[CANCER RESEARCH 51, 2451-2455, May 1, 1991]

Reactivity of Anti-CD15 Monoclonal Antibody PM-81 with Breast Cancer and

Elimination of Breast Cancer Cells from Human Bone Marrow by PM-81 andImmunomagnetic Beads1

James J. Vredenburgh, Wendy Simpson, Vincent A. Mentoli, and Edward D. Ball2

Departments of Medicine [E. D. B., J. J. V.], Microbiology IW. S.], and Pathology [V. A. M.J, Dartmouth Medical School, Hanover, New Hampshire 03 756

ABSTRACT

The CD15 carbohydrate antigen, lacto-N-fucopentaose III is expressedon a variety of human cancer cells including acute myeloid leukemia,small cell carcinoma of the lung, and colorectal carcinomas. We havefound that cells from breast cancer cell lines and patient-derived tissueare strongly GDIS positive, as seen by binding to the PM-81 monoclonalantibody. In this report we show that monoclonal antibody PM-81 andimmunomagnetic beads can remove breast cancer cells from bone marrowand thus be used as "purging" agents for autologous bone marrow

transplantation. PM-81 and immunomagnetic beads removed up to 3 logof SK-BR-3 and MCF7 breast carcinoma cell line cells while minimallyaffecting normal hematopoietic progenitor cells. This technique may beuseful for purging marrow for autologous bone marrow transplantationin breast cancer.

INTRODUCTION

The polymorphonuclear leukocyte-associated CD 15 antigen,LNF-III,3 is a pentasaccharide which was first discovered inhuman breast milk (1). LNF-III is found on all mature neutro-

phils and the majority of monocytes (2, 3). Myeloid progenitorsexpress LNF-III at the level of the CFU-GM (4). In a series of>200 patients with AML, >90% of patients expressed LNF-III on the surface of their leukemia cells (5). LNF-III is also

found on the surface of small cell carcinoma of the lung, colon,stomach, liver, and ovarian cancer cells (6-9).

PM-81 is a mAb that reacts with LNF-III (10). Anti-CD 15mAbs are not all reactive with the same epitope and they donot express differential reactivity with LNF-III, as demonstrated in a series of experiments reported at the Fourth International CD 15 Workshop. PM-81 was the most reactive of apanel of 25 anti-CD 15 mAbs to AML cells (3). In studies ofCD 15 expression on other tumor cells we found that breastcarcinoma cell lines were uniformly positive for this antigen.Since finding that PM-81 binds to breast cancer cell lines, westudied the reactivity of PM-81 with 36 human pathologicalbreast tumor specimens and found PM-81 binding to be very

common. Based on this result we decided to test the hypothesisthat PM-81 in conjunction with immunomagnetic beads could

remove a low percentage of breast cancer cells mixed withnormal bone marrow to use for autologous bone marrowtransplantation.

Received 10/26/90; accepted 2/22/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grants CA 37868 from the National Cancer Institute,DHHS, and ACS 1N-157 from the American Cancer Society. J. J. V. was aTiffany Blake Fellow at the Dartmouth-Hitchcock Medical Center. E. D. B. is aScholar of the Leukemia Society of America.

2To whom requests for reprints should be addressed, at Department of

Medicine, Dartmouth Medical School, Hanover, NH 03756.3The abbreviations used are: LNF-III, lacto-N-fucopentaose III; CFU-GM,

colony-forming unit, granulocyte-monocyte; AML, acute myeloid leukemia; mAb,monoclonal antibody; BFU-E, erythroid burst-forming units.

MATERIALS AND METHODS

Monoclonal Antibodies. MAb PM-81 was developed by one of us (E.D. B.) and is an IgM mAb that reacts with the CD 15 antigen (11). PM-81 was purified from ascites fluid by gel filtration using a CL-6BSepharose column (12). Thy-1, an irrelevant IgM-negative control mAb,was used as a hybridoma supernatant and was obtained from theAmerican Type Culture Collection.

