RU2174013C2 - Composition for stimulation and enhancement of production of antibody that recognize ganglioside - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: invention proposes the composition for stimulation or enhancement of production of antibodies that are able to recognize ganglioside in person to whom composition is administrated. Composition has some amount of ganglioside (or its oligosaccharide moiety) conjugated with immunogenic protein and this amount is effective to stimulate or enhance production of antibodies in person. Composition has also adjuvant and pharmaceutically acceptable carrier. Invention proposes also method of use of composition for treatment or prophylaxis of cancer when gangliosides are located on surface or in stroma of cancer cells. EFFECT: enhanced effectiveness of composition. 34 cl, 14 tbl, 26 dwg

Description

 Gangliosides are sialic acid-containing glycosphingolipids, consisting of a complex carbohydrate moiety connected to a hydrophobic ceramide moiety. The carbohydrate chain enclosed within the outer leaves of the cell membrane is open to the extracellular matrix. The observed qualitative and quantitative changes in the composition of gangliosides in the process of cell differentiation and proliferation apparently reflect the state of malignant degeneration of cancer of neuroectodermal origin (Hakonori, 1985). Malignant melanoma cells express various complex gangliosides besides GM3, the main ganglioside in normal melanocytes (Carubia et a1., 1984). Altered ganglioside metabolism in melanoma causes additional expression of GD3, GD2, GM2, 9-0-acetyl-GD3 and GT3 (Hamilton et a1., 1993; Tsuchida et a1., 1987). Treatment of patients with monoclonal antibodies against GD3 led to inflammation at the tumor site, partial regression of metastases was periodically observed. This confirms that gangliosides are suitable targets for an immune attack (Houghton et a1., 1985). Generation of human monoclonal antibodies (MAb) reacting with GD3 from patients with melanoma (Yamaguchi et a1., 1987) confirms the idea that gangliosides are also potential immunogens.

 In a study designed to induce a humoral immune response against gangliosides in patients with active immunization melanoma, GM2 / BCG vaccines seemed most effective (Livingston et a1., 1987; Livingston et al., 1989). In a blind selection study of 122 patients with melanoma who were not ill after surgery, it was shown that of the 64 patients who received only BCG and the 58 patients who received GM2 / BCG, the majority of patients (86%) who received GM2 vaccine produced antibodies. Patients who produced antibodies for GM2 were significantly longer in health and had greater overall survival than patients with no antibodies. Comparison of the two types of trials showed that patients receiving the GM2 / BCG vaccine had a 17% increase in time without disease and a 9% increase in survival compared with the control group receiving BCG, although not one result was statistically significant (Livinfston et a1., 1993a). Unfortunately, the immune response was only for a short time, mainly with the formation of IgM and a moderate titer. This confirmed that GM2 was recognized as a T-cell-independent antigen due to the presence of carbohydrate antigens (Livingston et a1., 1989) and also that gangliosides are autoantigens expressed on the same normal tissue (Hamilton et a1., 1993). Similar approaches using vaccines GD2 and 9-0-acetyl-CD3 in patients gave occasionally low titers, no humoral immune response was found against GD3 (Livingston, 1991).

 New potent adjuvants were able in some cases to enhance the immune response against gangliosides, but specifically for autoantigens, such as those associated with ganglioside tumors, a different approach should be used. Based on classical Landsteiner experiments (Landsteiner and Chase, 1942) with hapten-carrier conjugates, covalent attachment of weakly immunogenic antigens to immunogenic carrier proteins was successfully used to enhance the immune response / For example, an immune response to carbohydrates other than gangliosides can be achieved by conjugation with suitable proteins -carriers. The binding of bacterial capsular polysaccharides to immunogenic proteins has shown a significant increase in the immune response and immunity (Eskola et a1., 1990). Recently, work on vaccinating patients with ovarian cancer with the synthetic Thompson Friedenreich tumor antigen conjugated with cochlear lymphocytic hemocyanin has revealed a humoral IgM and IgG immune response (MacLean et a1., 1992). An important discovery common to these studies was the isotonic conversion of the short-duration IgM immune response into a long, high affinity IgG immune response, indicating that T-cell-dependent pathways are activated against carbohydrates. This approach is now used for the antigen of the GD3 melanoma tumor in order to develop a method for the synthesis of vaccines for ganglioside-protein conjugates and to study the immunogenicity of various GDS-protein conjugates in mice.

