Proc. Nati. Acad. Sci. USAVol. 83, pp. 8694-8698, November 1986Immunology

Regression of cutaneous metastatic melanoma by intralesionalinjection with human monoclonal antibody to ganglioside GD2

(immunotherapy)

REIKO F. IRIE* AND DONALD L. MORTONDivision of Surgical Oncology, John Wayne Clinic, Armand Hammer Laboratories, Jonsson Comprehensive Cancer Center, University of California School ofMedicine, Los Angeles, CA 90024

Communicated by S. Hagiwara, July 18, 1986

ABSTRACT In this study we used human monoclonalantibody (Hu-mAb) L72 as an intratumoral injection of cuta-neous metastasis of melanoma to study its anti-tumor effects inhuman patients. Hu-mAb L72 was developed by transformingperipheral blood lymphocytes from a melanoma patient in vitrowith the Epstein-Barr virus, forming a human lymphoblastoidcell line that produces 2-5 gig ofIgM per ml. This IgM Hu-mAbwas shown to react specifically with ganglioside GD2 and havea strong cytotoxic effect on human melanoma cells in thepresence of complement. Patients with cutaneous metastaticmelanoma were given intralesional injections on a daily orweekly injection schedule. Regression was seen in all tumorsexcept in those of two patients whose tumors were shown tohave low antigenicity. Histopathological data showed tumordegeneration, fibrosis, free melanin, and some degree oflymphocyte or macrophage infiltration. One patient withmelanoma satellitosis treated with Hu-mAb showed completeregression with no sign of recurrence 20 months after the initialtreatment. With the exception of mild erythema, no side effectswere observed in any patient.

Immunotherapy has been a fast-evolving area of cancerresearch since the development of the first murine monoclo-nal antibody. These highly specific tools have permittedspecific lysis of malignant cells without the destruction ofnormal cells. However, the clinical application of the murinemonoclonal antibody has limitations. Patients inoculatedwith mouse monoclonal antibody may develop anti-mouse Igantibodies and side effects, including hypotension, dyspnea,fever, nausea, chills, diarrhea, bronchospasm, tachycardia,pruritus and urticaria, have been observed. Although somepatients, particularly those with advanced disease, have notdeveloped antibodies to murine monoclonal antibodies, theseside effects have made prolonged treatment with this anti-body difficult. Considering these limitations of murine mono-clonal antibodies it is only a natural progression to developanti-tumor monoclonal antibodies of human origin and in-vestigate these antibodies in passive immunotherapy. Wehave successfully developed a human monoclonal antibody(Hu-mAb) (L72) of the IgM class that specifically reacts withganglioside GD2 (1, 2). This antibody is secreted by humanlymphoblastoid cells that were derived from peripheral bloodB lymphocytes (PBL). The PBL were transformed in vitro byEpstein-Barr virus (EBV) and produce 2-5 ,ug/ofIgM per ml.The antibody has a strong cytotoxic effect on human mela-noma cells in the presence of complement (3). Its in vivoanti-tumor effects were demonstrated by using the athymicnude mouse model (4, 5).

In this study Hu-mAb L72 was tested as an intralesionalinjection into cutaneous nodules of recurrent malignantmelanoma to study its anti-tumor effect in patients. Eight

patients were so tested. Cutaneous melanoma was chosenbecause these tumors are easily measurable and 70% ofmelanoma patients have tumors expressing GD2 antigens (6).For patients with multiple tumors of similar size and depth,control injections were also given consisting of protein-adjusted human serum albumin or fresh autologous serum.Remarkable regression was observed in some Hu-mAb-treated tumors, without noticeable side effects. To the best ofour knowledge, the clinical application of Hu-mAb for thetreatment of cancer in humans has not been reported previ-ously.