Breast Cancer Specimens. Human breast cancer tissues were obtainedfrom routine surgical biopsies and resections at the Dartmouth-Hitchcock Medical Center. Fresh tissue was fixed in acetone, stored at 22°C

overnight, cleared in methylbenzoate and xylene, consecutively, andembedded in paraffin at 60°C(13). Sections of 4-5 urn width were

mounted on silanated glass slides prior to immunostaining. Alternatively, tissues were fixed in 10% buffered formalin, paraffin-embedded,sectioned at 4-5 >jm, and mounted on poly-L-lysine-coated slides for¡ninninoli¡sioche-m¡caistudies (14).

Immunohistochemistry. PM-81 immunoreactivity was assessed usingan alkaline phosphatase-labeled indirect antibody technique (15). Theformalin-fixed, paraffin-embedded tissue was deparaffinized throughxylene and alcohols, and the Amex-processed tissue was placed inacetone. A trypsin digestion step, 10-min at 22°C,was used for for

malin-fixed tissues only. Optimal staining was achieved with a 0.12-mg/ml concentration of primary PM-81 antibody. Predilli ted blockingagent, alkaline phosphatase-conjugated goat-anti-mouse immunoglob-ulin and a chromogenic substrate were supplied in the Bio/Can Scientific AS/AP immunostaining kit. Positive controls were provided byneutrophils in the breast tissue being studied. Negative controls wereperformed by omitting the primary antibody and substituting nonimmune serum.

Breast Cancer Cell Lines. The breast cancer cell lines, SK-BR-3,MCF7, and BT-20, were obtained from American Type Culture Collection (16-18). The cells have been maintained in continuous steriletissue culture. The SK-BR-3 cells were grown in McCoy's 5A medium

(Gibco, Grand Island, NY) and the MCF7 and BT-20 cells were grownin RPMI 1640 medium (Gibco). Both media were supplemented with10% fetal calf serum (Hyclone, Logan, UT) and the cells were grownin a humidified incubator with 6.5% CO2 at 37°C.

Immunomagnetic Beads. The immunomagnetic beads, Dynabeads R(Dynal, Oslo, Norway), are 4.5-mm polystyrene beads with 20% ironin the form 7-Fe2O3 and have a magnetic susceptibility of approximately10~2cgs units (19). Dynabeads covalently coated with sheep-anti-mouse

IgG were used.Bone Marrow. Bone marrow was obtained from the posterior iliac

crests of healthy paid volunteers with informed consent. The marrowcells were separated by Ficoll-hypaque centrifugation and used the sameday in these experiments. The range of bone marrow mononuclear cellsobtained after Ficoll-hypaque centrifugation was 1.0-5.0 x 107/ml of

bone marrow aspirated.Flow Cytometry. The surface antigen phenotypes of the breast cancer

cells and normal bone marrow cells were determined by flow cytometryas previously described (20). Briefly, the cells were incubated with themAb for 60 min at 0-4°C in the presence of human IgG (Sigma

Chemical Co., St. Louis, MO) to block Fc-mediated binding. The finalconcentration of PM-81 was 20 Mg/ml and 1 Mg/06 tumor cells wasused. After the cells were washed, fluorescein isothiocyanate-labeledgoat-anti-mouse IgG and IgM were added, and the mixture was incubated for 30 min at 0-4°C.The cells were washed again and resuspended

in phosphate-buffered saline-bovine serum albumin-0.1% azide. The

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REACTIVITY OF PM-81 WITH BREAST CANCER

percentage of positive cells and mean fluorescent intensity were determined on the Ortho 50 H cytofluorograph, and the results were compared to a positive and negative control mAb.

