AU603585B2 - Vaccine for stimulating or enhancing production of antibodies directed against GM2 - Google Patents
Vaccine for stimulating or enhancing production of antibodies directed against GM2 Download PDFInfo
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- AU603585B2 AU603585B2 AU74358/87A AU7435887A AU603585B2 AU 603585 B2 AU603585 B2 AU 603585B2 AU 74358/87 A AU74358/87 A AU 74358/87A AU 7435887 A AU7435887 A AU 7435887A AU 603585 B2 AU603585 B2 AU 603585B2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001169—Tumor associated carbohydrates
- A61K39/001171—Gangliosides, e.g. GM2, GD2 or GD3
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
AU-AI-74358/87 PCT WORLD INTELLECTUAXOL 0 MR IZ' N Internat8 i Br INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Puilication Number: WO 87/ 06840 A61K 39/385, 39/39 Al (43) International Publication Date: 19 November 1987 (19.11.87) (21) International Application Number: PCT/US87/01049 (74) Agent: WHITE, John, Cooper, Dunham, Griffin Moran, 30 Rockefeller Plaza, New York, NY 10112 (22) International Filing Date: 7 May 1987 (07.05.87) (US).
(31) Priority Application Numbers: 860,663 (81) Designated States: AT (European patent), AU, BE (Eu- 044,801 ropean patent), CH (European patent), DE (European patent), DK, FR (European patent), GB (Euro- (32) Priority Dates: 7 May 1986 (07.05.86) pean patent), IT (European patent), JP, LU (Euro- April 1987 (30.04.87) pean patent), NL (European patent), SE (ELropean patent).
(33) Priority Country: US Published co P (71) Applicant: SLOAN-KETTERING INSTITUTE FOR With international search report.
CANCER RESEARCH [US/US]; 1275 York Avenue, co New York, NY 10021 (US).
(72) Inventors: LIVINGSTON, Philip, 0, 450 East 63rd A 2 C Street, New York, NY 10021 OLD, Lloyd, AhD..E. 2 4 DEC 1987 600 West End Avenue, New York, NY 10024 CALVES, Michele, Jones 200 Carmen Street, East AUSTRALIAN Meadow, NY 11554 NATOLI, Edward, J.
5920 58th Road, Maspeth, NY 11378 OETT- C 197 GEN, Herbert, F, 48 Overlook Drive, New Canaan, -I DEC 1987 CT 06840 3 a CT06840(US)PATENT
OFFICE
(54) Title: VACCINE FOR STIMULATING OR ENHANCING AGAINST GM2 Ili I I Wo"i C Fr. I lH PRODUCTION OF ANTIBODIES DIRECTED c l I I I lCI I I Ill G 6 N I I OM(M| S I wa 48.
(57) Abstract II I s s m n n 'a a a o i Vaccine for stimulating or enhancing in a subject to whom the vaccine is administered, production of antibodies directed against GM2. The vaccine comprises an amount of purified GM2 effective to stimulate or enhance antibody production in the subject, an effective amount of a microbial adjuvant and a pharmaceutically acceptable carrier, Thit invention also provides a method for stimulating or enhancing in a subject production of antibodies directed against GM12. The method comprises administering to the subject an effeaive dose of the vaccine of this invention, The microbial adjuvant may be bacillus Calmette-Guerin or Salmonella minneseta R595. This invention further provides a method for treating cancer in a subject affected with cancer. The method comprises administering to the subject an effective dose of the vaccine of this invention, Finally, this invention provides a method for preventing cancer in a subject affected with cancer.
The method comprises administering to the subject an effective dose of the vaccine of this invention, IL.
U
PCT/US87/01049 wo 87/06840 VACCINE FOR STIMULATING OR ENHANCING PRODECTICN OF ANTIBODIES DIRECTED AGAINST GM2 Background of the Invention This application is a continuation'-in-part of U.S.
Serial No. 860,663, filed May 7, 1986, the contents of which are incorporated by reference int6 the present application.
This invention was made with government support under Grant Numbers CA-36120 and CA-08748 from the National Cancer Institute. The U.S. Government has certain rights in the invention.
Throughout this application, various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claimsL The disclosures of these publications in their entireties are hereby incorporated by reference into this applicatior in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Gangliosides are prominent cell-surface conjtituents of melanoma and other tumors of neuroectodermal origin.
Three gangliosides, the monosialoganglioside GM2 and the disiaogangliosides GD' and GD,, are of particular interest to tumor immunologists because of their potential as targets for passive immunization with moioclonal antibodies (mAbs) and for active immunization with cancer vaccines. Despite the presence of GM2, GD2, and GD3 in normal bc:.in and other tissues these gangliosides are immunogenic in mice and r ri_-il -i-I~-21i li--:C WO 87/06840 PCT/US87/010 4 9 -2humans; mouse mAbs have been generated against GM2 GD2 and GD3 and human sera and human mAbs with reactivity for GM2 GD2 (11, 13, 14) and GD3 (15) have been identified.
Over the past decade we have immunized sequential groups of melanoma patients with a variety of melanoma cell vaccines (16-20). These yaccine trials were based on our serological analysis of the humor-a, immune response of melanona patients to cell-surface antigens of autologous and allogeneic ,elanomas (21) and each vaccine was constructed to contain melanoma surface antigens known to be immunogenic in humans. Although vaccinated patients readily produced antibody to HLArelated alloantigens and heterologous serum components in the vaccine, only rarely was antibody elicited to more restricted melanoma antigens, such as class 1 (unique), or GD2 or other class 2 (shared) melanoma antigens. In parallel vaccine studies in the mouse, we have identified immmunizing procedures that facilitate the serological response to tumor antigens (22-24) In the case of GM2, immunization with GM2-expressing tumor *cells or purified GM2 only infrequently induced GM2 antibody in mice, whereas vaccines containing GM2 with adjuvants such as bacillus Calmette-Guerin (BCG) or Salmonella minnesota R595 were far more effective (24).
Livingston, et al. (22, 23) teach BCG and Salmonella useful as adjuvants in stimulating an immunogenic reaction. However, Livingston, et al. neither teach nor suggest the use of such adjuvants to stimulate or enhance antibodies against GM2, Further, Livingston, et al. do not disclose the use of, the Salmonella minnesota R595 as an adjuvant.
I
WO 87/0(840 PCT/US87/01049 -3- Tai, et al. identified the immunogenicity of GM2 in studies of melanoma patients However Tai, et al.
do not teach or suggest the use of BCG or Salmonella minnesota R595 to stimulate or enhance antibodies directed against GM2.
Irie, et al., U.S. Patent No. 4,557,931, issued December 10, 1985, disclose the use of GM2 in combination with an adjuvant to raise antibodies against GM2.
However, Irie, et al. use as adjuvants liposomes or serum albumin. Moreover, Irie, et al. do not teach or suggest the use of BCG or Salmonella minnesota R595 to stimulate or enhance antibodies directed against GM2.
In the present study, we have examined the immunogenicity of GM2-containing vaccines in stage III melanoma patients. Two types of vaccines were used: a whole-cell vaccine containing high levels of GM2 and vaccines containing purified GM2 with or without microbial adjuvants.
