armstrong, N. J., Linn, S. C., Van't Veer, L. J., and Weigelt, B. Mammosphere-derived gene set predicts outcome in patients with ER-positive breast cancer. J Pathol, 218: 316-326, 2009.

26. Hartman, M., Baruch, A., Ron, I., Aderet, Y., Yoeli, M., Sagi-Assif, O., Greenstein, S., Stadler, Y., Weiss, M., Harness, E., Yaakubovits, M., Keydar, I., Smorodinsky, N. I., and Wreschner, D. H. MUC1 isoform specific monoclonal antibody 6E6/2 detects preferential expression of the novel MUC1/Y protein in breast and ovarian cancer. Int J Cancer, 82: 256-267, 1999.




21

27. Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nat Rev Cancer, 9: 874-885, 2009.

28. Hamann, P. R., Hinman, L. M., Beyer, C. F., Lindh, D., Upeslacis, J., Shochat, D., and Mountain, A. A calicheamicin conjugate with a fully humanized anti-MUC1 antibody shows potent antitumor effects in breast and ovarian tumor xenografts. Bioconjug Chem, 16: 354-360, 2005.

29. Koumarianou, A. A., Hudson, M., Williams, R., Epenetos, A. A., and Stamp, G. W. Development of a novel bi-specific monoclonal antibody approach for tumour targeting. Br J Cancer, 81: 431-439, 1999.

30. Snijdewint, F. G., von Mensdorff-Pouilly, S., Karuntu-Wanamarta, A. H., Verstraeten, A. A., Livingston, P. O., Hilgers, J., and Kenemans, P. Antibody-dependent cell-mediated cytotoxicity can be induced by MUC1 peptide vaccination of breast cancer patients. Int J Cancer, 93: 97-106, 2001.

31. Troise, F., Cafaro, V., Giancola, C., D'Alessio, G., and De Lorenzo, C. Differential binding of human immunoagents and Herceptin to the ErbB2 receptor. Febs J, 275: 4967-4979, 2008.

32. Thomas, S. M. and Grandis, J. R. Pharmacokinetic and pharmacodynamic properties of EGFR inhibitors under clinical investigation. Cancer Treat Rev, 30: 255-268, 2004.

33. Du, X., Xiang, L., Mackall, C., and Pastan, I. Killing of resistant cancer cells with low Bak by a combination of an antimesothelin immunotoxin and a TRAIL Receptor 2 agonist antibody. Clin Cancer Res, 17: 5926-5934.

34. Kreitman, R. J. and Pastan, I. Antibody fusion proteins: anti-CD22 recombinant immunotoxin moxetumomab pasudotox. Clin Cancer Res, 17: 6398-6405.

35. Vincenzi, B., Santini, D., and Tonini, G. New issues on cetuximab mechanism of action in epidermal growth factor receptor-negative colorectal cancer: the role of vascular endothelial growth factor. J Clin Oncol, 24: 1957; author reply 1957-1958, 2006.

36. Hudis, C. A. Trastuzumab--mechanism of action and use in clinical practice. N Engl J Med, 357: 39-51, 2007.

37. Gualberto, A. Brentuximab Vedotin (SGN-35), an antibody-drug conjugate for the treatment of CD30-positive malignancies. Expert Opin Investig Drugs.

38. Lievre, A., Bachet, J. B., Boige, V., Cayre, A., Le Corre, D., Buc, E., Ychou, M., Bouche, O., Landi, B., Louvet, C., Andre, T., Bibeau, F., Diebold, M. D., Rougier, P., Ducreux, M., Tomasic, G., Emile, J. F., Penault-Llorca, F., and Laurent-Puig, P. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol, 26: 374-379, 2008.

39. Di Fiore, F., Blanchard, F., Charbonnier, F., Le Pessot, F., Lamy, A., Galais, M. P., Bastit, L., Killian, A., Sesboue, R., Tuech, J. J., Queuniet, A. M., Paillot, B., Sabourin, J. C., Michot, F., Michel, P., and Frebourg, T. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer, 96: 1166-1169, 2007.

40. Modi, S., D'Andrea, G., Norton, L., Yao, T. J., Caravelli, J., Rosen, P. P., Hudis, C., and Seidman, A. D. A phase I study of cetuximab/paclitaxel in patients with advanced-stage breast cancer. Clin Breast Cancer, 7: 270-277, 2006.

41. Okines, A. F. and Cunningham, D. Trastuzumab in gastric cancer. Eur J Cancer, 46: 1949-1959.




22

42. Clynes, R., Takechi, Y., Moroi, Y., Houghton, A., and Ravetch, J. V. Fc receptors are required in passive and active immunity to melanoma. Proc Natl Acad Sci U S A, 95: 652-656, 1998.

43. Ragupathi, G., Liu, N. X., Musselli, C., Powell, S., Lloyd, K., and Livingston, P. O. Antibodies against tumor cell glycolipids and proteins, but not mucins, mediate complement-dependent cytotoxicity. J Immunol, 174: 5706-5712, 2005.