MATERIALS AND METHODSHu-mAb to GD2. The human IgM monoclonal antibody

producing cell line L72 had been cultured in RPMI 1640medium containing 2-3% fetal bovine serum and antibiotics(streptomycin, penicillin, and Fungizone). Cell numberswere adjusted to 2 x 105 per ml every 3-5 days uponcollection of spent medium. After testing the antibody titer bythe immune adherence assay (1), spent medium from eachculture was pooled and centrifuged at 10,000 x g for 20 minto remove cell debris. Ten liters of cell-free spent mediumfrom the L72 culture was processed at one time for antibodypurification as described (3). The purified human IgM wasconcentrated to 5 mg/ml, filtrated through a 0.4-,um filter,and stored at -190TC until used. Sterility was tested byclinical bacteriology at the University of California, LosAngeles Medical Center.

Patient Selection. Eight patients participated in the project.All patients had been diagnosed as recurrent melanoma withcutaneous metastasis, had not received any other treatment14 days prior to their first injection, and had a performancestatus of 10 (Karnofsky scale). The study sample consisted ofthree males and five females, ranging in age from 31 to 89years. Tumor size ranged from 0.2 to 4.0 cm in diameter. Withthe exception of patients 1 and 4, all patients had receivedsome therapy or combination of therapy at some periodbefore the trials. No previous therapy was effective in anycase.

Administration of Hu-mAb. Variable amounts of Hu-mAb(5 mg of human IgM per ml in 0.9% saline) were injectedintralesionally. The antibody doses were determined by thesize of the tumor: 100 Ag of antibody per 1 mm of tumor(diameter). Injections were given daily or weekly with26-gauge needles and clinical response was determined 3 or4 weeks after the last injection.

Detection of GD2 on Biopsied Melanoma. The amount ofGD2 expressed by biopsied melanoma cells was measured

Abbreviations: Hu-mAb, human monoclonal antibody; EBV,Epstein-Barr virus; ShE, sheep erythrocyte(s).*To whom reprint requests should be addressed at: the Division ofSurgical Oncology, 9th Floor, Louis Factor Building, UCLA Schoolof Medicine, Los Angeles, CA 90024.

8694

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 83 (1986) 8695

before and/or after treatment by thin-layer chromatography(TLC) and TLC scanner of purified gangliosides from mela-noma tissues. The ganglioside fraction was isolated andpurified by the method described previously (6). The quantityof GD2 in the purified fraction was determined by photoden-sity metric scanning (high-speed TLC scanner) at 580 nm (7).The quantity of sialic acid in the entire ganglioside fractionwas determined by the resorcinol HCl method (Roseman'smethod) (8).

Circulating Anti-GD2 Antibody Levels. Sera of patientstreated with Hu-mAb were tested for anti-GD2 antibodylevels by two immunologic techniques, the immune adher-ence inhibition assay (2) and enzyme-linked immunosorbentassays (ELISA) (9).

Detection of Human Anti-Human Monoclonal Ig. The pro-duction of human antibody in the patient's serum that reactsspecifically with the Hu-mAb was tested by the passivehemagglutination assay using Hu-mAb-coated sheep eryth-rocytes (ShE) as targets. One percent solution of ShE thathad been fixed with 1% glutaraldehyde was mixed with ahalf-volume of 2 mg of purified Hu-mAb per ml in thepresence of bis-diazotized benzidine (10), incubated at 370Cfor 15 min, and washed. This Hu-mAb-coated ShE wasfurther incubated with 2% human serum albumin to block theprotein binding site on ShE, washed, and stored at 40C untilused. Patients' sera to be tested were preabsorbed by nativeShE and immobilized bovine serum to remove natural anti-bodies to ShE and natural or induced antibodies to bovineserum antigens. Hu-mAb-coated ShE were incubated withthe absorbed sera and the degree of reaction was tested byagglutination. To determine whether the reactivity was due toanti-idiotype internal image responses, an inhibition assay

using purified GD2 was performed on the positive sera.Control antigens included gangliosides GM2, GD3, and GM1.