 The present invention provides a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, the production of antibodies that recognize ganglioside. This vaccine contains a ganglioside (or oligosaccharide moiety) conjugated to an immunogenic protein in an amount effective to stimulate or enhance antibody production in the subject, an effective amount of an adjuvant and a pharmaceutically acceptable excipient.

 The present invention also provides a method of stimulating or enhancing the production of antibodies that recognize a ganglioside in the body of the subject by administering to the subject an effective dose of a vaccine to stimulate or enhance the body of the subject receiving the vaccine, producing antibodies that recognize the ganglioside containing the ganglioside (or its oligosaccharide part ) conjugated to an immunogenic protein, in an amount effective to stimulate or enhance the production of antibodies in the body of the subject, effect vnoe amount of adjuvant and a pharmaceutically acceptable excipient.

Brief Description of the Figures
FIG. 1. Synthesis of CD3 protein conjugates after ozone digestion and reductive amination. The insert represents TLCHR GD3 before (A) and after (B) cleavage.

 FIG. 2A and 2B. Change of antibodies IgM (a) and IgG (b) antiserum on GD3-KLH from time to time. Each character in a shape refers to a mouse.

 FIG. 3A and 3B. Immuno thin-layer chromatography of three mouse antisera after GD3-KLH vaccination. The reactivity of IgG antibodies (a) and IgM (b) was tested for (A) human brain gangliosides, (B) neuroblastoma gangliosides, (C) melanoma gangliosides, and (D) GD3 antigen.

 FIG. 4. Immunoblot of four mice to show the specificity of the immune response. Pure ganglioside was applied to the dot blot and incubated with mouse serum.

 FIG. 5. Representative FACS analysis of mouse serum reactivity before (peak at 3) and after (peak at 50) immunization with GD3-KLH and QS-21, tested on the SK-VEL-28 melanoma cell line.

 FIG. 6A and 6B. Change in IgM (a) and IgG (b) antiserum antibodies to GD3-KLH over time. Each symbol of the figure refers to a person.

 FIG. 7. Detection of antibodies against GD2 in the sera of patients vaccinated with GM2 conjugate vaccine plus adjuvant, immunostaining in dot blots. Stains of standard gangliosides were applied to nitrocellulose strips (indicated on the vertical axis) and left to react with individual patient sera before vaccination and after vaccination with a peak titer of peroxidase-labeled goat anti-human IgM and IgG antibodies. The strips were distributed on a scale from 0 to +3. MAb 696 was used as a positive control for GM2.

 FIG. 8A-1 and 8A-2. The specificity of the peak titer sera of patients immunized with GM2-KLH + QS-21 was determined by thin-layer immune chromatography as described previously (3, ref. Third series of experiments). GM2 (A) and melanoma tissue ganglioside extract (B) were applied to TLC plates, incubated with individual patient sera and stained with peroxidase-labeled goat anti-human IgM or IgG antibodies. MAb 696 was used as a positive control for GM2, and resorcinol was used to stain gangliosides.

 FIG. 8B. Inhibition of patient serum IgG reactivity against GM2 and GD2. GM2 (A) and melanoma tissue ganglioside extract (B) were applied to TLCHR plates, incubated with patient serum No. 2 and stained with goat anti-human IgG antibodies labeled with peroxidase. 3 ml of the patient’s serum at a 1:50 dilution were preincubated with 150 μg GM2 or 150 μg GD2 until immune staining.

 FIG. 9A and 9B. IgM and IgG immune responses in patients with melanoma after immunization with GM2-KLH plus QS-21 vaccines. Sequential results in 6 patients receiving a dose of 100 μg QS-21 are shown in FIG. 9A and for 6 patients receiving a dose of 200 μg are shown in FIG. 9B. Note that one patient in each group received only 4 vaccinations and was excluded from the study due to the progress of the disease. The arrows indicate the time of injection of cyclophosphamide (Cy) and the GM2-KLH vaccine plus QS-21.

