L6 Monoclonal Antibody Binds Prostate Cancer

Robert T. O’Donnell,1,2* Sally J. DeNardo,1 Xu-Bao Shi,1,3 Gary R. Mirick,1Gerald L. DeNardo,1 Linda A. Kroger,1 and Frederick J. Meyers1

1Division of Hematology/Oncology, Department of Internal Medicine, University of CaliforniaDavis Medical Center, Sacramento, California

2Veterans Administration Northern California Health Care System, Martinez, California3Department of Urology, University of California Davis Medical Center,

Sacramento, California

BACKGROUND. Radioimmunotherapy (RIT) is a promising new modality for targeted,systemic delivery of radionuclides specifically to sites of androgen-independent metastaticprostate cancer. To be effective, RIT requires an antibody with specificity for malignant cellsand appropriate pharmacokinetics in the body.METHODS. Specific binding of the L6 monoclonal antibody to prostate cancer cell lines orcell lysates was determined by enzyme-linked immunoabsorbent assay (ELISA), solid-phaseradioimmunoassay, and immunofluorescent staining. Biodistribution, tumor uptake, andwhole body and blood clearances of 125I-L6 were determined in nude mice bearing humanprostate cancer xenografts.RESULTS. The L6 monoclonal antibody showed strong binding to the lysates of PC3 andDU145 prostate cancer cell lines, and 66% binding to live PC3 cells. The L6 antibody specifi-cally targeted prostate cancer in PC3 and DU145-tumored nude mice, where approximately10% of the injected dose of 125I-L6 bound to prostate cancer. Low-normal organ uptake wasfound, and the blood clearances were similar in each group of tumored mice.CONCLUSIONS. The L6 monoclonal antibody targets human prostate cancer xenografts innude mice and has low-normal organ uptake. Therefore, further study of the radiolabeled L6monoclonal antibody for RIT of prostate cancer is warranted. Prostate 37:91–97, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: radioimmunotherapy; immunotherapy; biodistribution; prostate cancer

INTRODUCTION

Prostate cancer will be diagnosed in as many as210,000 Americans in 1997 [1]. As the population agesand screening becomes more widespread, the diagno-sis of prostate cancer will become even more preva-lent. Androgen ablation is the only treatment that pro-longs life after metastasis has occurred, and even thenthe 2.5-year survival is only 50% [2]. After prostatecancer escapes hormonal control, the malignant cellscan remain sensitive to radiation therapy; however,dissemination of the cancer precludes external beamradiotherapy to all disease sites.

Radioimmunotherapy (RIT) is the specific, targeteddelivery of radiation to disseminated cancer cells byanti-tumor monoclonal antibodies (MoAb). Character-istics of a useful MoAb for RIT include: high avidityand specificity for malignant cells, high-density cell

surface target antigen restricted to cancer cells, andlack of circulating antigen target. Inherent biologic ac-tivity of the MoAb may also be useful in enhancing theeffect of targeted radiation.

Studies using radiolabeled MoAb have shown theability to detect metastatic prostate cancer, but thera-peutic trials have met with little success. The anti-prostatic acid phosphatase, murine IgG1 MoAb PAY276 labeled with DTPA-chelated indium-111, detected

Contract grant sponsor: Cancer Research Institute; Contract grantnumber: 57310; Contract grant sponsor: National Cancer Institute;Contract grant number: PO1 CA47829; Contract grant sponsor: De-partment of Energy; Contract grant number: DE-FG03-84ER60233.*Correspondence to: Robert O’Donnell, M.D., Ph.D., Radiodiagnosis& Therapy, 1508 Alhambra Blvd., Sacramento, CA 95816. E-mail:rtodonnell@ucdavis.edu.Received 17 December 1997; Accepted 29 April 1998

The Prostate 37:91–97 (1998)

© 1998 Wiley-Liss, Inc.

