(CANCER RESEARCH 49, 7045-7050. IX-cvmbcr 15. I9H9]

Characterization of IgG and IgM Antibodies Induced in Melanoma Patients byImmunization with Purified G>i2Ganglioside1

Philip O. Livingston,2 Gerd Ritter, Pramod Srivastava,1 Maureen Padavan, Michele J. Calves, Herbert F. Oettgen,

and Lloyd J. OldMemorial Sloan-KelteriiiK Cancer Center, .Vir York, \ew York 10021

ABSTRACT

The ganglioside (.%, is a differentiation antigen expressed on the cellsurface of human malignant melanomas and other cancers of neuroecto-dermal origin. We have previously reported that immunization withpurified GM¡combined with Bacillus Calmette-Guérinas adjuvant andpretreatmcnt with low-dose cyclophosphamide induced production ofantibodies against ( •¿�•>,•¿�in five of six patients. \Ye have now extended ourstudy and analyzed the induced antibody response in greater detail. Priorto immunization, high-titer antibodies against GM..were not detected.After immunization, high-titer IgM antibodies were induced in 17 of 24patients, and high-titer IgG antibodies in eight cases. Additional treatment of 12 patients with cimetidine, a histamine H2 receptor antagonistreported to have antisuppressor cell activity, had no effect on GM;antibody titers. Antibodies against asialo-G^ were present in all patients,and antibodies against (.%,, were present in 33% of patients, before andafter immunization. Antibodies induced by immunization were specificfor <IM•¿�-though some reacted predominantly with /V-acetyIneuraminicacid GMZ(<-M•),and some reacted with ».M/and ¿Y-glycolylneuraminicacid ( IM.(\eu( .<•<.x, ). The pattern of reactivity with <.M is consistentwith the response to T-cell-independent antigens: both IgM and IgGantibody responses against GM: were short lived; peak titers seen afterinitial and secondary vaccinations were similar; and delayed-type hyper-sensitivity responses to skin test challenges with (,%,. were not detectedin any patients. However, the IgG response typed as predominantly IgGland IgG3, not IgG2 as might be expected for carbohydrate antigens(which are generally T-cell-independent antigens). Because IgGl andIgG3 antibody responses are usually to T-cell-dependent antigens, thehumoral immune response elicited by <>M vaccination has both T-cell-dependent and T-cell-independent characteristics. These IgM and IgGresponses against this ncuroectodermal differentiation antigen expressedon melanoma cells have been induced without evidence of neurological orother toxicity.

INTRODUCTION

The gangliosides Gi>.,,4Gn:, and GM: are expressed on the

cell surface of human malignant melanomas. Treatment withmonoclonal antibodies against <¡,,;and Gl>: has resulted inregression of melanoma métastases( 1-3), suggesting that thesecell surface gangliosides are potential targets for melanomatherapy. In our attempts to develop immunogenic cancer vaccines, we have recently focused on GM: because (a) its distribution on various cell lines [defined by anti-GM; monoclonalantibody 5.3 (4)] and in various normal tissues (defined byextraction and thin-layer chromatography) was more restricted

Received 4/6/89: revised 8/28/89; accepted 9/18/89.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.

1This work was supported by grants from the Perkin Foundation, the DiamondFoundation, the National Cancer Institute (CA-40532. CA-08748). and a fellowship from the Deutsche Forschungemeinschaft (R:483/l-l).

2To whom requests for reprints should be addressed.3Present address: Department of Pharmacology. Mount Sinai School of

Medicine. City University of New York. New York. New York 10029.4The abbreviations used arc: GD1 (and other gangliosides) according to the

system of Svennerholm (30); BCG. Bacillus Calmette-duerin; ELISA, enzyme-linked immunosorbent assay; Cy, cyclophosphamide; Ci, cimetidine: DTH. dc-laycd-typc hypcrsensilivity (skin tests); i.d.. intradermally: NeuAcGM¡./V'-acetyl-neuraminic acid GM2:NeuGcGM:. .Y-glycolylneuraminic acid M,; NGcGM¡.

than G|)2 or GDI (4), (b) immunization with whole cells expressing a high level of GM: induces GM: antibodies in some patients(5-7), and (c) we were able to prepare purified GM: in sufficientquantity for vaccine construction and testing. Based on preclin-ical studies (8), we conducted sequential trials in small groupsof patients with Stage III melanoma indicating that BCG wasa highly effective adjuvant, and that patients pretrcated withlow-dose Cy developed higher titers of anti-G\T: antibody thanthose not receiving Cy (6, 9). The antibodies induced werepredominantly IgM antibodies capable of lysing G^-expressinghuman tumor cells in the presence of human complement.High-titer IgG antibodies against GM: were not detected.