Immunomagnetic Bead Separation. A mixture of 10% breast cancercells and 90% bone marrow cells were incubated with the mAb for 60min at 0-4°C.The amount of PM-81 added/106 total cells was 1 jig,

and the final concentration was 20 Mg/ml. The cells were then washedthree times, and the Dynabeads were added at a ratio of 25 beads: 1breast cancer cell. The mixture of mAb-coated cells and immunomag-netic beads was incubated for 30 min on an orbitron rotator at 4°C.

The mixture was then placed in the Dynal Magnetic Particle Concentrator for 2 min and the fluid aspirated and called the supernatant. Thesupernatant was mixed with the same number of beads and the immu-nomagnetic separation was repeated. The log separation was determined by limiting dilution cultures as described below.

Limiting Dilution Cultures. The breast cancer cells were grown inlimiting dilution assays, as previously described (21). The cells remaining in the supernatant were serially diluted in wells of microtiter trays(6 wells) (Becton Dickinson, Lincoln Park, NJ) in powers of 10. Inorder to establish standards for the assay, the breast cancer cells werecounted with a hemacytometer and known numbers of cells in powersof 2 were diluted in wells of microtiter trays (96 wells) (Becton Dickinson). Each well in the 6-well trays contained 1.0 ml filtered, conditioned media, 0.5 ml fetal calf serum (Hyclone), and 0.5 ml McCoy's

media for the SK-BR-3 cells or RPMI 1640 media for the MCF7 cells(Gibco). Each well in the 96-well trays contained 0.2 ml filtered,conditioned media, 0.1 ml fetal calf serum, and 0.1 ml media. The trayswere placed in a humidified incubator with 6.5% COj at 37°C.A cluster

of >20 cells was scored as positive, and the trays were scored on day 7under an inverted microscope.

Bone Marrow Colony-forming Unit Cultures. In order to determinethe effect of the mAb and immunomagnetic bead separation on thenormal hematopoietic progenitor cells, colony-forming unit culturescontaining methylcellulose were performed as described before (22).Bone marrow cells were incubated with the mAb, followed by theimmunomagnetic beads, as described above, and the cells remaining inthe supernatant were cultured. In addition, an aliquot of bone marrowcells was kept on ice and not treated, which served as the standard.Hematopoietic cells (6 x IO5) were mixed with methylcellulose (0.9%

final concentration), fetal calf serum (30%), bovine serum albumin(10%), L- glutamine (2 HIM), 2-mercaptoethanol (5 mivi), 15 ng ofinterleukin-3 and granulocyte-monocyte colony-stimulating factor (Im-

munex, Seattle, WA), and 2 units of erythropoietin (Amgen, ThousandOaks, CA) and plated in duplicate into two 35-mm Petri dishes. After

the dishes were incubated in a humidified incubator with 6.5% CCh at37°C,colonies were counted on day 14 under an inverted microscope.

RESULTS

Immunohistochemistry. The reactivity of PM-81 with 36 malignant breast tumors was determined by an alkaline phospha-tase-labeled indirect antibody technique. Thirty of 30 primaryand 6 of 6 metastatic breast carcinomas showed PM-81 reactivity (Fig. 1). Staining of breast carcinoma tissue was observedwith both formalin-fixed and Amex-processed tissue but moreintensely with the latter. The staining was cytoplasmic andmembranous (Fig. 1), the lateral membrane being most ofteninvolved, as compared to 5 normal and fibrocystic disease breasttissue cases in which the staining involved the luminal membrane (Fig. 2).

Flow Cytometry. The percentage of reactivity and mean fluorescent intensity of each mAb with the three breast cancer celllines, SK-BR-3, MCF7, and BT-20, and bone marrow cells asdetermined by cytofluorography are shown in Table 1. PM-81reacted strongly with all three cell lines, particularly the SK-BR-3 and MCF7 lines.

Limiting Dilution Cultures. Culturing as few as one SK-BR-3

and eight MCF7 cells gave rise to growth in the limiting dilutioncultures (data not shown).