prostate cancer metastasis in 19 of 25 patients, and thepercentage with imaged disease increased along withthe amount of antibody infused [3]. 111In-DTPA-PSA399, a murine, IgG, anti-prostate-specific antigenMoAb, was tested as a radioimmunodetection agent,and despite the potential for binding to circulatingprostate-specific antigen (PSA), it was able to detectmetastatic foci in 9 of 10 patients [4]. The 7E11-C5.3murine IgG1 MoAb was developed by immunizingmice with the human prostate cell line LNCaP; its tar-get antigen is a 100-kDa glycoprotein with some ho-mology to the transferrin receptor [5]. 111In-CYT-356(ProstaScint, Cytogen Corp., Princeton, NJ), employ-ing the 7E11-C5.3 MoAb conjugated with the linker-chelator 64K-DTPA, identified soft-tissue depositsof metastatic prostate cancer in 15 patients [6]. In asubsequent therapeutic trial, 58% of patients had de-tectable uptake of 111In-CYT-356 in at least one site;however, no complete or partial responses wereachieved by treatment with 90Y-CYT-356 [7]. CC49, amurine IgG1, binds to TAG-72, a high molecularweight mucin antigen found on many adenocarcino-mas. 131I-CC49 was used for prostate cancer RIT, andalthough 6 of 10 patients had pain relief, none met thestudy criteria for a PSA or radiographic response, andall developed an immune response against the anti-body [8]. KC4 is a murine IgG3 MoAb that reacts witha high molecular weight membrane and cytoplasmicantigen found on a variety of adenocarcinomas; 90Y-KC4 was administered to 7 patients with prostate can-cer, and mild to moderate pain relief was achieved insome patients [9].

To improve therapeutic efficacy of RIT for prostatecancer, new antibodies with beneficial characteristicsneed to be identified by examining their ability to spe-cifically bind to prostate cancer cell lines in vitro, andin animal models. L6 is a murine IgG2a MoAb ob-tained after immunization of mice with human nons-mall-cell lung cancer cells. L6 binds to an abundant,nonshed, cell-surface, tumor-associated antigen ex-pressed on breast, lung, colon, and ovarian adenocar-cinomas [10]. L6 possesses tumoricidal capacity, mani-fested by antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity(CDC) [11]. The antigen recognized by L6 and chi-meric L6 (ChL6) is an approximately 24-kDa proteinbelonging to a family of integral membrane proteins,including CD63 and CO-029, which are overexpressedon tumor cells. Several proteins with homology to theantigen recognized by L6 may have a role in regula-tion of cellular proliferation [12]. L6 has been used inRIT for breast cancer patients and targets metastaticdisease when an appropriate preload of unlabeled L6is given to coat a target on vascular endothelium, thusprolonging blood clearance of the radioimmunocon-

jugate [13]. Other than the vascular endothelium ofsmall vessels, adenocarcinoma cells are the onlyknown target of L6 [14]. Six of 10 patients with L6-reactive breast cancer had clinically measurable tumorresponses to 131I-ChL6 following L6 or ChL6 preload[15].

The biological activity of L6, i.e., ADCC, CDC, in-creased vascular permeability, and elevated serumIL-2 receptor levels, has been demonstrated in breastcancer patients treated with 131I-L6 [16]. Since L6 hasshown promise for treatment of breast cancer, its re-activity with prostate cancer cell lines was determinedas a prelude to clinical studies.