We have now extended vaccination with the Cy-BCG-GM:vaccine to a larger group of patients, defined the specificity andsequential pattern of induced antibodies in greater detail, andidentified high-titer IgG antibody against G\l:, in addition toIgM antibody, in the serum of some patients. Unexpectedly,the predominant subclass of these IgG antibodies was not IgG2,the subclass usually associated with carbohydrate antigens, butIgGl andIgG3.

MATERIALS AND METHODS

Patients. Patients with melanoma métastasesrestricted to regionalskin and lymph nudes were considered eligible if the skin métastasesand regional lymph nodes had been resected within the last 4 mo andif they were free of detectable melanoma. None of the patients hadreceived prior chemotherapy or radiation therapy. Patients were examined at 6-wk intervals, including neurological examination, and chestX-rays, liver function tests, and urinalysis were performed at 3-mointervals. Blood for serological studies was obtained at 2-wk intervals.

Gangliosides. NeuAc GM; (GM2)and GD; were prepared by treatingCM, and Goib with /i-galactosidase (G. W. Jourdian, University ofMichigan. Ann Arbor, Ml) according to published methods (10). GM,was purchased from Supelco(Bellefonte, PA). NeuGcGM: was extractedfrom C57BL/6 mouse liver as previously described (4). GI>, was a giftfrom Fidia Research Laboratories (Abano Terme. Italy), and GDIHwaspurchased from the same source. GM, was purified from dog crythro-cytes as previously described (4).

Serological Procedures. The ELISA for GN1;antibodies (4) was performed with rabbit anti-human IgM. anti-human IgG, or Protein Aconjugated with alkaline phosphatase (Zymed Laboratories, San Francisco, CA). Readings obtained with normal sera were subtracted fromreadings obtained with the patients' sera before and after vaccination.

Antibody titer was defined as the highest serum dilution yielding acorrected absorbance >0.190. IgG and IgM antibodies were separatedon DEAE-Sepharose columns equilibrated with 100 niM sodium phosphate buffer, pH 6.8; antibodies were eluted with O.I M NaCI in thesame buffer, and on Sepharose CL-7B gel filtration columns in phosphate-buffered saline containing 0.35 M additional salt (final NaCI)concentration, 0.5 mol). Fractions were tested by ELISA for antigenbinding activity as well as antibody class. IgG subclass determinationwas performed with subclass-specific murine monoclonal antibodiespurchased from AMAC, Inc. (Westbrook, ME). ICN Biochemicals.Inc. (Irvine, CA), Southern Biotechnology Associates (Birmingham.AL), or kindly provided by Dr. Mitchell Scott (M. S.) (WashingtonUniversity School of Medicine. St. Louis, MO). These contained 1 mg/ml except in the case of HP60I6 which contained 37.3 mg/ml. The

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INDUCTION AND CHARACTERIZATIONOF GM! ANTIBODIES

Table 1 SeroloKic response in patients receiving ( y plus H( 6-6'M;.-summary of ELISA and immune slain results

Treatment"100

/¿gIO«MB100

rt100f|100m100„¿�g300Mg.100„¿�B.100MB.100Mg.100

Mg.100

MB200Mg/Ci200Mg/Ci200Mg/Cia»Mg/ci200Mg/C1200MB/Ci200Mg/Ci200Mg/Ci200MB/Ci200Mg/C¡200Mg/Ci200

Mg/CiPatient

no.1234567X•1IIIII121314IS1617IX192021222324ELISA(anti-GBeforevaccinationIgM200001000104004002001010001000000IgG0(III(I00000000000000000000results

M2tiler)Immune

slain results"' *

(IgM/IgG)After

vaccinationIgM80160SO20320801604080206401601608080400160160SOso(116(10IgG20SO10(140160001602008016001600101600000800GM22+.1+/2+1

+0342-H/2+3+02+/2+03+3+/2+3+/I

+3+2+/3+002+/.1+2+3+2+0I+/2+0NGcGM21

+3+003+1+/1

+2+(12+/1

+02+3+/1

+2+3+2+002+/2+2+1

+2+(1(I0CM,.1+0002+0(10II01+01+1+1

+2+0(12+000(I0GDJ000II1+01+(I0(10(I1+1+000001+000

" All patients »erepretreated with cyclophosphamide and received BCG-GM2 vaccines with the indicated quantity of GM2. Some patients also received p.o. Ci

during the initial immuni/ations. as indicated.* Dot blot immune stains graded as O, 1+, 2-f, or 3+ as indicated in Fig. 2.