WO 87/06840 PCT/US87/010 4 9 -4- 3ummary of the Invention This invention provi4(s a vaccine for stimulating or enhancing in a subject to whom the vaccine is administered, production of antibodies directed against GM2.
The vaccine comprises an amount of purified GM2 effective to stimulate or enhance antibody production in the subject, an effective amount of a microbial adjuvant and a pharmaceutically acceptable carrier. The microbial adjuvant may be bacillus Calmette-Guerin or Salmonella minnesota R595.
This invention also provides a method for stimulating or enhancing in a subject production of antibodies directed against GM2. The method comprises administering to the subject an effective dose of the vaccine of this invention.
This invention further provides a method for treating cancer in a subject affected with cancer. The method comprises administering to the subject an effective dose of the vaccine of this invention.
Finally, this invention provides a method for preventing cancer in a subject affected with cancer. The method comprises administering to the subject an effective dose of the vaccine of this invention.
WO 87/06840 PCT/US87/01049 Brief Description of Figures Figure 1 shows the GM2 antibody response of stage III melanoma patients after immunization with a whole-cell vaccine or purified GM2 ganglioside vaccines. Each curve represents the response of an individual patent.
Arrows indicate time of Cy injection or vaccine injection. The adjuvant used in the booster vaccine was the same as the adjuvant used in the initial vaccines, except in cases in which BCG was replaced by R595.
Figure 2 shows the detection of GM2 antibody in sera from vaccinated melanoma patients by immunostaining.
Identical TLC plates were stained with resorcinol (far left) or were allowed to react with patients vaccinated with GM2/BCG: GM2 antibody titers by ELISA 1:160 and 1:320. D and E, sera from two patients treated with Cy and vaccinated with GM2/BCG: GM2 antibody titers by ELISA 1:80 and 1:160. F, serum from a patient vaccinated with GM2/R595: GM2 antibody titer by EL13dA 1:20.
WO 87/06840 PCT/US87/01049 Detailed Description of the Invention This invention provides a vaccine for stimuiating or enhancing in a subject to whom the vaccine is administered, production of antibodies directed against GM2.
The vaccine comprises an amount of purified GM2 effective to stimulate or enhance antibody production in the subject, an effective amount of a microbial adjuvant and a pharmaceutically acceptable carrier. Preferably, the subject is a human being and the GM2 is bound to the microbial adjuvant by a hydrophobic bond between the lipid portion of the GM2 and the cell membrane of the microbial adjuvant.
The pharmaceutically acceptable carrier may be any well known carrier. However, the presently preferred carrier is a phosphate buffered saline solution.
The preferred microbial adjuvant is bacillus Calmette- Guerin. However, the microbial adjuvant may also be Salmonella minnesota R595.
A range of the amount of purified GMA may be ejmoployed.
However, the preferred amount present in the vaccine is an amount between about 50 micrograms and about 300 micrograms. Additionally, a range of thee amount of Salmonel], minnesota R595 may be employed. However, the preferred amount present in the vaccine is an amount between about .2 mg and about 1.5 mg. Further, a range of the amount of bacillus Calmette-Guerin may be employed. However, the preferred amount present in the vaccive is an amount between about 105 viable units and about 3 x 107 viable units.
L I A i WO 87/06840 PCT/US87/01049 -7- This invention also provides for administering a vaccine of this invention to a subject afflicted with cancer. The antibody produced in the subject upon administration of the vaccine effectively treats the cancer.
This invention still further provides for administering a vaccine of this invention to a subject susceptible to cancer. The antibody produced in the subject upon administration of the vaccine effectively prevents the cancer. A subject susceptible to cancer incl ue subjects predisposed to cancer as well as subjects who have previously had cancer.
The cancer treated or prevented by th e vaccine of this invention may be of neuroectoderrmal origin. Specifically, the cancer of neuroectodermal origin may be a melanoma.
This invention also discloses a method for stimulating or enhancing in a subject production of antibodies directed against GM2. The method comprises administering to the subject an effective dose of the vaccine of this invention.
This invention further discloses a method for treating cancer in a subject affected with cancer. The method comprises administering to the subject an effective dose of a vaccine of this invention.
This invention still further discloses a method for preventing cancer in a subject affected with cancer.
The method comprises administering to the subject an effective dose of a vaccine of this invention.
L- *I WO 87/06840 PCT/US87/01049 -8- The vaccine may be administered subcutaneously, intradermally or intramuscularly. Further, the vaccine may be administered in a single dose or by. a single dose followed by a booster dose. The booster dose is preferably administered 12-16 weeks aftet the inital dose.
Preferably, the methods of this invention employ the GM2 bound to the microbial adjuvant by -a hydrophobic bond between the lipid portion of the GM2 and the cell membrane of the microbial adjuvant. Additionally, the microbial adjuvant may be Salmonella minnesota R595 or bacillus Calmette-Guerin.
The methods of treating or preventing cancer may involve a cancer of neuroectodermal origin. Specifically, the cancer of neuroectodermal origin may be a melanoma.
Finally, an effective amount of cyclophosphamide may be administered to the subject prior to administering the vaccine. A range of time prior to administering the vaccine may be employed. However, the perferred time for administering the cyclophosphamide is between about 3 days and about 7 days prior to administering the vaccine. Further, a range of the amount of cyclophosphamide may be employed. However, the preferred amount i's between about 1 mg/m 2 and about 500 mg/m 2 Materials and Methods Animal Trials WO 87/06840 PCT/US87/0W4 -9- Animals. Female BALB/c-C57BL/6 F 1 (B6) mice, 2 to 5 mo of age, were obtained from The Jackson Laboratory, Bar Harbor,'ME.
Tumor cells. The derivation by Berkelhammer et al.
(26) of the JB-Rf cell line, and by ourselves of the JB-RH-16 subclone used for serological analysis, as i well as the sero.ogical and biochemical analysis characterizing this subclone as an exceptional expressor of GM2, have been described 26). All references to JB-RH refer to this JB-RH-16 subclone.
Serological assays. Mice were bled from the retroorbital sinus at 2 wk intervals after vaccination, and serum samples for serological testing (approximately 0.1 ml) were stored at -20 0 C. The immune adherence (IA) and complement-dependent cytotoxicity assays (with the use of rabbit complement) that detect primarily immunoglobulin M (IgM) and the protein A and i anti-IgG assays that detect primarily IgG, were performed as described (22, 37, 38). The results of the 1i PA, anti-IgG, and IA assays were analyzed microscopically and were expressed as the highest antibody titer resulting in 20% of the target cells having indicator red cells attached to 50% or more of their cell perimeters.
Inhibition tests with the use of purified gangliosides to inhibit IA reactivity were also performed as described Various quantities of ganglioside were dissolved in chlorofcrm:methanol 2:1, were aliquoted into dilution tubes (6 x 50 mm). an were dried in a desiccator. Thirty microliters of serum were allowed to react with the gangliosides at 4°C for 1 hr and the, were tested i,n an IA assay on JB-RH cells.
L i WO 87/06840 PCT/US87/01049 Binding capacity of R595 and bacillus Calmette-Guerin (BCG) for GM2. Fifty micrograms of GM2 were mixed with various quantities of BCG and R595 and were lyophilized. The resulting powder was resuspended as described for vaccine production, was sedimented by centrifugation at 15,60,0 x G, and thin layer chromatography (TLC) was performed to detect GM2 in supernatant and pellet.