MATERIALS AND METHODS

Monoclonal Antibodies

L6 was obtained in a sterile, pyrogen-free form (On-cogen, Seattle, WA), and labeled with 125I by the chlo-ramine-T method [11,17]. 125I-L6 purity was examinedby high-performance liquid chromatography (HPLC),using a Beckman HPLC system (Beckman Instru-ments, Inc., Palo Alto, CA) with a gel filtration column(Beckman, SEC-3000) and cellulose acetate electropho-resis (CAE), using a Gelman system (Gelman Sciences,Inc., Ann Arbor, MI) in which the CAE strip is spottedand developed at 11 min (for nonprotein-bound radio-isotope separation) or 45 min (for aggregate separa-tion). The CAE strips were scanned for radioactivityon an Ambis gel scanner (Ambis Systems, San Diego,CA) [17]. Br96 (Bristol Myers Squibb, PharmaceuticalResearch Institute, Seattle, WA) is a murine IgG2aMoAb that targets the Le-y related glycoprotein foundon a number of adenocarcinomas [14]. Lym-1 (DamonBiotech, Needham Heights, MA) is highly selective formalignant B cells and binds to a discontinuous epitopeon HLA-DR10 [18].

Cell Lines

Prostate cancer cell lines DU145 and PC3 weregrown in tissue culture, using Iscove’s modified Dul-becco’s medium, and LNCaP cells were grown inRPMI-1640 with 0.12 mM MEM nonessential aminoacids, 1.2 mM MEM sodium pyruvate, and 1.2% (v/v)antibiotic/antimycotic and 20% fetal calf serum (FCS).When confluent, cells were harvested by trypsin-EDTA digestion. Raji cells, a human lymphoblastoidcell line, were grown in RPMI-1640 with 10% FCS andused as a negative control. The human prostate cancercells lines PC3, DU145, and LNCaP were derived fromtumor metastatic to lumbar vertebrae [19], brain [20],and lymph nodes [21], respectively.

92 O’Donnell et al.

Prostate Cancer Cell Lysates

Cells were harvested, pelleted, and resuspended inlysis buffer (10 mM Tris-HCl, 1 mM MgCl2, and 1 mMCaCl2, pH 7.4). After three cycles of freeze-thawing,the suspension was homogenized in a Wheaton (Mill-ville, NJ) 40-ml glass Dounce tissue grinder. Proteinconcentration was determined by optical density (OD)280 nm and adjusted to 1 mg/ml. Aliquots of 200 mlwere dispensed into 96-well Pro-Bind assay plates(Falcon #3915, Becton Dickinson, Lincoln Park, NJ) orMicroTest III Flexible assay plates (Falcon #3911). Theplates were allowed to dry at room temperature andstored at −20°C until used in ELISA or immunoreac-tivity assays.

ELISA

Coated Pro-Bind plates were blocked with 5% bo-vine serum albumin (BSA). L6 (100 ng/ml) was addedand incubated for 1 hr at room temperature, and thenwashed. HRP-anti-mouse IgG was added, and afterincubation and washing, the substrate (40 mg O-phenylenediamine and 60 ml H2O2 in 100 ml of 0.1 Mcitric acid buffer, pH 5) was added. After the reactionwas stopped, the OD 490 nm was read using a platereader.

Solid-Phase Radioimmunoreactivity Assay

Coated plates were blocked with 5% BSA in phos-phate-buffered saline (PBS), and then 100-ml aliquotscontaining 100 or 10 ng/ml of 125I-L6 were added totriplicate wells and incubated for 1 hr at 37°C. Equalamounts of each concentration were added to 12 ×75-mm tubes for 100% counts per minute (cpm) deter-minations. In some experiments, 20 mg/ml of unla-beled L6 or Lym-1 were preincubated in the coatedwells to assess specificity of binding (blocking). Afterwashing with 1% BSA in PBS, the wells were cut out,counted in a gamma well counter for 1 min, and thepercent bound was determined.

Live Cell Assay

Cells were harvested, centrifuged, and resus-pended in RPMI-1640 with 15% FCS, and then incu-bated for 1.5 hr at 37°C. Triplicate tubes with 1, 2, 3,and 5 × 106 cells were pelleted, and 100 ml of 125I-L6were added. The tubes were incubated for 30 min atroom temperature. Two milliliters of PBS were addedto each tube, and the cells were pelleted. The super-natants and pellets were counted in a gamma wellcounter, and the percent bound was determined.