12 16 20 24 28 32

WEEKS

Fig. I. IpM and IgCi antibody responses in melanoma patients after immunization with purified GM:-BCG vaccines. Curves for the 8 patients with IgG responsesare shown. Arrmrs indicate time of C'y and vaccine injections (all patients), and line indicates duration of Ci treatment (Patients 13 to 24).

lowest dilution producing no reactivity with pretreatment or otherknown negative sera was used (specified in Table 2). Goat anti-mouseIgCìconjugated with alkaline phosphatase (Zymed Laboratories, Inc.,San Francisco, C'A) was used as the third antibody at a dilution of

1:200. Reagents for dot blot immune stains (11, 12) were peroxidase-conjugatcd goat anti-human IgM and goat anti-human IgG (Tago,Burlingame. CA) diluted 1:500. Dot blots were graded as negative, 1+,2+, or 3+ as indicated in Fig. 2.

Skin lests for DTI I. Six. 12, and 25 ¿<gof CM?suspended in 0.05 mlof normal saline were injected i.d. Skin tests for DTH against mumpsantigen, tuberculin purified protein derivative, and other recall antigenswere performed and interpreted as previously described (13, 14).

BCG-GM: Vaccine. Viable units of BCG (IO7, Tice strain; Universityof Illinois. Chicago. IL), or 3 x 10" units in the case of patients showing

strong reactions to BCG, were suspended in distilled water by sonicationand added to tubes containing 100. 200. or 300 /jg of dried purifiedNcuAcGv,,. The suspension was lyophiliz.cd. and the residue was suspended in phosphate-buffered saline shortly before vaccine administration. Following this procedure, all GM: was attached to BCG, presumably by hydrophobic bands, as we have previously reported (8). Patients

received three or four i.d. vaccine injections into extremities with intactlymphatic drainage at 2-wk intervals on a rotating basis. In addition, abooster injection was administered 2 to 3 mo after the initial series ofvaccinations to all but three patients whose melanoma had recurred atthat time.

Cy and Ci Administration. Cy (Cytoxan; Mead Johnson and Co..Evansville, IN) (200 mg/m2) was administered i.v. to all patients 4 to

6 days prior to the first vaccine injection. Ci (Tagamet; Smith, Kline &French, Cidra, PR) (200 mg 3 times daily plus 400 mg at bedtime) wasadministered p.o. to 12 patients beginning 5 days before the firstimmunization and continuing until 2 wk after the third immunization.

RESULTS

Vaccine Administration and Side Effects. Twenty-four patients were immunized with the BCG-GM; vaccine. The initialtwo groups of six patients each received 4 vaccinations containing 100 (this group has been reported previously, Ref. 6) or 300/jg of GM: at 2-wk intervals. As no difference in the serological

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INDUCTION AND CHARACTERIZATION OF GM! ANTIBODIES

STD j Immune Stain

H I! ,1STD ImmuneStain

GM3

ASIALOGM2

GM2

NGCGM2

GM1

GD3

GD2

2 6 9 12 13 15 18 23

Patient NumberFig. 2. Detection of GM; antibody in sera from 8 vaccinated melanoma patients

by dot blot immune staining. Ganglioside standards were applied to silica gelstrips and stained with oroinol/sulfuric acid or to nitrocellulose strips and allowedto react with sera from individual patients and pcroxidasc-labclcd goat anti-human IgM antibody. Reactions are graded as follows: Patient 2: GMj. 3+;NeuGcGw!. 3+: Patient 6: GM¡.2+; NeuGcGM2. l+: Patient 9: GM2. 2+: Neu-GcGM2, 2+; Patient 12: GM2. 3+; NeuGcGM¡.3+; Patient 13: GM2, Ì+:Neu-GcGM2. 2+; GM,, 1+; Patient 15: GM2. 2+; NeuGcGM¡.2+; GMi. l+: GDI, 1+:Patient 18: GM2,2+; NeuGcGM2. 2+: Patient 23: GM2. 1+. Sera from all patientsreact with asialo-GM2. The trace-positive spots seen with GI».(here are assumedto be artifactual as they are not consistent findings, they cannot be inhibited withGDI. no Gm reactivity is seen by immune thin-layer Chromatograph}1 on silicagel. and KI.ISA tests on {;,,, and melanoma cells expressing GT),are negative.