Preparation of vaccines: vaccine containing JB-RH cells. JB-RH was grown in tissue culture with medium containing 5% fetal calf serum. On the day of vaccination, cells were dislodged mechanically, irradiated with 10,000 rad from a Cobalt 60 source, and counted.
Cells (5 x 107) were mixed with 50 micrograms of MPLA in 0.2 ml normal saline, as described (22).
Ganglioside content of this vaccine was determined by extracting the cells as described performing TLC, and quantitating the gangliosides by densitometric scanning JB-RH cells (5 x 107) contain 60 micrograms of GM2 Traditional vaccines. Equal volumes of GM2 in saline and commplete Freund's adjuvant (DIFCO Laboratories, Detroit, MI) were emulsified as descirbed
MPLA
and BCG cell wall skeletons (BCG CWS) (Ribi Immunochem, Hamiliton, MT) were added to intralipid (Cutter Laboratories Inc., Berkely, CA) and were used to suspend the dried ganglioside.
Vaccines containing bacteria. J-5 E. coli and Salmo- AiIl minnesota (American Type Culture collection, Rockville, MD) and Salmonella minnesota mutant R595 (kindly provided by Dr. Jerry McGhee, University of WO 87/06840 PCT/US87/01049 -11- Alabama) were boiled in 1% acetic acid as described were washed, and were stored frozen before use.
The day before vaccination, these bacteria or BCG (Tice strain: University of Illinois Medical Center) were resuspended in distilled water by sonication and were added to tubes containing dried GM2. The suspension was lyophilized and on the day of vaccination was resuspended in normal saline shortly before administration.
Liposome vaccines. Liposome preparation 1 was prepared by mixing 200 micrograms of Gr2 with 9.3 mg of lecithin, 9.3 mg of sphingomyelin, and 18.4 mg of cholesterol (Sigma Chemical Co., St. Louis, MO). Liposomes 1 were formed by sonication by using a probe sonicator with a microtip and an energy level of 7 for 5 sec (Model 185:Bronson Sonic Power Co., Danbury, CT).
Liposome preparations 2a and 2b were prepared with 200 micrograms of GM2, phosphatidyl choline (800 micrograms), cholesterol (600 micrograms), and dicetyl-phosphate (30 micrograms) Liposomes 2a were prepared with a probe sonicator as described for liposomes 1, whereas liposomes 2b were formed by sonication in water bath sonicator for 5 min (Bransonic 12; Bransonic Cleaning Equipment Company, Shelton, CT). Adjuvants to be incorporated into the liposomes were added to the GM2 before sonication. These adjuvants included the lipoidylamine CP-20, 961 (Pfizer, Groton, CT) the muramyldipeptide (MDP) succinimidester analog CGP 17107 supplied courtesy of Professor D.G. Braun, CIBA-Geilgy, Basel, switzerland, endatoxin (highly purified lipopolysaccharide) derived from Salmonella abortus egui and provided by Dr. C. Galanos, Freiberg, Germany, and MPLA.
La' i 4- WO 87/06840 PCT/US87/01049 -12- Cyclophospbamide Cy (Cytoxan: Mead Johnson and company, Evansville, IN) was administered at a dose of mg per kg i.p. 3 days before the 26,irst vaccination.
Administration of vaccines. In each experiment, five mice were immunized with a given vaccine. Mice were 4 selected randomly from the same shipment. Vaccines were administered subcutaneously in a total vol. of 0.1 ml per mouse. Two or more vaccinations containing micrograms of GM2 were given at 1 mo intervals, with the exception of vaccines containing complete Freund's adjuvant (which produced marked induration and draining ulcers), no toxicity or morbidity was detected a~s a consequence of the administration of any vaccine.
Human Trials Pati~ents. For the studies involving vaccination with purified GM2t patients with AJCC stage III mnelanoma metastases restricted to r. nLskin and lymph nodes) were considere eli mor and regi 4 ,,a1 lymph nodes had boev rL$ iw 4 moaths and if they were free ot de,,ecUtW.. "L The0 with Clark's level IV or V primary_ Oela.!Oma orr palpable regional lymph node rnotastases, who were scheduled 5 or regional lymph node dissection. In *hetie c.ases, thle initial vaccine was administered at 10, l ddys 0ior to surgery. None of the Patients h4a6 0,eiV~d, chemotherapy or radiation h-rapy.
Jined at 6-week intervals. Ch est liv~ ver 'iction tests and uriralysis were P~ wtoith intezvals. Blood for soIologi, Ai- at 2-'week intervals.
WO 87/06840 PCT/US87/01049 -13- Gangliosides. GM2 was prepared by treating GM1 with beta-galactosidase Joul;dian, Michigan State University, Ann Arbor, MI) according to published methods GDla, GDlb and GT1 were purchased from Supelco (Bellafonte, PA) GD2 was generously provided by 3 Herbert Wiegandt (University of Marburg, Federal Republic of Germany). Ganglioside extraction, identification, and quantification were performed as described Serological Procedures. Typing of cell lines for expression of cell-surface gangliosides with mAbs 5-3, 3F8, and R24 was performed as described 3, The enzyme-linked immunosorbent assay (ELISA) was performed with rabbit anti-human IgM. anti-human IgG, or protein A conjugated to alkaline phosphatase (Zymed Laboratories, San Francisco). Antibody titer was defined as the highest serum dilution yielding an OD greater than or equal to 0.190. Complement-dependent cytotoxicity assays (22) were performed with normal human serum (diluted 1:3) as the complement source.
Reagents for ITLC (Gelman) were peroxidase-conjugated goat anti-human IgM and goat anti-human IgG (Tago, Burlingame, CA) diluted 1:500.
Whole-Cell Vaccines. Procedures used for establishing human astrocytoma cell line SK-MG-14 and human melanoma cell line SK-MEL-31 have been described The JB- RH mouse melanoma cell line was established by J.
Berkelhar. er et al. (26) and a subclone, JB-RH-16, selected for high GM2 expression was established in our laboratory Methods for culturing and harvesting cells for vaccine production have been described (19) Cells were irradi4ted with 10,000 rads (1 tad 0.01 gray) from a 6 0 Co source and frozen viable in dimethyl L I l WO 87/06840 PCT/US87/01049 -14sulfoxide. On the day of vaccination, cells from each line were thawed rapidly, washed three times in phosphate buffered saline (PBS) pooled in roughly equal numbers, and injected. The median total number of cells per vaccine was 2.2 x 108 suspended in 1 ml of PBS, with approximately 40% viability (as judged by trypan blue exclusion). No bacterial adjuvant was used. Two or three vaccinations were administered at intervals immediately prior to lymph node dissection, two or three additional vaccinations were given at 4-weeks after surgery for a total of five vaccinations. The vaccine was administered intradermally on a rotating basis involving all extremities.