Immunofluorescent Staining

Cells were harvested, washed, and resuspended to1 × 106 in PBS. Ten microliters of cells were applied toslides, air-dried, fixed with cold acetone, and washedwith PBS. MoAb (50 mg/ml) in PBS was added to cellsand incubated for 1 hr in a humidified box at 37°C.After washing with 0.05% Tween-20 in PBS, FITC-anti-mouse IgG diluted 1:32 was applied to each cell circleand incubated for 45 min at 37°C, and then washed. Adrop of 50% glycerol in PBS was applied to each cellcircle, and a coverslip was applied prior to fluorescentmicroscopy.

125I-L6 Biodistribution

Cells (3 × 106 in 100 ml) were implanted subcutane-ously into each of 10 male nude mice 3 days after400-cGy whole-body irradiation. After 3 weeks, thePC3 and DU145 tumors were of sufficient size to per-form biodistribution; LNCaP did not produce tumors.125I-L6 (20 mCi/17 mg), along with 100 mg of unlabeledLym-1, was injected in the tail veins of tumored mice.Blood samples were obtained from a tail vein at 5 min,and at 1, 24, 48, 72, and 96 hr after injection. Whole-body activity was counted at these times, using twoopposed isoresponsive and standardized sodium io-dide detectors [22]. All mice were sacrificed on day 4by cervical dislocation. Organs were removed, cleanedof excess blood, weighed, and counted in a gammawell counter.

RESULTS

The L6 and Br96 anti-adenocarcinoma MoAb, andthe Lym-1 anti-lymphoma MoAb (negative control),were tested against lysates of the DU145, LNCaP, andPC3 prostate cancer cell lines to assess their ability anddegree of binding in an ELISA assay. L6 showedstrong binding to each cell-line lysate, binding best toPC3. The average OD for L6 binding to PC3 cell lysatewas 1.019, vs. 0.132 and 0.222 for DU145 and LNCaP,respectively. L6 exhibited more binding than Br96,which bound best to LNCaP, with an OD of 0.057.Lym-1 exhibited negligible binding to the lysates(Table I).

Next, solid-phase radioimmunoassays were doneusing the three prostate cancer cell lines as targets of125I-L6. Table II shows that the percent 125I-L6 boundto the prostate cancer cell lysate target was between27.9–53.2%; once again, PC3 was targeted best. L6binding was not substantially blocked by Lym-1, butwas blocked by cold L6, demonstrating specific bind-ing. L6 did not bind to the Raji control cell-line lysate.

L6 Antibody Binds Prostate Cancer 93

In a live cell assay, similar results were found tothose in the solid-phase radioimmunoassay. The per-cent binding of 125I-L6 to PC3 was 66%, whereas thepositive control, human breast tumor (HBT), had 82%binding, and Raji lymphoma cells, the negative con-trol, had <5% binding. Binding of L6 and Lym-1 toPC3 cells was compared by immunofluorescence afterstaining with FITC-anti-mouse IgG. Strong positivefluorescence was seen with L6 to PC3 but not to thenegative control Raji; the control arm detected strongfluorescence after Lym-1 binding to Raji, but not toPC3 (Fig. 1).

125I-L6 biodistribution in nude mice bearing PC3 orDU145 cells was performed (LNCaP cell xenograftsdid not grow well in our system). Tumored mice wereinjected i.v. with 20 mCi 125I on 17 mg of L6. The whole-body and blood-clearance rates were similar for PC3and DU145 tumored mice (Fig. 2).

The percent injected dose/g of 125I-L6 in DU145and PC3 tumors at 96 hr was 10.6% and 9.5%, respec-tively (Fig. 3 and Table III). These biodistribution datashowed that 125I-L6-specific targeting occurred, thatlarge amounts of radiolabeled-L6 could target DU145and PC3 tumors, and that there was low accumulationin normal organs.