RESULTS

Summary of Eight Patients' Responses to Hu-mAb Treat-ment. A total of 21 melanoma nodules developing in eightpatients was injected with Hu-mAb with or without comple-ment (fresh autologous serum). Seven nodules were injectedwith human serum albumin and two were injected withcomplement alone as controls. The size of the tumor nodulesinjected ranged from 2 x 2 mm to 34 x 40 mm in diameter.The antibody dose administered was dependent upon tumorsize and ranged from 80 gg to 2.5 mg per nodule injection.Antibody was given either weekly or daily, with an injectionfrequency of two to four times. Thus, a total dose adminis-tered per nodule ranged from 160 gg to 7.4 mg. Clinicalresponse was evaluated 4 weeks after the last treatment. Ofthe 21 nodules treated with antibody, 16 responded, 10completely regressed, 5 partially regressed, and 1 exhibiteda minor response. Three nodules stabilized and 2 did notrespond. None of the control groups, either receiving humanserum albumin or complement alone, responded to thetreatment, and all grew during the study. The results aresummarized in Table 1.To examine the differences in GD2 antigenicity on tumor

cells between patients, cutaneous melanomas were biopsiedfrom four patients before treatment and gangliosides werepurified and evaluated by using TLC and TLC scanner. Forpatients 8 and 3, who were responsive to immunotherapy,TLC clearly indicated the presence of GD2 (Fig. 1). Theamount was 5 and 4.3 nmol/g of wet tissues, respectively.

Table 1. Treatment of cutaneous recurrent melanoma with Hu-mAb to GD2 by means of the intralesional route

Patient Location of treated Antibody AdministrationNo. Age, years Sex melanoma nodules Size, mm dose, mg interval Response1 74 Female Lower leg 4 x 4 0.6 Weekly x 3 Stabilization

2 x 2 0.6 Weekly x 3 Complete3 x 4 0.64* Daily x 4 Complete3 x 3 0.64* Daily x 4 Complete3 x 3 0.64 Daily x 4 Complete3 x 3 0.64 Daily x 4 Complete122 10.9 Six injections Complete

nodulest in 15 days2 53 Male Neck 7 x 10 0.6 Weekly x 3 Stabilization

Scalp 16 x 17 2.4 Weekly x 3 Minor3 89 Female Foot 4 x 5 0.6 Weekly x 3 Partial

2 x 3 0.6 Weekly x 3 Complete2 x 5 0.32* Daily x 2 Complete5 X 5 0.32* Daily x 2 Complete3 x 3 0.16* Daily x 2 Complete

4 43 Female Chin 10 x 12 1.8 Weekly x 3 StabilizationRight axilla 34 x 40 6.0 Weekly x 3 Progression

5 76 Male Neck 20 x 40 7.4 Weekly x 3 Progression6 31 Female Left axilla 11 x 15 4.8 Daily x 3 and Partial

weekly x 17 35 Male Upper thigh 16 x 19 4.5 Daily x 4 Minor8t 77 Femalet Left axilla 12 x 15 1.7 Daily x 2 Partial

Upper thigh 15 x 17 4.4 Daily x 2 and Partialweekly x 1

Upper back 13 x 13 1.8 Daily x 2 PartialComplete response is defined as the complete regression of the tumor nodule. Partial response is a reduction of treated tumor by at least 50%.

Minor response is a reduction oftreated tumor by at least 25% in diameter (width x length/2) (but less than that for partial response). Stabilizationis objective change of treated tumor by <25%. Progression is the increase in size of treated tumor by at least 25%. Clinical response was evaluated4 weeks after the last injection. Side effects were not detectable.*Fresh autologous serum was admixed with antibody for complement supplement.t1-4 mm in diameter.tPatient 8 was followed only 10 days after the last treatment.

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GM3

GMI - _

GD

t

GD2 -

GTlb-

Stds Pt.8 Pt. 3 Pt.5 Pt. 4

FIG. 1. TLC of gangliosides purified from four cutaneous mela-noma specimens [patients (Pt.) 8, 3, 5, and 4] from left to right. Stds,standard gangliosides. The clinical responses of patients 4 and 5 wereminimal, whereas patients 3 and 8 responded remarkably to theHu-mAb treatment.