Immunomagnetic Bead Separation. The log tumor cell separations from mixtures of 90% normal bone marrow mononu-clear cells and 10% breast cancer cells as determined by limitingdilution cultures are shown in Table 2. In three experiments,PM-81 alone resulted in a 3 log reduction in breast cancer cellsof both cell lines tested.

Normal Hematopoietic Colony-forming Unit MethylcelluloseCultures. The effect of PM-81 and the immunomagnetic beadson hematopoietic progenitor cells determined by colony-forming units is demonstrated in Table 3. Despite the reactivity ofPM-81 with most of the bone marrow cells, hematopoietic

- <^ ^

>—„- v ***•ife«rJÄ*r* >.»•»•:/

V

,:'•- %^5'^ itmtk s»-l

Fig. 1. Normal breast showing minimal •. SPM-81 immunoreactivity limited to the luminal membrane (x 630). ^^^»^

.

* ~

••-.r2452

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REACTIVITY OF PM-81 WITH BREAST CANCER

Fig. 2. Infiltrating ductal carcinoma showing intense cytoplasmic and membranous PM-81 immunoreactivity in a high-grade tumor (x315). •v •«.•*

,

Table 1 Reactivities of PM-81 and a negative control antibody with three breast carcinoma cell lines, SK-BR-3, MCF7, and BT-20, and normal hone marrowdetermined by cytofluorography

MonoclonalantibodyThy-1

PM-81SK-BR-3

cells%

positive5.2

±0.399.7 ±0.2MFI°104

±60937 ±22MCF7

cells%

positive6.2

±0.880.9 ±6.4MFI295

±6791 ±42BT-20

cells%

positive7.7

±1.157.9 ±2.4MFI307

±26774 ±60Bone

marrowcells%

positive2.7

±2.299.6 + 0.1MFI61

±36978±14

" MFI. mean fluorescent intensity.4 Mean ±SEM.

Table 2 Log separation of breast cancer cells from a mixture of normal bonemarrow cells and breast cancer cells using monoclonal antibodies and

immunomagnetic heads

Table 3 Effect of the monoclonal antibody and immunomagnet head separationon normal hematopoietic progenitor cells

MonoclonalantibodyPM-81>Thy

IDilution":10:IOO:

1.000:10.000:10:100:

1,000:10.000:100.000:1.000.000SK-BR-3

cellsLog

sepa-Growth*ration++

3.7+c—+++

1.3+±d—MCF7

cellsLog

sepa-Growthration++

3.0+—+++

0.5++±f

' Dilution of cells in limiting dilution assay.* —.no growth: +. growth.f One of 3 wells had growth.rfTwo of the 3 wells had growth.

progenitor cells were not reduced by the immunomagneticseparation with PM-81.

DISCUSSION

We have determined the reactivity of an anti-CD 15 mAb,PM-81. with 30 human breast cancers and three breast cancercell lines. PM-81 reacted with all of the breast cancers using analkaline phosphatase indirect immunohistochemical technique.PM-81 reacted strongly with the three breast cancer cell lines,as determined by cytofluorography. PM-81 in conjunction withimmunomagnetic beads effectively removed breast cancer cellsfrom a mixture of 90% normal bone marrow and 10% breast

mAbControl

Thy-lPM-81No.

ofseparations2

22CFU-GM"322341%

change*-28+

28BFU-E155

18%

change-66

+20" Mean number of colonies in methylcellulose cultures/300.000 marrow cells.* % change determined by

Mean no. colonies sample

Mean no. colonies controlx 100.

Analysis of variance revealed that there was no significant effect on the numbersof CFL'-GM when the cells were treated with PM-81 (compared to control, P =0.09: compared to Thy-1. P = 0.06) or the numbers of BFU-E (compared tocontrol. P = 0.3: compared to Thy-1. P = 0.006).

cancer cells. The immunomagnetic separation with PM-81 didnot adversely affect normal bone marrow progenitor cells.