DISCUSSION

The choice of a MoAb is critical for successful tar-geting of systemic radiation to disseminated cancercells. Uptake of 125I-L6 into DU145 and PC3 xenografts(Fig. 3) was comparable to tumor uptake at 5 days inhuman breast tumor-3477 (HBT-3477)-bearing nudemice: 13.0%, 7.4%, and 13.8% injected dose (ID)/g for125I-L6, 125I-ChL6, and 90Y-DOTA-peptide-ChL6, re-spectively [11,23]. BrE-3 and m170 are mouse IgG1MoAb that react with cell-surface antigens on a num-ber of adenocarcinomas [14]; tumor uptake of BrE-3and m-170 in PC3-bearing nude mice was only 4.6%and 3.1% ID/g, respectively. In an imaging study, 10mCi of 131I-L6 were given to 10 women with meta-static breast cancer, and good tumor targeting wasfound after administration of an optimal cold L6or ChL6 preload [13]. Clinical imaging studies with

TABLE II. Blocking of 125I-L6 Binding to ProstateCancer Cell Lines in a Solid-Phase RIA*

125I-L6(100 ng/ml)

125I-L6(10 ng/ml)

Noblock

Lym-1blocka

L6blocka

Noblock

Lym-1blocka

L6blocka

DU145 35.2 31.7 5.5 37.1 34.7 6.5LNCaP 27.9 19.4 3.8 33.1 29.0 9.2PC3 53.2 55.0 22.3 50.2 53.2 20.2Raji 0.2 0.8 0.8 0.0 0.3 0.6

*Values are for percent bound.aUnlabeled antibody, 20 mg/ml.

TABLE I. Binding of MoAb to Prostate CancerCell Lysates

MoAb

200 mg of lysate

PC3 DU145 LNCaP

L6 1.019 ± 0.034a 0.132 ± 0.010 0.222 ± 0.024Br96 0.020 ± 0.006 0.010 ± 0.006 0.057 ± 0.016Lym-1 0.010 ± 0.010 0 ± 0 0.018 ± 0.011

aAverage OD 490 for 100 ng of MoAb.

Fig. 1. Immunofluorescence demonstrated specific binding of L6to PC3 cells. A: Intense FITC-labeled anti-mouse IgG immunoflu-orescent staining of PC3 cells after incubation with murine L6MoAb. B: No FITC-labeled anti-mouse IgG immunofluorescentstaining was seen in the control Raji lymphoma cells after incuba-tion with murine L6 MoAb.

94 O’Donnell et al.

131I-L6 and 131I-ChL6 found no normal organ targeting[13,15], and L6 antigen expression on the vascular en-dothelium of small vessels [14] could be blocked by anL6 preload [13]. Low levels of circulating L6 antigenwere found in 3 of 10 breast cancer patients prior toimaging with 131I-L6 [13].

L6 and ChL6 have the same antigen-binding sites,but in the chimeric version the mouse G2a and kappa-constant regions were replaced with human G1 andkappa. L6 and ChL6 have been chosen for use in clini-cal trials to exploit their biologic activity. In vitro, L6and ChL6 mediate ADCC with human macrophagesand natural killer cells, and CDC with human serum[24]. Within several hours of L6 or ChL6 infusion, su-perficial tumors in several patients became inflamed,and complement was activated [15]. Soluble interleu-kin-2 receptor levels were elevated in patients receiv-ing 150 mg or more of L6 or ChL6 [16]. Althoughunlabeled L6 and ChL6 produce immune system ac-tivation, therapeutic efficacy has been shown onlywith their radiolabeled counterparts [16,25]. Nineteenpatients with L6-reactive adenocarcinomas receivedL6 (5–400 mg/m2/day for 7 days); one patient withbreast cancer had a complete response [24]. Eighteenpatients with advanced cancer were given 4–6 infu-sions of ChL6 (350–700 mg/m2), and no clinical re-sponses were seen [26]. In HBT-3477 tumored nude

mice, administration of unlabeled ChL6 (315 mg) hadno therapeutic benefit: response rates were 13%, 18%,and 79% for unlabeled ChL6, untreated mice, and 90Y-DOTA-peptide-ChL6, respectively [25].