44. Gilewski, T., Adluri, S., Ragupathi, G., Zhang, S., Yao, T. J., Panageas, K., Moynahan, M., Houghton, A., Norton, L., and Livingston, P. O. Vaccination of high-risk breast cancer patients with mucin-1 (MUC1) keyhole limpet hemocyanin conjugate plus QS-21. Clin Cancer Res, 6: 1693-1701, 2000.

45. Ding, C., Wang, L., Marroquin, J., and Yan, J. Targeting of antigens to B cells augments antigen-specific T-cell responses and breaks immune tolerance to tumor-associated antigen MUC1. Blood, 112: 2817-2825, 2008.

46. Ramanathan, R. K., Lee, K. M., McKolanis, J., Hitbold, E., Schraut, W., Moser, A. J., Warnick, E., Whiteside, T., Osborne, J., Kim, H., Day, R., Troetschel, M., and Finn, O. J. Phase I study of a MUC1 vaccine composed of different doses of MUC1 peptide with SB-AS2 adjuvant in resected and locally advanced pancreatic cancer. Cancer Immunol Immunother, 54: 254-264, 2005.

47. Lepisto, A. J., Moser, A. J., Zeh, H., Lee, K., Bartlett, D., McKolanis, J. R., Geller, B. A., Schmotzer, A., Potter, D. P., Whiteside, T., Finn, O. J., and Ramanathan, R. K. A phase I/II study of a MUC1 peptide pulsed autologous dendritic cell vaccine as adjuvant therapy in patients with resected pancreatic and biliary tumors. Cancer Ther, 6: 955-964, 2008.

48. Karanikas, V., Hwang, L. A., Pearson, J., Ong, C. S., Apostolopoulos, V., Vaughan, H., Xing, P. X., Jamieson, G., Pietersz, G., Tait, B., Broadbent, R., Thynne, G., and McKenzie, I. F. Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein. J Clin Invest, 100: 2783-2792, 1997.




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Figure 1 Scheme of MUC1 proteins used for immunizing mice and subsequent screening for anti-MUC1 SEA module alpha/beta junction monoclonal antibodies Schema of isoforms structures The cleaved full-length MUC-TM protein containing both the beta-chain and the alpha chain with its tandem repeat array and the cleaved MUC1-X protein. The domains are color schemed as follows: signal peptide- purple; N-terminal 30 amino acids (N30)- yellow; sequences flanking the tandem repeat array- green; central tandem repeat array- light blue; N-terminal part of SEA domain- red; C-terminal part of SEA domain- hatched red and white; transmembrane domain- dark blue and cytoplasmic domain- pink. The SEA domain comprised of the interacting alpha and beta subunits is indicated by the dashed black circles. Screening of hybridoma supernatants for antibodies that selectively recognize the SEA module alpha/beta junction was carried out with an ELISA screen using wells precoated with MUC1-Xex protein (see Methods). The series of seven anti-MUC1 antibodies (the DMB series, red lettering), as well as the previously described (18) IgM DMC209, bind the SEA module. Figure 2 Flow cytometric analyses of DMB anti-MUC1 SEA module alpha/beta junction monoclonal antibodies with MUC1-TM expressing cells Parental nontransfected mouse mammary tumor DA3 cells and the same cells transfected with cDNA coding for and expressing full-length MUC1-TM. Columns designated DA3-PAR and DA3-MUC1 were reacted with the DMB series of anti-MUC1 SEA module alpha/beta junction monoclonal antibodies. Flow cytometry with the fluorescently labeled secondary antibody alone (red tracing), and with anti-MUC1 antibody followed by secondary antibody (green tracing) are as shown. Colo357 cells were similarly reacted with the 7 mAbs, either in the absence or presence of competing soluble extracellular domain of the MUC1-Xex protein [column Colo357 and column Colo357 + MUC1-Xex]. Figure 3 Binding of anti-junctional antibodies to MCF7 side population cells An MCF7 breast cancer cell population showing some characteristics of cancer stem cells was isolated as described by Finn (22). MCF7 cells were stained with the fluorescent dye Hoechst 33342. Due to their increased efflux of the dye, a cone-shaped side population (SP) of MCF7 stem cells ( 5.1% of the overall population) was isolated and characterized (top right plots). Reaction with IgG1 isotype control resulted in no cell shift (left lower panels). Reaction with 10μg/ml of either DMB-4B4 or DMB-4F4 resulted in near-total shifts in both the MCF7 side cell side population (orange color, middle and right lower panels ) as well as in 'mature' MCF7 breast cancer cells (green color; middle and left lower panels).