 FIG. 10A and 10B. Detection of antibodies against GM2 by immunostaining on dot blots of sera of 10 patients vaccinated with GM2-KLH. Ganglioside standards were applied to nitrocellulose strips (as indicated on the left) and incubated first with sera and then, after washing, with peroxidase-labeled goat anti-human IgM or IgG antibodies. The results of detection of sera of two patients of each of their 5 groups who received different doses of QS-21 (as shown above) are presented. Serum before (a) and after immunization (b) are given for each patient. Mouse monoclonal antibodies 696 and 3F8 are IgM and IgG antibodies (respectively) against GM2 and GD2. IgM antibodies against GM1 were detected in the sera of most patients before and after vaccination. Anti-GM2 IgM and IgG antibodies were not detected prior to vaccination in any of these patients. After vaccination, IgM and IgG antibodies against GM2 were detected in the sera of all patients. Reactions were graded 0, 1+, 2+, or 3+. An example of the degree distribution of the reaction for this analysis is as follows: Patient 1 (100 μg QS-21) IgM (before / after vaccination): KLH 1 + / 2 +, GM3 0/0, GM2 0/3 +, GM1 1 + / 1 +, GD3 0/0, GD2 0/1 +, GD1b 0/0.

 FIG. 11A and 11B. IgM - immune responses in patients with melanoma after immunization with the GM2 / BCG vaccine. Consistent results are given for 5 patients treated during the initial 4 months of the regimen (group A) and 5 patients treated during the last 4 months of the regimen (group B). The arrows indicate the time of injection of cyclophosphamide (Cy) and the GM2 / BCG vaccine.

 FIG. 12. Detection of antibodies on GM2 by immunostaining on dot blots in the sera of 10 patients with melanoma vaccinated with GM2 / BCG. Ganglioside standards were applied to nitrocellulose strips (as shown on the left) and incubated first with sera and then, after washing, with peroxidase-labeled goat anti-human IgM antibodies. GM2-MEL refers to purified GM2 extracted from melanoma biopsy samples; all other gangliosides, including GM2, were obtained from the bovine brain. The number of patients (6-58) is indicated and the serum is given before (a) and after (b) immunization for each patient. 696 and 3G6 are mouse IgM monoclonal antibodies against GM2 and GD2, respectively. Antibodies against GM2 were detected in serum after vaccination of 10 patients. Antibodies against GM1 were detected in serum before and after immunization of patient 53 and in serum after vaccination of patient 57. Reactions were graded 0, 1+, 2+ or 3+. Examples of the degree distribution of the reaction are as follows: Patient 8: GM2 3+, GM2-MEL 2+; patient 54: GM2 3+, GM2-MEL 1+; and patient 57: GM2 3+, GM2-MEL 3+. The lowest reactivity against GM2-MEL compared to GM2 was found in sera after vaccination, and murine monoclonal antibodies 696 reflect the smallest amount of GM2-MEL ganglioside applied to the strips.

 FIG. 13. Distribution by Kaplan-Meier of disease-free patients and surviving patients with antibody production after GM2 vaccination. Patients were classified as positive if GM2 reactivity was a) 2+ or 3+ on dot blots and ELISA titer was not lower than 1/20 or b) 1+ on dot blots and ELISA titer not lower than 1 / 80.

 FIG. 14. Kaplan-Meier distribution of 59 disease-free and surviving patients randomized to receive BCG and 57 patients randomized to receive GM2 / BCG, including 6 patients who produced antibodies to GM2 before immunization (5, who were then given BCG and 1 to which GM2 / BCG was administered).

 FIG. 15. Kaplan-Meier distribution of 64 disease-free patients and surviving patients randomized to receive BCG and 58 patients randomized to receive GM2 / BCG.

), в сравнении с 57 пациентами с отрицательной реакцией на антитела против GM2, рандомизированными для получения GM2/BCG (□ или ■). Пациенты разделяют на две группы: пациенты с одним положительным лимфатическим узлом (Δ или □) и пациенты с двумя или более положительными лимфатическими узлами (

или■).FIG. 16. Kaplan-Meier distribution of 59 non-ill and surviving patients negative for anti-GM2 antibodies randomized to BCG (Δ or

), compared with 57 patients with a negative reaction to anti-GM2 antibodies randomized to receive GM2 / BCG (□ or ■). Patients are divided into two groups: patients with one positive lymph node (Δ or □) and patients with two or more positive lymph nodes (

or ■).

 Throughout this application, various references are given in parentheses. The description of these publications is thus fully incorporated into the present application by reference for a more complete description of the state of the knowledge field to which the present invention relates. A full bibliography of these references can be found at the end of this application prior to the claims.