Purified GM2 Vaccines. To prepare GM2 vaccines without adjuvants, 100 micrograms of GM2 was dissolved in 1 ml of PBS. For vaccines containing BCG, 10 7 viable units of BCG (Tice strain, University of Illinois), or 3 x 106 units in the case of patients showing strong reactions to BCG, were suspended in distilled water by sonication and added to tubes containing 100 micrograms of dried GM2. The suspension was lyophilized and suspended in PBS shortly before vaccine administration q.
minnesota mutant R595 (kindly provided by Jerry McGhee, University of Alabama) was boiled in 1% acetic acid for 1 hour as described wahsed, dried, and stored frozen. For vaccine preparation, 0.5 mg of R595 was suspended in PBS by sonication and added to GM2 in the same manner as described for BCG. Patients immunized w th GM2 vaccines received four vaccinations intradernially at 2-week intervals on a rotating basis to uninvolved extremities. In addition, some patients immunized with GM2 vaccines containing BCG or R595 received a booster imunization between 3 and 5 months after the fourth vaccination.
J
WO 87/06840 PCT;US87/01049 Results Animal Trials Sera were obtained from mice before and at regular intervals after vaccination, and were tested by the IA and PA assays cn JB-RH target cells. No sera were reactive before vaccination. The reactivity of sera obtained after two vaccinations in eight separate experiments are shown in Table I. All reactions were detected by the IA assay (detecting IgM); no IgG reactions were induced. Three broad categories of vaccines were tested: traditional vaccines such as irradiated whole cells, GM2 alone, or GM2 incorporated into complete Freund'r adjuvant, GM2 attached to or mixed with bacteria, and Gb.. in liposomes.
Traditional vaccines. GM2 administered in saline, in complete Freund's adjuvant, and in intralipid mixed with MPLA and BCG CWS, either with or without Cy, resuLted in serologic responses in only nine of 65 mice (median titer 1/20, see Table Immunization with x 107 irradiated JB-RH. cells (known to contain 60 micrograms GM2) plus MPLA in Cy-pretreated mice induced a serologic response in four of 20 mice (median titer 1/20).
Bacterial adjuvants. E. coli was found to be an ineffective adjuvant (resulting in only two of 10 responser) Slmonella .Jnsota and BCG were moderately effective adjuvants (10 of 25 and eight of 15 responses, iespectively) and the R595 mutant of Salmonella minnesota was a highly effective adjuvant (27 of responses, mredian titer 1/64 including nine mice with titers greater than 1/128) for inducing a serologic WO 87/06840 PCr/US87/01049 -16- Table 1 Serological response in IA tests on JB.RH after tui 0M2 uacclnartons Pretreanmen N No. M" Media 0M2 Dow Na. Mie Vacine Groups 106h1y Respoding Tir" MON (titer m 1/20) (reciprocall Unvaccinated 0 10 0 JB-RHISx 10) MPLA 60 20 5 Saline 50 10 2 24 Saline 50 25 3 Complete Freund's adjuvant 50 10 3 MPLA OG CWS 50 100 MPLA BCG CWS so 10 1 E coy(0.5 mg) 50 10 2 32 E. cdll(5 mg) 50 10 0 S. mlnn. (0.5 mgl 50 5 3 27 S. minn. (51 MPLA (50) 5 0 S. oninn. (5 mgj 50 5 0 S. mll't. (0.5 50 20 7 27 S. minn. 50 20 5 S. minn, RS505 0.05 50 5 2 S. Mi)nn. R595 10.5j 50 30 27- 64 S. Minn, R595 50 15 7 S. minn. R595(5 50 15 2 120 8CG 10' so 5 2 BCG O 050 1k 5 8 BCG to 50 5 1 BCG 1o so 5 2 160 Llposomes I +MPLA(50 so 5 0 Llposomes1 50 5 0 Llposomes I MPLA (50) 50 15 3 Lposones I CP2o.961 50 5 1 64 Llpoames 2a so 5 0 LUposomes !a MPLA (501 so 25 16 Liosomes 2 NIP 50 5 1 64 ilposomes2a 4N4 PLA(50) 50 10 3 L1poewtes 2b 50 5 1 LUposmes 2a endotaxin 50 +S 0 lposomes 2b+ MPLA 1501 50 15 5 lposomes2b+MPLA IN so 5 I Llpomes 2b +CP20.961 so 5 1 StatlstlreAl ulgntifcince by the f!*er euct test compared with JBRH MPLA Cy. p 0 00 1, compared with R595 Cy. p 0.003: cQtfi;Ared with BCG 104 Cy. p 0.009; compared With l1posomes 2a MPLA CY. p a 0.026.
WO 87/06840 PCT/US87/01049 -17response to GM2 (see Table The p values for these compared with GM2 plus saline were 0.03, 0.01, and less than 0.001, respectively. The optimal dose of R595 was mg, and Cy pretreatment significantly augmented vaccine immunogenicity.
To determine whether binding of GM2 to adjuvant was required for optimal effect, the binding capacity of R595 and BCG for GM2 was determined. At-a dose of micrograms GM2 and 0.3 mg R595, trace amounts of GM2 were detected in the supernatant; at higher doses of R595 all of the ganglioside was in the pellet and at lower doses of R595 most of the ganglioside was in the supernatant. The R595 dose found most immunogenic here (0.5 mg) coincides therefore with the least amount of R595 capable of binding the full 50 micrograms of GM2 to its surface. BCG differed from R595 in that approximately 5% of the bacteria were viable and that at the highest dose tolerated by the mice (107) about of GM2 was not attached to the BCG cell pellet (15,600 x G, 30 min), but was found in the supernatant.
This persisted over a range of BCG:GM2 ratios. To determine whether GM2 in the supernatant was free or attached to subcellular BCG components, the mixtures were sujected to ultracentrifugation (300,000 x G, 180 S 25 min), and TLC plates were prepared from both pellet and the supernatant. At a ratio of 50 micrograms GM2:10 6 viable BCG U, 90% of the GM2 was in the pellet. At higher BCG doses, all BCG was in the pellet and at lower doses most was in the supernatant. Immunostaining of these plates with anti-GM2 monoclonal antibody 5-3 confirmed that the bands observed were indeed GM2. These results show that in the most immunogenic BCG and R595 vaccines, most GM2 is bound to the bacteria or bdcterial products, the bacteria are maximally 4 I; L1 WO 87/06840 PCT/US87/0104 -18saturated with GM2, and the vaccines contain little free GM2.
Liposome vaccines. Liposomes 1 and 2b were not very immunogenic (three of 15 and five of 15 responses, respectively; see Table but liposomes 2a were moderately immunogenic (16 of 25 responses, p 0.001 compared with GM2 plus saline). The difference between liposome preparations 1 and 2 was their lipid content, whereas the difference between 2a and 2b was the sonication method; liposomes 2a, which were sonicated more vigorously, are assumed to be smaller. Our results indicate that it is both the composition of- the liposomes and the way in which they are formed that determines their immunogenicity. MPLA was required in the liposomes and could not be substituted for by the lipoidylamine CP20,961 or MDP. Endotoxin at a dose of micrograms (the highest dose tolerated by the mice) was not an effective substitute, but MPLA at this dose was also not effective. Higher titler IgG antibody titers such as these were not detected again in the subsequent four experiments.. Cy appeared to enhance the immunogenicity of the liposomes 2a-MPLA vaccine (16 of 25 vs three of 10 responses), but the difference was not statisticlly significant (p 0.13 by the Fisher exact test).