Clinical studies for breast cancer using L6 and ChL6have shown that adenocarcinomas can respond to RIT.Six of 10 patients with metastatic breast cancer, whosetumors demonstrated in vitro reactivity with L6, hadclinically measurable tumor responses to 131I-ChL6

Fig. 2. Whole-body clearances (s, PC3; d, DU145) and bloodclearances (h, PC3; j, DU145) of 125I-L6, as seen in PC3 andDU145 tumored nude mice (mean ± standard deviation). Similarblood and body clearances were demonstrated in the tumoredmouse models.

Fig. 3. Biodistribution of 125I-L6 in prostate cancer-bearing nudemice. The percent injected dose/g (%ID/g) of 125I-L6 was similar inPC3 (h) and DU145 (j) tumored nude mice (mean ± standarddeviation). Tumor targeting was demonstrated, with 10.6% and9.5% for DU145 and PC3, respectively. Similar concentrations innormal organs were seen in the DU145 and PC3 tumored mousemodels. Substantially more targeting of tumor was demonstratedthan localization in normal organs.

TABLE III. Ratios of 125I-L6 Tumor Uptake to 125I-L6Uptake in Normal Organs of PC3 and DU145

Tumored Mice

Tumor:normal tissuePC3

tumored miceDU145

tumored mice

Tumor:blood 1.6 1.5Tumor:liver 3.5 3.5Tumor:spleen 2.0 1.9Tumor:lung 2.7 2.9Tumor:kidney 5.0 5.3

L6 Antibody Binds Prostate Cancer 95

following L6 or ChL6 preload [15]. The infusion of L6and ChL6 induced an inflammatory response at thetumor site, enhancing delivery and efficacy of the sub-sequently administered radiolabeled antibody [16].Because of the biologic activity of L6 and the increasedvascular permeability at the tumor site, a higher per-cent ID localized to the tumor and bound specificallyto tumor antigens. Since the biologic behavior of L6and its therapeutic potential have already been deter-mined in breast cancer patients [13,15,16], it was rea-sonable to proceed with preliminary studies in pros-tate cancer animal models.

Molecular mechanisms of tumor cell killing in-duced by RIT may be important for prostate cancertherapy. Low-dose-rate irradiation (5–25 rad/hr), asdelivered by targeted MoAb therapy, may enhancetumor-cell sensitivity to programmed cell death [27].We studied genetic expression of apoptosis-relatedgenes in HBT-3477 xenografts in nude mice treatedwith 90Y-DOTA-peptide-ChL6 [28,29]. Expression pat-terns of TP53 and PIC1 mRNA observed in the xeno-grafts soon after treatment heralded tumor regression;this tumor regression seemed, at least in part, the re-sult of an apoptotic cell-death pathway. Analysis bypoly-ADP-ribose polymerase (PARP) cleavage, per-formed on tumor cells from HBT-3477-bearing nudemice treated with 90Y-DOTA-peptide-ChL6, revealeda fragmentation pattern characteristic of apoptosis[30]. These data also indicated that genetic responsesoccurred when relatively low radiation doses were de-livered continuously, as in RIT. Given the lack oftherapeutic efficacy of unlabeled L6 or ChL6 in miceand patients, the ability of radiation to induce apop-tosis [27], and the continuous low-dose-rate radiationcharacteristic of RIT [31], it is most likely that radiationrather than factors unique to a particular antibody wasthe cause of apoptosis.

CONCLUSIONS

This study showed that the L6 MoAb binds to anepitope on human prostate cancer cell lines in vitroand in vivo. RIT employing the L6 MoAb or its hu-manized counterpart chimeric L6, alone or in combi-nation with other agents, may be an effective new mo-dality for treatment of metastatic prostate cancer. Thegood binding and specificity demonstrated by L6,along with its ability to activate antitumor biologicalresponses, make L6 a good candidate for use in an RITclinical trial.

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