 The present invention provides a vaccine for stimulating or enhancing a subject to which the vaccine is administered, producing antibodies that recognize a ganglioside containing a ganglioside (or its oligosaccharide portion) conjugated to an immunogenic protein in an amount effective to stimulate or enhance the production of antibodies in the subject's body. , an effective amount of an adjuvant and a pharmaceutically acceptable excipient.

 The oligosaccharide portion of the ganglioside can be formed by cleavage of the ganglioside, or it can be synthesized directly. As used herein, the term “immunogenic protein” means a protein that, when conjugated to a ganglioside or its oligosaccharide moiety, stimulates or enhances antibody production in a subject.

 In one embodiment, the subject is a human.

 The present invention also provides the above vaccine in which the ganglioside or its oligosaccharide moiety is conjugated to cochlear lymph hemocyanin or a cochlear lymph hemocyanin derivative.

 Snail lymph hemocyanin is a well-known protein. A cochlear lymphocytic hemocyanin derivative can be obtained by direct binding of at least one immunological adjuvant, for example monophospholipid A, or non-ionic block copolymers, or a cytokine with cochlear lymphocytic hemocyanin. Cytokines are well known to specialists in this field of science. An example of a cytokine is interleukin 2. There are other known interleukins that can be combined with snail lymph hemocyanin to produce a snail lymph hemocyanin derivative.

 In one embodiment of the above vaccine, the adjuvant is QS-21.

 There are other known adjuvants that may be suitable for the present invention. They can be of the QS-21 class or chemicals similar to QS-21, which can likewise be used in accordance with the present invention.

 The present invention also provides the above vaccines in which the ganglioside is selected from the group consisting of GM2, GM3, GD2, GD3, GDS-lactone, O-acetyl-GD3 and GT3.

 In one preferred embodiment of the present invention, the ganglioside is GM2. In another example, the ganglioside is GD3. In yet another example, the ganglioside is GD2.

 In accordance with the present invention, various effective amounts of conjugated ganglioside or its oligosaccharide moiety can be used. A person skilled in the art can conduct simple titration experiments to determine what effective amount is required for effective immunization. An example of such a titration experiment is the injection of various amounts of a conjugated ganglioside or conjugated oligosaccharide moiety to its subject and then determining an immune response.

 In one embodiment of the invention, an effective amount of a conjugated ganglioside or conjugated oligosaccharide moiety thereof is between about 1 μg and about 200 μg.

 In another embodiment of the invention, an effective amount of a conjugated ganglioside or conjugated oligosaccharide moiety thereof was between about 50 μg and about 90 μg. In yet another example of the invention, an effective amount of a conjugated ganglioside or conjugated oligosaccharide moiety is about 70 μg.

 In another embodiment of the invention, an effective amount of a conjugated ganglioside or conjugated oligosaccharide moiety is between about 1 μg and about 10 μg. In a more specific example, an effective amount of a conjugated ganglioside or conjugated oligosaccharide moiety is between about 7 μg and about 10 μg. In one example, an effective amount of a conjugated ganglioside or conjugated oligosaccharide moiety thereof was about 7 μg.

 In addition, an effective amount of adjuvant can also be determined in a similar manner, i.e., by administering various amounts of adjuvant with conjugates and establishing an immune response to determine how much adjuvant is effective. When QS-21 is used as an adjuvant, an effective amount of QS-21 can also be determined in a similar manner.

 In a preferred embodiment, an effective amount of QS-21 is between about 10 μg and about 200 μg. In one example, an effective amount of QS-21 is about 100 μg. In another example, an effective amount of QS-21 is about 200 μg.

 The present invention also provides a vaccine for stimulating or enhancing the body of the subject to which the vaccine is administered, producing antibodies that recognize a ganglioside containing a ganglioside (or an oligosaccharide portion thereof) conjugated to an immunogenic protein in an amount effective to stimulate or enhance antibody production in the subject’s body, an effective amount of an adjuvant and a pharmaceutically acceptable excipient, wherein the subject is afflicted with cancer and antibodies produced by the subject upon administration and vaccines that effectively treat cancer.