Specificity of observed responses. Initially, selected sera with high titer reactivity on JB-RH-16 were tested by ELISA assays against a panel of purified gangliosides to determine specificity. This analysis was complicated, however, by natural antibodies against GM1 detected before and after vaccination in some mice and "sticky sera" from some mice that resulted in positive reactions in wells containing various gangliosides or PW CT/US87/01049 WO 87/06840 -19no ganglioside at all. Because we were not able to block this later artifact with bovine serum albumin or normal mouse serum, we analyzed the specificity of five high titer sera against JB-RH cells by inhibition tests (see Table II). We found that the IA reactivity with JB-RH cells was completely inhibited only by GM2; GDIa, GTI, GD3, and GM3 did not inhibit this reactivity. GMI, however, partially inhibited reactivity of two sera from BCG GM2-treated mice.
I 10 Sera reactive by IA also mediate complement-dependent cytotoxicity (CDCX). Initially, sera reactive in IA tests were also tested for CDCX. Titers of reactivity were similar. In one such experiment, sera from five mice immunized with GM2 alone were nonreactive in both assays, whereas those from four of the five GM2 plus R595 mice were strongly reactive in both assays; median titer by CDCX for 50% kill was 1/40 and by IA, 1/64.
We have detected no significant discrepancies between titers obtained by CDCX and IA, but have relied more heavily on IA because it is more rapid and requires less serum.
Attempts to obtain an IgG response and delayed-type hypersensitivity (DTH). Although sera induced by various vaccines in these experiments were able to mediate potent CDCX with rabbit complement on JB-RH, no consistent spontaneous conversion to an IgG response was observed. In an attempt to obtain such a conversion, in ore series of experiments 10 mice were revaccinated subcutaneously with R595-GM2 2 mo after the last vaccination. The median IA titer (IgM) on JB-RH cells in this group increased from 1/80 to 1/160, but no PA or anti-IgG assay reactivity (IgG) was observed. Three additional groups of four mice were given "booster" Pcr/US87/01049 WO 87/06840 Table 2 Inhibilon by purffied gangilosides of IA reactlulty in sera of mice immunized with 012 uaccines Vam~nes R593 (1142 plus 04 pGus 042 plus nhiblUng Alone R595 83G Llpmom. 2& 2 3 4 5 6 Recipropt ,4nu body titer bsy IA o JB.RH None 0 160 320 160 80 80 GM2 10 20 20 2 20 40 20 1 20 80 40 GMI 10 80 160 20 20 40 160 40 20 2 40 160 1 80 160 160 40 ODla 10 80 320 160 40 80 160 160 40 s oC 2 160 d0 1 160 160 160 80 GTI l 40 160 80 80 80 160 80 80 1 GM3 10 80 160 160 40 160 160 160 40 1 160 320 160 40 GD3 10 80 160 160 40 160 160 160 40 1 160 320 160 40 L A WO 87/06840 PCT/US87/01049 WO 87/06840 -21vaccinations with 50 micrograms of GM2 administered i.v. either alone or with R595 or BCG 2 mo after the last vaccination. Once again, the median IA titer increased slightly (from 1/40 to 1/80) but no PA or anti-IgG reactivity was detected. These same groups of mice were also tested for DTH reactivity by footpad injection of 1, 5, and 25 micrograms of GM2. No reactivity was detected.
Human Trials Vaccine characteristics and Seriological Response of Vaccinated Patients. Table 3 summarizes the characteristics of the whole-cell vaccine constructed from three cell lines: a mouse melanoma cell line and melanoma and astrocytoma cell lines of human origin. These cell lines were selected for high surface expression of GM2, as indicated by reactivity with a mouse mAb detecting GM2 Five-vaccines containing purified GM2 were tested, one with GM2 alone and four with BCG or R595 as adjuvants. In two of these trials patients were pretreated with low-dose cyclophosphamide (Cy) (200 mg/m 2 3 days prior to the initial vaccination.
The whole-cell vaccine, though containing no bacterial adjuvant, resulted in induration and erythema (greater than 8cm in diameter at 48 hr) in 5 of 6 patients and low-grade fever (less than 39 0 C) in 4 patients. Four Spatients experienced tenderness and swelling in the draining lymph nodes and 5 of the 6 patients showed prominent hyperplasmia in the resected lymph nodes.
These reactions in skin and lymph nodes, which increased with each vaccination, were not seen in our previous trials with human melanoma cell vaccines. It seems likely, thers-ore, that this heightened inflammatory reaction is attributable to an antimouse response.
PCT/US87/0 1049 WO 37/06840 -22- Table 3 0112. 0fl2 AND GD3 COMPOSITION Of WHOLE CELL VACCINE Cell Surface EFpression of Gangliosides Monoc lonal Antibody 5-3 (aGM2) 3F8 (oGD2) R24 (WD3) Cell Lines JB-RH-16 SK-MG- 14 SK-KEL-31 Ant~ibody Titers (reciprocal) 65 ,000 0 65 ,000 25,000 22,000 2.5 x 106 11,000 Ganglioside content of cell lines (ujg/10 7 cells) GM2 GD2 GD 3 12.4 0 0 6.9 1 .0 Ganlioside concenc of combined vaccine (ug/2.2 x 108 cells) 173.8 54.3 22.7 -i li~ WO 87/06840 PCT/US87/01049 -23- Vaccination with GM2 alone or GM2/R595 was well tolerated; no side effects were detected. GM2/BCG vaccines resulted in low-grade fever (less than 39°C) and marked local ulceration in 5 of 11 patients, requiring a decrease in the BCG dose (3 x 10 organisms) or use of R595 in place of BCG for the booster vaccination. No neurologic or other detectable abnormalities were associated with GM2 vaccination.
j Figure 1 and Table 4 show the results of ELISAs for GM2 antibody in serum ,rom normal individuals and from nonvaccinated and vaccinated melanoma patients. The frequency and titer of GM2 antibody in normal individuals and nonvaccinated melanoma patients were similar: 80% were negative and only one normal individual had a titer above 1:40. The whole-cell vaccine induced GM2 antibody in high titer (1:80 or greater) in 5 of 6 vaccinated patients. No GM2 antibody was induced in patients immunized with GM2 alone. Addition of BCG to the purified GM2 vaccine resulted in. GM2 antibody production, particularly in patients pretreated with Cy or given a booster immunization 12-16 weeks after the last vaccine injection. The effect of Cy was also evident in the case of GM2 vaccines with R595 as the adjuvant; 2 of 6 patients pretreated with Cy produced GM2 antibody, whereas no GM2 antibody was detected in patients not treated with Cy. R595, in contrast to BCG, was not effective as an adjuvant in booster immunizations. No increase in GM2 titers was found in 4 Cy-treated patients given the GM2/R595 vaccine and booster immunizations with GM2/R595 (14 weeks after the last vaccine injection). Because of increasing local inflammatory and systemic reactions induced by BCG in somte patients, of the 11 patients initi&lly vaccinated with GM2/BCG .ii L, F_ -r PCT/US87/01049 WO 87/06840 -24- Table 4 CM2 ANTIBODY TITERS (ELISAi OF NORMAL INDIVIDUALS.