The LNF-III antigen has been found on a number of adeno-carcinomas and small cell carcinoma of the lung (6-9). We arenot aware of reports concerning the LNF-III antigen on breastcancers or of the reactivity of anti-CD 15 mAb with breast cancertumors or cell lines, despite the original description of the LNF-III antigen in human breast milk (1) and the known presenceof LNF-III in the serum of patients with breast cancer (23). Anumber of related glycosphingolipids have been found of breastcancer tumors and cell lines (24-26). The expression of theLNF-III antigen is significantly higher on malignant tissues,compared to normal tissue, which may be immunologically andphysiologically important. The glycosphingolipids, of whichthe LNF-III antigen is a specific type, may possess immuno-

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REACTIVITY OF PM-81 WITH BREAST CANCER

moilulatory properties and inhibit immune-mediated destruction of tumor cells expressing these antigens (27, 28). Alternatively, the glycosphingolipids are embryonic antigens, and theirincreased or novel expression on tumor tissues compared tonormal tissue may simply represent genetic derepression, similarly to the expression of carcinoembryonic antigen or a-fetoprotein on malignant cells.

A number of mAbs reactive with breast cancer have beendescribed (reviewed in Ref. 29). Most of the mAbs react withsome normal human tissues and other malignant human tissues,particularly cancers of the ovary, uterus, and prostate. The useof mAbs as diagnostic tools for immunohistopathology or ra-dioimmunodetection of métastasesor the selective targeting ofanticancer agents requires tumor-specific mAbs. However, tooptimize the in vitro purging of malignant cells from bonemarrow, a mAb must react with all of the malignant cells andnot react with hematopoietic stem cells. The reactivities of themAbs with other types of malignant cells and normal cells, evencommitted bone marrow cells, have no impact on the use of themAbs for in vitro purging of breast cancer cells. PM-81 may beuseful for in vitro bone marrow purging of breast cancer cells,which would allow intensive chemotherapy to be administeredwith the purged autologous bone marrow providing the necessary hematological support.

Intensive chemotherapy followed by autologous bone marrowtransplantation has provided potentially curative therapy forwomen with advanced breast cancer (30, 31). However, mAbsdetect bone marrow infiltration with low numbers of tumorcells in 30-60% of women with advanced breast cancer (32,33). Bone marrow purging with mAbs and immunomagneticbeads (34), gradient fractionation (35), or chemotherapy (36)effectively removes contaminating breast cancer cells and sparesnormal hematopoietic progenitor cells to varying degrees inmodel systems. Our results demonstrate that PM-81 aloneremoves 3 log of breast cancer cells from normal bone marrowand does not decrease CFU-GM or BFU-E and could be usedin vitro to purge the bone marrow of breast cancer cells priorto an autologous bone marrow transplantation. There is extensive experience with PM-81 in purging of bone marrow in AML(37). We have treated 50 AML patients with high-dose chemotherapy and autologous bone marrow transplantation usingPM-81 and AML-2-23 (anti-CDU mAb) plus rabbit complement-purged bone marrow. There are approximately 30% long-term survivors, and >95% of the évaluablepatients have engrafted. The AML experience supports the safety of purgingbone marrow with PM-81. Immunomagnetic beads may offertherapeutic advantages and decreased toxicity compared tocomplement-mediated purging. We have generally found complement to be nonspecifically toxic to cell lines of solid tumorssuch as lung and breast carcinoma and inconsistent when usedwith mAbs.4 Thus, the immunomagnetic bead procedure allowsthe expansion of a well-tested mAb-based purging method to

breast cancer.

ACKNOWLEDGMENTS

The cytofluorograph was a gift of the Ripple Foundation.

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1991;51:2451-2455. Cancer Res   James J. Vredenburgh, Wendy Simpson, Vincent A. Memoli, et al.   Bone Marrow by PM-81 and Immunomagnetic BeadsCancer and Elimination of Breast Cancer Cells from Human Reactivity of Anti-CD15 Monoclonal Antibody PM-81 with Breast

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