But for two patients, who were unresponsive to im-munotherapy (patients 4 and 5), TLC showed no sign ofGD2(Fig. 1).Most injected lesions were biopsied and subjected to

pathological examination 28 days after the last injection. Alltumors injected with Hu-mAb showed some degree of lym-phocyte infiltration, and degeneration occurred in all tumorsexcept those of patients 4 and 5. The lack of degeneration inthe tumors of patients 4 and 5 is consistent with the lowantigenicity observed with the TLC as described. Fibrosiswas evident in all tumors except those of patients 2 and 8,whereas necrosis was observed in all tumors except those ofpatients 4 and 5. The tumors that received control injectionsshowed typical dermal metastasis of melanoma and noincreased lymphocyte infiltration. No sign of degradationwas evident in any of these control tumors.A Complete Regression of Melanoma Satellitosis. Patient 1,

a 74-year-old woman, had been treated with a wide excisionof her primary melanoma (Clark's level III, 1.36 mm) on theanterior surface of her left lower extremity in March 1984 anda second wide excision from a recurrence adjacent to theprimary lesion in June 1984. The patient received no post-operative treatment. In October 1984, the patient developedmultiple cutaneous melanoma on her left lower leg around theprimary site (melanoma satellitosis). In November 1984, thepatient entered into the project. Initially two treatment planshad been scheduled. The first plan consisted of one injectioneach week for three times. The second was a single dailyinjection for 3 days. A third treatment schedule would bedetermined by using the results of the first two. Because ofthe extent of satellitosis, the patient had many lesions ofsimilar size and depth. With these similar tumors present,control injections were used to show any effects unrelated tothe immunologic tumor destruction by Hu-mAb.

Treatment 1. For the weekly injections the six largesttumors (0.2-0.4 mm) were chosen. Of these lesions, threewere used as controls and injected with human serumalbumin and three were injected with Hu-mAb. Two lesions,each injected either with Hu-mAb or human serum albumin,were biopsied on day 7 after the last injection for histopath-ological evaluation. The lesion injected with Hu-mAbshowed partial tumor necrosis and a high degree of infiltra-tion of lymphocytes and macrophages. No degradation oftumor cells or lymphocyte infiltration was seen in the controltumor. Twenty-eight days after the last injection the remain-ing four lesions were biopsied. Histopathological datashowed one of the experimental lesions contained no malig-nant cells and had a multinucleated giant cell response to

foreign material. The other experimental lesion and both ofthe control lesions showed typical cutaneous malignantmelanoma.

Treatment 2. The second treatment consisted of a singleHu-mAb injection each day for 3 days administered on days0, 1, and 4. For this treatment, eight of the largest tumors(0.2-0.4 mm) were selected and injected in pairs. Of thesetumors, two were injected with 640 pkg of Hu-mAb and twowere injected with 640 gg ofHu-mAb and complement (fromfresh autologous serum diluted 1:5). The remaining fourtumors were used as controls, with two injected with 640 Agof complement alone and two injected with 640 jug of humanserum albumin alone. After the second injection, all fourtumors injected with Hu-mAb had mild erythema and edemaand were slightly ulcerated. The control lesions were stillintact and showed no sign of regression. On day 4, theHu-mAb-injected lesions had developed eschars, whereasthe control tumors continued to show no sign of regression.On day 24, all eight tumors were biopsied. Histopathologicaldata showed no evidence of melanoma in the four Hu-mAb-treated lesions. The lesions contained fibrosis, telangiectasis,free melanin, and scattered lymphocyte infiltration. In con-trast, tumors treated with human serum albumin or comple-ment exhibited a typical cutaneous malignant melanoma (Fig.2). The results of treatment 1 and 2 are summarized in Table2.

Treatment 3. During this 3-month experimental period(November 1984-February 1985) lesions untreated grew andmany new lesions appeared. A total of 122 lesions >1 mm indiameter was counted, with :60 more lesions <1 mm also inthe same area (Fig. 3A). With the results oftreatment 2, it wasdecided that the remaining tumors >1 mm (122) would beinjected on a daily basis. Depending on the size of the tumor,between 10 and 60 1Lg ofHu-mAb was injected intralesionallyfor 3 days (days 0, 1, and 4), with some larger tumorsreceiving an additional three injections on days 11, 12, and 14.Total antibody dose reached 10.9 mg. By day 14, many smalllesions had disappeared, and larger tumors had flattened orhad eschars (Fig. 3B). Even the tumors that were <1 mm indiameter that had not been injected were regressing. With theexception of mild erythema, no side effects were observed.On day 26, all eschars were gone and the tumors remainedflattened (Fig. 3C). On day 42, a biopsy was taken of threerepresentative lesions. Histopathological examinationshowed no tumor. Currently (July 1986), the patient is in goodhealth and has not shown any sign of relapse.Anti-GD2 Antibody Titer Analysis. All patients' sera were

tested for anti-GD2 antibody response throughout the trialperiod. No significant increase in antibody titer was observedin any patient at any time during the trials.