UNTREATED MELANOMA PATIENTS IMMUNIZATION WITH A WHOLE CELL AND MELANOMA PATIENTS AFTER AN EAOAPTET
FE
VACCINE OR PURIFIED GM2 VACCINES VACCINE OK PURIFIED GM2 VACCINES No, Patients With a Given Titer Statistical Significance**** Total No, Patients 0 (Reciprocal) 20 40 80 160 320 UNTREATED Normal Individuals Stage =I Melanoma Patients* VACCINATED WITH WHOLE CELLS SK-MEL-31, SK-MG-14 and JB-RR-16 VACCINATED WITH PURIFIED GM2 GM2 Alone GM2/R.595 Cy GM2/R595*** GM2/BCG*** Cy GM2/BCG*** 6 1 0 0 3 2 0 <.001 1,000 1.000 ,009 <,001 Serum from two patients excluded from evaluation because of non-specific reactivity (refs. 11,24), Peak titer observed after vaccination, Including booster vaccine.
Pishers exact test of number of vaccinated patients with titers 2 1/80 compared with 92 untreated controls.
WO 87/06840 PCT/US87/01049 vaccine received booster injections of a GM2/R595 vaccine. Four of the 6 patients given the GM2/BCG booster immunization showed a strong rise in GM2 titer: no increase in GM2 titers was found in the 5 patients given the GM2/R595 booster immunization.
Specificity Analysis of Sera from Vaccinated Patinets.
Sera from the 19 patients with anti-GM2 titers of 1:40 or greater were tested for reactivity with GDla, GM1, GD2, GD3, and GM3 by ELISA and ITLC. Reactivity was restricted to GM2, with the exception of secum from 1 patient in the whole cell vaccine trial that recognized GD2 at a titer of 1:40. All sera with a titer of 1:80 or higher were also analyzed by ITLC. Reactivity was restricted to GM2. Figure 2 shows ITLC tests with four seru having an anti-GM2 titers of 1:80 or higher by ELISA. Sera with lower anti-GM2 titers could not generally be analyzed by ITLC. GM2 antibodies in vaccinated patients belonged to the IgM class; tests with an IgG indicator system revealed no IgG anti-GM2.
Complement-Dependent Cytotoxicity of GM2 Antibodies.
Sera from patients developing high titers of GM2 anti'body after vaccination were found to be cytotoxic for GM2 positive target cells in the presence of normal human serum as complement source. GM2-negative target cells were not lysed. Table 5 shows the relation between anti-GM2 titers detected by ELISAs and cytotoxicity tests (with SK-MG-6 astrocytoma cells, ref A positive correlation was seen between antibody titers in both assays.
i WO 87/06840' WO 8706840PCTr/US87/01049 -26- Table CM2 ANTIBODIES IN4 SERA. OF VJAC CINATT- EDMLjA.'0A ?ATTENTS'; CO?~kISON, OF TITERS DETE.MINED BY ELISA AN'D CYTOTOXIC TESTS W'ITi HL"W.' C0MEUXEST GM2 Antibody Titer By ELISA (Reciprocal) Number of Patients Optical Density (Range) At 1/40 Serum Dilution Me*dian Cyjtotoxicivj Titer (Range) 20%Z Lysis 50% Lysis End Point End ?oinrt .04 (.01-,06) ,12 (.08-.23) (.21-.35) (.38-,84) .66 (.41-1.45) .83 (1,40,1.415)
N.D.
(5-80) (20-40) (80-320) (320)
N.D.
(10-80) (20,40) PCT/US87/01049 WO 87/0684t -27- Discussion Anima, Trials The purpose of these studies was to ident'.,y methods for consistently immunizing mice against GM2. Immunization with irradiated whole cells expressing GM2 and mixed with a suitable adjuvant would have been predicted to be the method of choice. Although no other studies have compared the relative immunogenicity of a ganglioside on tumor cells and in vaccines containing purified ganglioside, such studies have been done with protein or glycoprotein antigens by using DTH or tumor rejection as end points (39-41). In these studies, antigens expressed on irradiated whole cells have consistently been more immunogenic than the same antigens purified. Our results show, however, that with regard to antibody production against GM2, whole cell vaccines are not very immunogenic, inducing an immune response in only occasional mice. Our results also show that GM2 alone or in complete Freund's adjuvant is not immunogenic, but that GM2 incorporated into certain li.posome preparations or mixed with bacteria such as BCG or especially acid-hydrolyzed R595 is much more immunogenic. In fact, GM2 presented in this way is significantly more immunogenic than the same amount of GM2 expressed on whole tumor cells. A consistent cytotoxic antibody response against tumor cells expressing GM2 was induced, a finding we have used to produce a new series of monoclonal antibodies against GM2. In comparing these results with those of investigators working with DTH or tumor rejection and protein or glycoprotein antigens, the conclusiions only appear to be contraditory. Vaccines containing GM2 alone or with complete Freund's adjuvant are not more immunogen- WO 87/06840 PCT/US87/01049 -28ic than those expressing GM2 on irradiated tumor cells either. The difference is in the way the antigen is presented, and this has never been thoroughly explored by using protein or glycoprotein tumor antigens.
Use of acid-hydrolyzed mutant Salmonella minnesota strain R595 as a vehicle for increasing the immunogenicity of glycolipids was initially described by Galanos et al. They found that acid-hydrolyzed R595 or complete Freund's adjuvant in terms of increasing the immunogenicity of the lipid A component of bacterial lipopolysaccharides. This approach has since been used by Hakomori and Young et al. to augment the immunogenicity of asi'alo GM2 (42) and othe. glyolipid blood group antigens (43) and to produce a series of monoclonal antibodies against blood group and tumor glycolipids (44, 45). We show here that R595 can also be applied to inducing antibodies against gangliosides such as GM2. Since the studies of Galanos et al. (27) with lipid A, little work has been done on identifiying alternative methods for argumenting the immunogenicity of glycolipids. In the studies we have expanded the variety of approaches tested for inducing antibodies against glycolipids by identifying BCG- and MPLA-containing liposomes as two other vehicles worthy of additional study. The results of Galanos et al. (27), together with our findings that acid-treated R595 was superior to wild-type Salmonella minnesota or j. coli and that vaccines containing the highest GM2 to R595 ratio were also the most immunogenic, suggest the role that acid-treated R595 plays. Lipopolysaccarides are made up of two distinct regions, the hydrophylic polysaccharide portion consisting of 0-specific chains and basal core, and the hyd'ophobic lipid portion lipid A. Our previous studies with whole cell vaccines WO 87/06840 PCT/US87/01049 WO 87/06840 PCT/US87/01049 -29- 22) and those described here with liposomes have identified lipid A as the single most potent adjuvant tested. The R (rough) mutant 595 contains lipid A but no O-specific chains, and the core polysaccharide consists only of 2-keto-3-dioxyoctonate, which is removed by acid hydrolysis, as originally described by Galanos The cell surface of acid-treated R595 is therefore highly hydrophobic and ideal for binding and concentrating added glycolipids such as GM2. GM2 is concentrated on the surface and oriented in such a way that cerranide is imbedded and polysaccharide (the antigen) exposed and in close proximity to bacterial lipid A.