24

Figure 4 Immunoreactivity of DMB mAbs with MUC1-Xex, SEA module and MUC1 4G-SEA module proteins [I] ELISA plates were coated with one of three recombinant bacterial MUC1 proteins- extracellular domain of the MUC1-X protein (dotted lines), MUC1 SEA module (dashed lines), or the uncleaved MUC1 4G-SEA module protein (dashed and dotted lines). The indicated monoclonal antibodies were applied and bound antibody was detected as described in Methods. The three monoclonal antibodies DMB-5F3, DMB-4F4 and DMB-7F3 show different patterns of binding for the three MUC1 proteins. [II] Both the MUC1 SEA module (SEA, lanes 1)and the mutant uncleaved MUC1 SEA module (SEA-4G, lanes 2) were resolved by SDS-PAGE and stained with Coomassie blue stain (panel A). This revealed the separate alpha and beta subunits of the SEA module (lanes 1, indicated by alpa and beta) or the alpha and beta subunits connected by the four- glycine residue linker (alpha-4G-beta). Identical gels were western blotted and probed with monoclonal antibodies BOS10D2, DMB5F3, DMB4F4 or DMB4B4 (panels B-E, respectively). The epitope recognized by the BOS10D2 antibodies has been previously reported (26) and is indicated in the lower panel. Color scheme of the protein segments is the same as in Fig. 1. Figure 5 Titration analysis of DMB monoclonal anti-MUC1 SEA module alpha/beta junction antibodies to human breast cancer cells expressing MUC1 and affinity analysis MUC1+ Human ZR75 breast cancer cells were reacted with antibodies DMB4B4, DMB4F4, and DMB5F3 at final concentrations of 4 and 1 micrograms/ml (left and center panels, respectively), and antibody binding was assessed by flow cytometry. Antibody DMB5F3 was shown to have the highest affinity. Its reactivity with ZR75 cells was assessed at decreasing concentrations (as indicated, right panel), down to 62 nanograms/ml, equivalent to a 0.4nM concentration of DMB5F3 (right panel). Surface Plasmon Resonance (SPR) binding assays for the determination of DMB5F3 full mAb and Fab fragments (lower panels A and B, respectively) were performed as described in Methods. Figure 6 Activity of recombinant chimeric DMB5F3 (chDMB5F3) monoclonal antibody synthesized in CHO cells. Recombinant antibody (chDMB5F3, see Methods) secreted into CHO culture medium was assessed by dot-blotting dilutions (ten fold to eighty fold) followed by detection with HRP-conjugated anti-human Fc (Panel B). To assess absolute amounts of chDMB5F3, a parallel dot blot was performed with known amounts of human IgG (Panel A). The activity of the chDMB5F3 recombinant antibody was compared to that of mouse hybridoma DMB5F3 mAb by ELISA (p anels C and D, respectively). Flow cytometry analyses were performed with the chDMB5F3 antibody at the indicated concentrations using MUC1-negative parental DA3 cells (DA3-PAR) and mouse cell transfectants (DA3-TM) expressing human MUC1-TM (E and F, respectively). MUC1+ cells were incubated with DyLight649-labeled DMB5F3 mAb at 4oC and at 37oC (panels G and H, respectively) and photographed by confocal laser microscopy. Whereas at 4oC antibody remained at the cell surface, at 37oC DMB5F3 was all but completely internalized into the cell. Panel I- Cytocidal activity of chDMB5F3:ZZ-PE38 immunotoxin conjugate was tested




25

(Panel G) on DA3-PAR, DA3-TM and human breast cancer T47D cells by applying varying levels of chDMB5F3 antibody, as indicated on the x-axis, together with ZZ-PE38. Cell viability (y-ordinate) was assessed using the alkaline phosphatase assay (Methods).

Figure 7 Specificity and affinity of chDMB5F3 immunotoxin(IT) for cell killing of cancer cells- comparison with Erbitux and Herceptin immunotoxins (IT). Human adenocarcinoma cells were incubated at 40C (A) followed by incubation at 370C (A') with DyLight650-labeled DMB5F3 mAb- internalization of the antibody is clearly seen at the 370C incubation. Unambiguous membrane labeling was seen following incubation of fixed human adenocarcinoma cells with either chDMB5F3 (B, green label, detection with Alex488-labeled goat anti-human Fc) or Erbitux (B', red label, detection with R-Phycoerythrin-labeled goat anti-human Fc). Human Colo357 pancreatic and ZR75 breast cancer cells (panels C and D, respectively) were incubated with chDMB5F3, Erbitux or Herceptin ZZ-PE38 immunotoxin conjugates, and cell viability (y-ordinate) was assessed using the alkaline phosphatase assay. Addition of 50microg/ml of soluble MUC1 Xex to chDMB5F3 immunotoxin abrogated cell killing (dotted orange lines). In contrast, addition of MUC1-Xex competitor did not abrogate cell killing mediated by immuntoxin conjugates formed with either Erbitux (panel C, compare dotted blue and solid blue lines) or Herceptin (panel D, compare dotted green and solid green lines).




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Published OnlineFirst April 16, 2012.Cancer Res Edward Pichinuk, Itai Benhar, Oded Jacobi, et al. cellsAntibody targeting of cell-bound MUC1 SEA domain kills tumor

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