Side Effects. Although mild erythema was present at theinjected area in some cases, no other side effects weredetected. No patients had nausea or fever or complained ofany pain during the treatment.Anti-Human Ig Responses. Sera preabsorbed (as described

in Materials and Methods) were diluted at 1:10 and 1:40 andtested against Hu-mAb-coated ShE. The sera from fivepatients gave either a mild or strong agglutination in thepassive hemagglutination assay during the course of treat-ment. When these sera were retested after preincubation ofHu-mAb-coated ShE with purified GD2 antigen, two of thefive sera became negative in passive hemagglutination assay,indicating that these two sera were directed against anti-idiotype antibodies of internal image of the antigen GD2 (11,12). These responses occurred within 1 and 3 weeks after theinitial injection of Hu-mAb. The reactions between Hu-mAb-coated ShE and the remaining three sera were not inhibitedby preincubation of Hu-mAb-coated ShE with GD2; theresults indicate that internal images of GD2 are not involved

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B

''"

Z,

-.M r

T~J

d'.~~~~~*~~~~~~4 4"~ ~ ~ ~ ~ ~ ~ ~ IN

FIG. 2. Tissue sections of involved area. Biopsies were taken 20 days after the last Hu-mAb injection (24 days after the initial injection) (B)and human serum albumin injection (A). Tissues were stained with hematoxylin/eosin. (x22.4.)

in the reactions. These observations will be the subject of amore extensive report.

DISCUSSIONIf the concept of an "ideal" anti-tumor therapy had to bedefined, the definition would probably include ". . . a tumor-specific therapy that has no side effects for the patient."Hu-mAb to GD2 may fulfill that definition because it appearsto have destroyed a malignant tumor without serious sideeffects. In this pilot study we demonstrated the anti-tumoreffects of anti-GD2 Hu-mAb in patients with recurrentcutaneous melanoma. With the exception of mild erythema,

Table 2. Treatment of melanoma satellitosis by intralesionalinjection of Hu-mAb to GD2

Lesions regressed/Injection interval lesions tested

Treatment 1Hu-mAb Weekly x 3 2/3*HSA Weekly x 3 0/3*

Treatment 2Hu-mAb Daily x 3 2/2Hu-mAb + C Daily x 3 2/2C Daily x 3 0/2HSA Daily x 3 0/2

Treatment 3tHu-mAb Daily x 3-6 122/122-

HSA, human serum albumin; C, complement.*One of the lesions was evaluated 7 days after the last injection.tAll tumors received three injections except for some larger tumors,which were injected an additional three times.tComplete regression.

no adverse effects were detected in any of the eight patients.A maximal dose of 14.7 mg was given.To compare different injection schedules, two patients (1

and 3) were given weekly and daily injections. Althoughtumors treated on both schedules regressed, it was apparentthat the daily injections accelerated tumor destruction. Afterobserving both schedules it appeared that the weekly injec-tions allowed the tumor time to grow. Because this was a pilotstudy, we did not attempt to detect the optimal dosage ofantibody in relation to tumor size or antigenic expression.However, we have observed in one patient that 320 4g ofHu-mAb was sufficient to eradicate a 5-mm-sized melanomaexpressing 4.3 nmol of GD2 per g of wet tissue.