Liposomes are another method of concentrating glycolipids on a membrane with an orientation that enhances immunogenicity and of bringing them into close proximity to selected adjuvants. Our results show the Simportance of both liposome composition and size. They also show that one adjuvant, MPLA, was most effective in liposome preparations. As opposed to our studies with GM2-coated R595 and BCG in which the variables were limited and quickly addressed, our studies with liposomes have only served to suggest additional areas for study. We are currently investigation the effect of lipid composition, liposome size and charge, unilamellar vs multilamellar structure, incorporation of the antigen and lymphokines on to the liposome surface as opposed to within the liposome or both, and the use of various single adjuvants or combinations of adjuvants.
Many o£ the mice immunized in these studies continued to produce antibody more than 6 mo without evidence of neurologic or othet coxicity. This is reassuring, I I i_ i _i WO 8706840 PCT/US87/01049 WO 87/06840 because GM2 is present on a small subpopulation of human and (presumably) B-6 astrocytes Although experimental autoimmune encephalomyelitis (EAE) is generally induced by immunization with the basic protein of myelin in a suitable adjuvant such as complete Freund's adjuvant, Nagai et al. (46) have reported that immunization of rabbits with GDIa or GM in complete Freund's adjuvant resulted in a syndrome similar to that described for EAE: hind leg paralysis and sometimes death. Furthermore, Rapport et al. (47) have described grand mal seizures in rabbits after intracerebral injections of anti-GM1 antibodies, but not injecitons of antibodies against various other unrelated antigens. Although EAE is generally short lived, requiring continuing immunizations for progression of the disease, and although there have been no reports of autoimmune phenomena associated with anti- GM2 antibody activity, we observed immunized mice Sclosely for as long as a fu.1 year to confirm that there was no association between anti-GM2 antibody and autoimmune disease.
Human Trials The identification of melanoma cell-surface antigens that are immunogenic in the host of origin has been the object of our analysis of sera -cytotoxic T cells (28,29), and mAbs (15, 30) derived from melanoma patients. Three general categories of melanoma cellsurface antigens that are immunogenic or potentially immunogenic in the autologous host have been defined; these range from highly restricted antigens that are detected only on autologous melanoma cells [class 1 (unique) antigensI, to antigens present on a subset of melanomas as well as a limited range of other cell WO 87/06840 PCT/US87/0 1049 -31types (class 2 antigens), to antigens that are widely distributed on melanomas and other cell types (class 3 antigens) Biochemical 'characterization of these melanoma antigens is limited, but class 1 (unique) antigens appear to be glycoproteins (31) and one of the best-studied class 2 melanoma antigens is the ganglioside GD2 (13, 14). As reactivity against these antigens is found in only a small percentage of melanoma patients, we have attempted to induce antibodies to class 1 Or class 2 antigens using vaccines of irradiated cells expressing these antigens (16-20). These human trials have not been successful and prompted us to define conditions required for a consistent humoral immune response to tumor antigens in the mouse, including the class 1 antigens of Meth A sarcoma (22, 23) and the ganglioside GM2 (20, 24). Adjuvants and pretreatment with low doses of Cy were important factors in the mouse studies, and results of the present human trials indicate their important in melanoma patients.
Irie, Tai, Morton and colleagues have also identified the immunogenicity of GM2 in their studies of melanoma patients (11, 12). They isolated stable cultures of Epstein-Barr virus-transformed B cells from a melanoma patient that produced a mAb to GM2 In addition, Tai et al. have immunized melanoma patients with melanoma cell vaccines containing a mixture of gangliosides and found that reactivity against GM2 was induced in 10/26 patients As in the present study, reactivity against GD2 was only rarely detected (2/26 patients) i: their series and no antibody agaist GD3 or GM3 was ftund.
We and others have detected low levels of GM2 antibody in some normal individuals (10) and nonvaccinated stage WO 87/06840 PCT/US87/01049 -32- III melanoma patients. Natural growth of melanoma in the skin and regional lymph nodes does not appear to be a potent stimulus for generating GM2 antibody, since antibody levels in stage III melanoma patients are no higher than in normal individuals. The fact that most melanoma patients can be induced to develop high levels of GM2 antibody after vaccination is surprising in view of the presence of these gangliosides in brain and other tissues of neuroectodermal origin. The other finding of GM2 vaccines is that high titers of GM2 antibody had no demonstrable ill effect on these patients.
There is a suggestion from the present study that melanoma recurrence is delayed in patients developing GM2 antibody. Evaluation of 31 vaccinated patients in this study observed for 15 months showed that 5 of 14 patients with GM2 titers less than or equal to 1:20 are disease free, as compared to 14 of 17 patients with GM2 titers greater than or equal to 1:40.
I WO 87/06840 PCT/US87/01049 -33- Ref erences 1. Cochran, F.B. Jr., Yu, R.K. &Ledeen, (1982) J. Neurochem, 39, 773-779.
2. Natoli, JL: Livingston, Cordon-Cardo, Pukel, Lloyd, Wiegandtf Szalay, Oettgen, H. F. Old, L.J. (1986) Cancer Res.
46, 4116-4120 3. Cheung, N. K.V. Saarinen, Neely, J.E., Landmeier, Donovan, D. Coccia, P. F. (1985) Cancer Res. 45, 2642-2649.
4. Cheresh, Harper, Schulz, G Reisfeld, R.A. (1984) Proc. Natl. Acad. Sci. USA 81, 5767- 5771.
Dippold, W. G. Lloyd, K.0. Li, L. T. Ikeda, H. Oettgen, H. F. Old, L.J. (1980) Proc. Natl Acad.
Sci. USA 77, 6114-6118.
6. Pukel, C. S. Lloy d, K.0. Travassor, L. R. Dippold, Oettgen, H.F. Old L.J. (1982) J.Exp Med.
155, 113 3-1147 7. Nudelman, Hakomori, Kannagi, Levery, Ye1h, M. H. Hellstrom, K. E. H Hel 1s t r i, 1.
(1982) J. Biol. Chem. 257, 12752-12756.
8. Cheresh, D.A. Klier, F.G. (1986);~ J. Cell Biol.
102,, 1887-1897.
9. Arnon, F. Crisp, E. Kelley, R. Ellison, G.W., IMYers, L.W. rourtellotte, W.W. (1980) J. Neurol.
Sci. 46, 179-186.
WO087/06840 PCT/1,JS87/0 1049 -34- Richards, R. L. Alving, C. R.
Ser., 461-473.
11. Tai, Cahan, Tsuchida, Irie, R.F. Morton, D.L. (1985) 607-612.
(1980) ACS Syinp.
Saxton, R.E., Int. J. Cancer 12. Tait Paulson, Cahan, L.D. Irie, R.F.
(1983) Proc. Natl. Acad. Sci. USA 80, 5392-5396.
13. Watanabe, Pukel, Takeyaia, Lloyd, K.0.,F Shiku, H.,I Li, L. T.C. Travassos, t.R. Oettgen, H.F. Old, L.J. (1982) J.Exp. Med. 156, 1884-1889.
14. Cahan, Irie, Singh, Cassidenti, A.
Paulson, J.C. (1982) Proc. Natl. Acad. Sci. USA 79, 7629-7633.
15. Yamaguchi, Furukawa, K.,f Fortunato, S.R., Livingston, Lloyd, Oettgen, H.F. old, L.J. (1987) Proc. Natl. Acad. Sci. USA, in press.
16. Livingston, P9O41 Watanabe, Shiku, Ho, Houghton, A. No, Al bi no, Takahashi, To, Resnick, Michitsch, Pinsky, Oettgen, H.F. I& old, L.J. (1982) Int. J. Cancer 30, 413-422.