response was detected between the two doses, and no increasein serological titer occurred after the fourth vaccination (asopposed to the subsequent booster vaccination), the remaining12 patients received three vaccinations containing 200 ¿igofGM: at 2-wk intervals. In addition, these 12 patients weretreated with Ci as described above. No side effects were observedas a consequence of Cy or Ci administration. Nine of 12 patientsreceiving Cy-BCG-GM? and 6 of 12 patients receiving Cy plusCi-BCG-GM: experienced low-grade fever (less than 39°C)and/

or marked local ulcérationrequiring a decrease in the dose ofBCG to 3 x IO6viable units for the last vaccine injection. No

other side effects were observed. In particular, there were noneurological abnormalities.

Absence of DTH to GM2. Skin tests for DTH to GM: andrecall antigens were performed after the initial series of threeto four vaccinations. All patients were reactive with at least onerecall antigen, and all developed prominent DTH reactions toBCG, but none showed delayed hypersensitivity to GM2.

Antibody Response to GMJ Vaccination. Prior to vaccination,GM2antibodies were detected only rarely in the patients' serum

and at low titer, 1:40 and 1:20 in two cases each. The remaining20 prevaccination sera showed antibody titers of 1:10 or less.The results of ELISAs on postvaccination sera are summarizedin Table 1. High-liter (1:80 or greater) IgM antibodies weredetected in the sera of 9 of 12 patients receiving Cy-BCG-GM2and of 8 of 12 patients receiving Ci plus Cy-BCG-GM2. Threeof the 7 patients who did not produce high-liter GM2antibodies

(Patients 8, 10, and 24) received only the initial series ofimmunizations but no booster immunizalion because diseaseprogression required olher irealmenl. High-liler IgG antibody

GM3

AsíaloGM2

GM2

NGcGM2 •¿�

•¿�IIGM3 Asialo GM2 NGc None

GM2 GM2

InhibitingGangliosidesFig. 3. Analysis of GM2specificity of sera from Patient 2 by dot blot immune

stain inhibition. Ganglioside standards were applied to silica gel strips and stainedwith orcinol/sulfuric acid, or to nitrocellulose strips and allowed to react withsera (None) or sera that had been mixed with 10 /jg of GM1.asialo-GM2. GM2.orNeuGcGM2. Dot blots were stained with peroxidase-labeled goat anti-human IgMantibody. GMI reactivity was inhibited only by GM2.and NcuGcGM2 reactivity wascompletely inhibited by GM2and in this experiment only partially by NeuGcGM2.Asialo-GM! reactivity was inhibited only by asialo-GM2-

was detected in 4 of 12 patients in each group. Although thenumbers of patients are small, GM: doses of 100 and 300 ngper vaccine appeared to be equally effective in inducing antibodyproduction. Similarly, addition of Ci did not result in a consistent increase in IgM or IgG antibody tilers. Sequential IgMand IgG GN12antibody tilers for Ihe eighl patients producinghigh-liter IgG antibodies are shown in Fig. 1. Although high-tiler IgG anlibodies were reslricled lo palienls who also produced high-liter IgM antibodies, a consisteni lemporal relalion-ship belween IgM and IgG lilers was noi seen. In mosl cases,IgG anlibodies were delecled after Ihe fourih (boosler) immunizalion, bul lwo palienls (Palienls 15 and 18) produced high-liler IgG anlibodies before high-liler IgM anlibodies were de-

leclable.Specificily Analysis of GMZAntibodies. Pre- and poslvaccin-

alion sera from all patients were tesled for reaclivily againsl apanel of gangliosides including GM,, NeuAcGM2, NeuGcGM:,asialo-GM2, GMI, GI>.,,and Gi>2in dol blol immune slains. NoGM: reaclivily was delecled prior lo immunizalion. Sample dolblol immune slains for IgM anlibodies wilh sera from ihe eighlpalients who showed both IgM and IgG reactivily againsl GM2are shown in Fig. 2, and ihe results of post immunizationimmune slains wilh sera from all patients are summarized inTable 1. No reaclivily wilh GMI or GD, was delecled. All serawilh GM2anlibody lilers of 1:80 or grealer by ELISA were alsoreaclive by immune slain. Several sera were also reaclive wilhGMI or GD2, and all sera were reaclive wilh asialo-GM2. bulihese anlibodies were noi induced by Ihe immunizalion. Theywere shown lo be presenl prior lo immunizalion.