Before starting this project many studies were done toensure that the anti-tumor effect (or lack of) was a directresult of the Hu-mAb. Two examples of preclinical trialsinclude (i) an experiment showing that antibody withoutcomplement did not have any anti-tumor effect in vitro (3, 4)and (ii) a series of trials confirming that melanoma cell linesexpressing no or mimimal GD2 were not affected by theantibody/complement in vitro as well as in in vivo experi-ments using nude mice (4, 5). These two tests exclude thepossibility that the antibody itself destroys the tumor wheninjected intralesionally and they exclude the possibility thatthe antibody is binding nonspecifically. Also, to investigatewhether the injection itself or the protein in the serum was

responsible for destruction of the tumor, controls were

established when many similar tumors were present. Thesetumors were unaffected by the control injections.Because of the specificity of Hu-mAb, it would be prefer-

able to know the antigenicity of tumors prior to treatment.Predetermination would allow us to manipulate differentcombinations of Hu-mAb to conclusively show their speci-

A

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FIG. 3. A patient with melanoma satellitosis was treated withHu-mAb to GD2 by means of intralesional administration. (A) Threedays before treatment (February 4, 1985). (B) February 22, 1985. (C)March 24, 1985. (D) July 1, 1985. The patient is currently (July 1986)free of disease.

ficity. In these initial clinical trials the anti-tumor effect ofHu-mAb to GD2 proved to be quite effective, but the dataclearly indicate that all tumors did not respond identically tothe Hu-mAb. Though only 70% of all melanoma tumorsexpress GD2, GD2-negative melanoma could express theother immunogenic ganglioside, GM2 (6). Virtually all biop-sied melanomas express either GD2 or GM2. Since humanmonoclonal anti-GM2 antibody has been produced by EBVtransformation techniques (13), a combination therapy ofanti-GD2 and -GM2 Hu-mAb should be considered to en-hance the therapeutic effects on melanoma.As noted previously, not one patient had any significant

side effects during or after the treatment. This was probablynot only due to the small doses tested but also to the specificbinding capacity that the Hu-mAb possesses. In fact, manypatients with melanoma possess natural or induced antibod-ies to GD2 without suffering from any autoimmune disease orneurologic symptoms (14, 15). This observation suggests thatlarger doses of Hu-mAb to GD2 can be safely given system-ically to patients, to prevent hematogenous or lymphaticmetastasis or to suppress the growth of established tumor.We have shown that this IgM antibody can bind to melanoma

cells grown at various sites in nude mice (16, 17). Systemi-cally injected Hu-mAb exhibited anti-tumor effect on estab-lished melanoma nodules in nude mice as well (5). Inaddition, if larger dose-systemic injections are given, theymight induce a high idiotypic antibody response that couldstimulate the third antibody production that is directedagainst GD2 on melanoma. The reason(s) for the regressionofuninjected lesions in patient 1 during the course ofthe thirdtreatment is unknown at this time. The most likely explana-tion is due to diffusion of antibody through the skin from theinjected to uninjected lesions. However, another possibleexplanation may relate to the formation of an anti-idiotyperesponse against our Hu-mAb, with a resultant secondaryanti-tumor effect.

This initial series of clinical trials using Hu-mAb in anti-tumor therapy is encouraging. These results will hopefullylead to increased development of Hu-mAb-producing celllines that will allow us to test the therapeutic effectiveness ofsystemic administration of multiple monoclonal antibodiesagainst defined antigens. Antigenic typing of each patient'smetastatic melanoma will make possible the selection ofcombinations of monoclonal antibodies for more specificindividualized therapy.We thank Lan Sze and Ming Jien for their assistance with the

antibody production and their excellent technical support, AnneNizze for antibody injection and tumor measurement, and Drs.Armando Guiliano, Barry Mann, and James Huth for the clinicalfollowup. We also express our appreciation to Dr. Alistar Cochranand the staff of the Department of Pathology at the University ofCalifornia, Los Angeles, for their pathological evaluation, Dr.Mitshuriro Hachidafor his independent pathological evaluation, Drs.Tetsuya Tsuchida and Iwao Ando for their ganglioside analysis andevaluation, Ms. Jane Shaw for the photography, and Peter Chandlerfor his assistance in the preparation ofthe manuscript. This work wassupported by Grants CA29605, CA30647, and CA12582, awarded bythe National Cancer Institute, Department of Health and HumanServices, by the Joyce and Ben Einsenberg Foundation, and byFriends of the John Wayne Clinic and Wayne Enterprises.

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