17. Livingston, Takeyania, Pollack, M.S., Houghton, Albino, Pinsky, Oettgen, H.F. Old, L.J. (1983) Int. J. Cancer 31, 567-575.
18. Livingston, Albino, Chung, T#J.C., Real, Houghton, Oettgen, H.F. Old, L.
J. (1985) Cancer 55, 713-720.
WO 87/06840 PCT/US87/01049 19. Livingston, Kaelin, E.f Pinsky, C. M., Oettgen, H.F. Old, L.J. (1985) Cancer .56, 2194- 2200.
20. Livingston, P.O. (1985) UCLA Symp. Mol. Cell. Biol.
New Ser. 27, 537-548.
21. Old, L.J. (1981) Cancer Res. 41, 361-3-68.- 22. Livingston, DeLeo, Jones, Oettgen, H.F. Old, L.J. (1983) J. rImmunol. 131, 2601.
23. Livingston, Jones, DeLeo, Oettgen, fl.F. Old, L.J. (1985) J. Iminunol. 135, 1505-1509.
24. Livingstont Jones Calves, M. Natoli, J., Jr. (1.987) J. Inununol., in press.
Watanabe, T. Livingston, P.O. (1981), in A.ACHT Tissue Typing Manual, eds. Zachary, A.A. Braun, W.E. (AACHT, New York), pp. 1-21-1-1-21-4.
26. Berkelhamner, Oxenhandler, Hook, R.R., Jr., Hennessy, J.M. (1982) Cancer Res. 42, 3157- 3163.
27. Galanos, Luderitzz, 0. &Westphal.# 0. (1971) Eur. J. Biochein. 24, 116-122.
28. Livingston, Shiku, Seant Pinsky, Cettgen, H.F. SOld, L.J. (1979) Int. J.
Cancer 24, 34-44.
-1 WO 87/06840 PCT/US87/0 1049 29. Knuth, Danowski, Oettgen, H.F. &Old,' L.J.
(1984) Proc. Natl. Acad. Sci. USA 81, 3511-3515.
Houghton, Brooks, Cote, Taormina, Oettgen, H.F. Old. L.J. (1983) J. Exp. Med.
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31. Re al Jules Mattes, Houghton, A.N.e Oettgen, H. F. Lloyd, K.O0. Old, L;J. (1984) J.
Exp. Med. 160, 1219-1233.
32. Svennerholm, L. (1964) J. Lipid. Res. 5, 145-155.
33. Cheresh, ED.A., Honsik, Staffileno, L.K., Jung, G. Reisfeld, R.A. (1985) Proc. Natl. Acad.
Sci. USA 82:5155.
34. Livingston, Natoli, Calves, M.J., Stockertf Oettgen, H.F. Old, L.J. (1987) Proc. Natl. Acad. Sci. USA. In press.
Houghton, Mintzer, Cordon-Cardo, C.,f Welt, Fliegel, B.f Vadhan, Carswell, ,f Melamed, M. R.,f Oettgen, H.F. Old, L.J. (1985) Proc. Natl. Acad. Sci. USA 8 2:1,242 36. Theung, N-K, Berger, No, Coccia, Kallick, Lazarus, Miraldi, Saarinen, U. Strandjord, S. (1986) Proc. Am. Assoc. Cancer Res.
27 :318 37. Shiku, Takahashi, Oettgen, H.F. old, L.J.
(1976) J. Exp. Med. 144:873.
WO 87/06840 PCT/US87/01049 37- 38. Ueda, R. Shiku, Pfreund .iMeta.(1979) J. Exp. Med. 150:564.
39. Saunde'vs, Kahan, B.D. &Pellis, N.R. (1985) Cancer Imm~unol. Inununother. 19:22.
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Claims (13)
1. A vaccine for stimulating or enhancing in a subject to whom the vaccine is administered, production of antibodies directed against GM2 comprising an amount of purified GM2 effective to stimulate or enhance antibody production in the subject, an effective amount of the microbial adjuvant cdcillus Calmette-Guerin and a pharmaceutically acceptable carrier.
2, The vaccine of claim 1, wherein the subject is a human i being.
3. The vaccine of claim 1, wherein the GM2 is bound to the microbial adjuvant Bacillus calmette-querin by a hydrophobic bond between the lipid portion of the GM2 and I the cell membrane of the microbial adjuvant Baccillus I calmette-querin.
4. The vaccine of claim 1, wherein the effective amount of GM2 is an amount between 50 micrograms and 300 micrograms.
The vaccine of claim 1, wherein the effective amount of Bacillus calmette-querin is an amount between 10 s viable i* units and 3 x 107 viable units. i
6. A method for stimulating or enhancing in a subject production of antibodies directed against GM2 comprising *administering to the subject the vaccine of claim 1 in a dose effective for stimulating or enhancing the antibodies. i
7. A method for delaying the recurrence of melanoma in a subject susceptible to melanoma comprising administering to the subject the vaccine of claim 1 in a dose effective j for delaying the recurrence of melanoma.
8. The method of claims 6 or 7, wherein the GM2 is bound to the microbial adjuvant Bacillus calmette-querin.
9. The method of claims 6 or 7, wherein the GM2 is bound to the microbial adjuvant Bacillus calmette-cuerin by a hydrophobic bond between the lipid portion of the GM2 and the cell membrane of the microbial adjuvant Bacillus calmette-querin.
The method of claim 9, wherein the melanoma is of neuroectodermal origin. _@I I ir
11. The method of claim 6 or 7, wherein an effective amount of cyclophosphamide is administered to the subject prior to administering the vaccine.
The method of administered administering claim 11, wherein the cyclophosphamide between 3 days and 7 days prior the vaccine.
13. The method of claim 11, wherein the effective amount of cyclophosphamide is between 1 mg/m 2 and 500 mg/m2.-- DATED: this 17th day of August 1990 6. @6 6 6060 66 6 6* SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH Patent Attorneys for the Applicant: F,B. RICE CO. 66@6 6 6666 6666 66 66 6 66 6 66 S. 6
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US6432402B1 (en) | 1989-05-25 | 2002-08-13 | Sloan-Kettering Institute For Cancer Research | Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof |
US6805862B1 (en) | 1989-05-25 | 2004-10-19 | Sloan-Kattering Institute For Cancer Research | Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof |
US7585512B1 (en) | 1990-05-08 | 2009-09-08 | Thomas Jefferson University | Composition and method of using tumor cells |
CU22420A1 (en) * | 1993-12-29 | 1996-01-31 | Centro Inmunologia Molecular | Vaccine composition for the development of an answer against gangliosides and glycolilades and its use for cancer treatment |
US6149921A (en) * | 1993-12-29 | 2000-11-21 | Centro De Inmunologia Molecular | Vaccine compositions for eliciting an immune response against N-acetylated gangliosides and their use for cancer treatment |
AU727316B2 (en) * | 1995-06-07 | 2000-12-07 | Thomas Jefferson University | Hapten modified tumor cell extract and methods of treating or screening for cancer |
US5792455A (en) * | 1996-03-21 | 1998-08-11 | Sloan-Kettering Institute For Cancer Research | Anti-idiotypic antibody vaccines |
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