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INDUCTION AND CHARACTERIZATION OF GMJ ANTIBODIES

IgG IgM IgG

V> -a

C>J O)E ~ '"

? 5

a.o41DC

Serum from patient 3

X Serum from patient 5

B 10 0 10 20 30 40 SO 60

Fraction Number

DEAE-Sepharose Sepharose CL-4B

Fig. 4. GM! reactivity of sera from Patients 12 and 18 after separation intoIgM and IgG fractions. Sera were passed over DEAE-Sepharose or SepharoseCL-4B columns to separate IgM and IgG fractions by charge and size, and thentested on purified GMj by ELISA. GM¡reactivity of sera from Patients 12 and 18resides in both IgG and IgM fractions.

Inhibition tests were used to analyze the specificity of vaccine-induced GM2antibodies in more detail. A representative experiment using the immune stain procedure is shown in Fig. 3.The major reactivity is with NeuAcGM2, with some reactivityagainst NeuGcGM2. While in this particular experiment Neu-GcGM2 reactivity was only partially inhibited by NeuGcGM2, inother experiments this inhibition was complete, while GM2reactivity was unchanged or only slightly diminished. TheseGM2antibodies did not react with GM,, GD2, asialo-GN12,or anyother ganglioside. Results of dot blot inhibition tests showedthat the sera of all of the 17 patients with IgM NeuAcGM2

antibodies also reacted with NeuGcGM2. In each case, Neu-AcGM: and NeuGcGN,: reactivity was completely inhibited byNeuAcGM2, but NeuAcG^ reactivity was not inhibited or onlypartially inhibited by NeuGcGM2 (Fig. 3). This observationsuggested two possible explanations. One possibility was thattwo populations of antibodies with different specificities wereinvolved, one restricted to NeuAcGM2 and the other includingboth NeuAcGM2 and NeuGcGM2. Alternatively, it was possiblethat all induced antibodies had the same specificity which hada higher affinity for NeuAcGM2 than NeuGcGM2. To addressthis issue, quantitative inhibition tests were performed with serafrom four patients. Complete inhibition of IgG and IgM reactivity with GM2 was obtained with 1 ng of GM2 but was notobtained with 10 /¿gof NeuGcGM2 (data not shown), suggestingthat some of the induced antibodies had a very low affinity forNeuGcGM2 or were specific for NeuAcGM2 at the exclusion ofNeuGcGM2.

Subclass Identification of IgG Antibodies against (¡\,:.Foursera containing IgG antibodies to GM2as shown by ELISA weresubjected to fractionation by size and charge on Sepharose CL-6B and DEAE-Sepharose columns. A representative experiment involving two of the sera is shown in Fig. 4. Both seracontained IgG and IgM antibodies which could be separated onthe basis of size and charge. The two other sera were tested inthis way with similar results. All eight sera containing IgGantibodies against GM2were tested by ELISA using a panel ofIgG subclass-specific monoclonal reagents as second antibodies.The results are shown in Table 2. The IgG antibodies in theserum are either predominantly of the IgGl subclass (Patients6, 13, and 15), or predominantly IgG3 (Patients 2 and 9), orIgGl and IgG3 (Patients 18 and 23), or IgGl, IgG2, and IgG3(Patient 12).

Correlation of Antibody Titer with Prognosis. We have previously reported that evaluation of 31 melanoma patients vaccinated with various GM2vaccines 15 mo after vaccination showeddelayed recurrence of melanoma in patients developing GM2antibody as compared with patients who did not produce antibodies in response to vaccination (6). We have now followed66 patients for at least 3 yr (Fig. 5). Evaluation at 3 yr showsthat 18 of 24 patients with GM2 antibody titers <40 havedeveloped progressive disease, and 14 have died. Of 42 patients

Table 2 Characterization by IgG subclass-specific monoclonal antibodies of IgG antibodies induced against G*

IgG subclass antibodiesELISA titer against <•-,,•of the following patients

SpecificityIgGIgGllgG2lgG3IgG4:Tiler:200-.2001:50:IOO:IOO:50:100:100:2000:50:100:100:100:50:IOO:100Antibody(source)O279

(AMACfJDC-IO(SBA)JL512(AMAC)JDC-1

(SBA)HG1I(MS)GOM1

(AMAC)COMI(ICN)AC3-AA11(SBA)HP6016(MS)ZG4

(AMAC)CC4-DC10(ICN)C3-8-80

(SBA)HP6047RJ4

(AMAC)AC3-BB2(ICN)C-27-l5(SBA)24010010000008080000006408008040000002000000940200100000080401000001280801040800040404040100000138080408080000020201000001516016020160800000000000018160800401000008040100000238040020100000204000000

" AMAC, AMAC. Inc. (Westbrook, ME); SBA, Southern Biotechnology Associates (Birmingham. AL); MS, Dr. Mitchell Scott (Washington University School of

Medicine, St. Louis, MO); ICN, ICN Biochemicals. Inc. (Irvine, CA).

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INDUCTION AND CHARACTERIZATION OF OM! ANTIBODIES

£Tiler < 40 24 PatientsTiter i 40 42 Patients

2

YEARSFig. 5. C'orrclalion between GM2antibody response after vaccination and time

to progression in melanoma patients with resected local métastases.

with GM: antibody titers of >40, 16 have developed progressivedisease and 13 have died. Comparison of the difference betweenprogression rates of these two groups at 1,2, and 3 yr of follow-up yields P values of 0.25, <0.02, and <0.01, respectively (x~

test). The two groups did not differ in known prognostic indicators, including the number of positive lymph nodes (median2, mean 4) or history of intransit disease.

DISCUSSION

We have confirmed and extended our observation that melanoma patients immunized with BCG-GM: after pretreatmentwith cyclophosphamide produce antibodies that react with GM:.Both the primary antibody response and the secondary responseafter booster immunization were short lived (median duration,6 to 8 wk), including the IgG antibody responses observed insome patients. Peak titers seen after initial and secondaryvaccinations were similar. Delayed-type hypersensitivity to GM2did not develop.

This pattern of reactivity is characteristic for a T-cell-inde-pendent immune response. One possible reason for the lack ofa T-cell response is the carbohydrate nature of the determinantson GM:; recognition of carbohydrate antigens is generally considered to be T-cell independent (15). A possible reason for thisinability of T-cells to recognize some carbohydrate determinatescould be that they cannot be processed and presented. If this isthe case, immunization with anti-idiotype antibodies shouldcircumvent the problem.

Unlike the commonly studied bacterial carbohydrate antigens, GM2 is a constituent of normal tissues, and this mayinfluence the immunoglobulin profile illicited by GM: immunization. In contrast to the indications that the anti-GM: responsewas T-cell independent, the subclasses of the anti-GM2 IgGantibodies induced were predominantly IgGl and IgG3. SinceIgGl and IgG3 are usually associated with T-cell-dependentantigens (16, 17), this might indicate that there is some T-cellinvolvement in GM: recognition. This involvement need not beantigen specific. Similar to the phenomenon of split tolerance(18-20), it may be due to nonspecific T-cell help induced byadjuvant, resulting in short-lived antibody responses after eachimmunization. Furthermore, there are some reports that immunization with carbohydrate antigens can induce T-cell-dependent immune responses resulting in protection from several

infectious agents (21-25), and transplants of murine mammary

adenocarcinoma (26) and B16 melanoma (27).We have previously compared the known expression of GM:

and other gangliosides in melanomas and normal tissues (28).Because GM: is found in small amounts in some normal tissues,immunization with GM: could illicit autoimmunity. No evidencefor autoimmune disease has been seen in any of the vaccinatedpatients. GM: has been found in moderate or large amounts(0.5 to 14 ng/g of tissue) in all melanomas (29), raising thepossibility that GM: antibody will be able to mediate antimela-noma immunity without inducing autoimmune disease (28).The suggestion that melanoma recurrence was delayed in patients developing GM: antibody after vaccination, made firstafter an observation period of 15 mo (6), still holds at 3 yr.This may only indicate that GM: antibody production afterimmunization, like 2,4-dinitrochlorobenzene skin test reactivity(14), identifies patients destined to do well. Although patients producing GM: antibodies and patients lacking antibodiesdid not differ in known prognostic indicators, the therapeuticeffects of the GM: vaccine need to be examined in a prospectivestudy. We have initiated a randomized controlled trial to determine whether administration of BCG-GM: vaccine after pretreatment with Cy is, in fact, of therapeutic value.

ACKNOWLEDGMENTS

We thank Dr. David E. Normansell and Dr. William O. Weigle forhelpful discussions and Christine Hanlon for assistance in preparingthe manuscript.

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7050

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1989;49:7045-7050. Cancer Res   Philip O. Livingston, Gerd Ritter, Pramod Srivastava, et al.   Ganglioside

m2Melanoma Patients by Immunization with Purified GCharacterization of IgG and IgM Antibodies Induced in

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