 The present invention also provides a vaccine for stimulating or enhancing the body of the subject to which the vaccine is administered, producing antibodies that recognize a ganglioside containing a ganglioside (or an oligosaccharide portion thereof) conjugated to an immunogenic protein in an amount effective to stimulate or enhance antibody production in the subject’s body, an effective amount of an adjuvant and a pharmaceutically acceptable excipient, the subject being susceptible to cancer and antibodies produced in the subject When given a vaccine, they effectively prevent cancer.

 The present invention also provides a vaccine for cancers in which cancer cells on the surface have gangliosides.

 The present invention also provides a vaccine for cancer in which gangliosides are found in the stroma of a cancerous tumor.

 The present invention provides a vaccine for cancers of epithelial, mesodermal or neuroectodermal origin. Examples of epithelial cancers are breast cancer and intrauterine cancer. An example of cancer of mesodermal origin is sarcoma. One example of cancer of neuroectodermal origin is melanoma.

 The present invention also provides a method of stimulating or enhancing the production of antibodies that recognize gangliosides in the subject's body by administering to the subject an effective dose of a vaccine to stimulate or enhance the subject receiving this vaccine, producing antibodies that recognize ganglioside containing the ganglioside (or the oligosaccharide portion thereof) conjugated to an immunogenic protein in an amount effective to stimulate or enhance antibody production in a subject, an effective amount of hell yuvanta and a pharmaceutically acceptable excipient.

 In an embodiment of the process, ganglioside is GM2.

 The present invention further provides a method for treating cancer in a subject affected by cancer, administering to the subject an effective dose of a vaccine to stimulate or enhance the subject to which the vaccine is administered, producing antibodies that recognize the ganglioside containing the ganglioside (or its oligosaccharide portion) conjugated with an immunogenic protein, in an amount effective to stimulate or enhance antibody production in a subject, an effective amount of an adjuvant and a pharmaceutically acceptable excipient, wherein the subject is affected by cancer; and antibodies produced by the subject upon administration of the vaccine effectively treat cancer.

 The present invention also provides a method for preventing cancer in a subject susceptible to such a disease by administering to the subject an effective dose of a vaccine to stimulate or enhance the subject to be given the vaccine to produce antibodies that recognize the ganglioside containing the ganglioside (or the oligosaccharide portion thereof) conjugated to immunogenic protein, in an amount effective to stimulate or enhance antibody production in a subject, an effective amount of an adjuvant and pharmaceutically an acceptable excipient, wherein the subject is susceptible to cancer and the antibodies produced in the subject upon administration of the vaccine effectively prevent cancer.

 The present invention is also directed to a method for using the above vaccine in which the ganglioside or oligosaccharide portion thereof is conjugated with cochlear lymph hemocyanin or a cochlear lymph hemocyanin derivative. The present invention is also directed to a method of using the above vaccine in which the adjuvant is QS-21.

 The present invention is also directed to a method of using the above vaccine for the treatment or prevention of cancer in which cancer cells have gangliosides on their surface.

 The present invention also aims to provide a method of using the above vaccine for the treatment or prevention of cancer, in which gangliosides are found in the stroma.

 The present invention is directed to a method of using the above vaccine for the treatment or prevention of cancer of epithelial or neuroectodermal origin. One such cancer of a neuroectodermal origin is melanoma.

 In the present invention, the term "pharmaceutically acceptable excipients" means any of the standard pharmaceutical excipients. Examples of suitable excipients well known in the art may include, but are not limited to, any of the standard pharmaceutical excipients, for example phosphate buffered saline, phosphate buffered saline containing Polysorb 80, water, emulsions, for example an oil-in emulsion -water, and various types of wetting agents.

 The vaccine of the present invention can be administered intradermally, subcutaneously or intramuscularly. You can also apply other methods well known to specialists in this field.

 In a preferred embodiment, the present invention is directed to a method for stimulating or enhancing antibodies that recognize gangliosides in a subject's body by administering to the subject an effective dose of a vaccine to stimulate or enhance the subject to which the vaccine is administered, producing antibodies that recognize ganglioside containing ganglioside (or oligosaccharide portion thereof) conjugated to an immunogenic protein in an amount effective to stimulate or enhance antibody production in The object, an effective amount of adjuvant and a pharmaceutically acceptable excipient, wherein the administering comprises administering the effective dose at two or more locations. The term "administration of an effective dose in two or more places" means that the effective dose is divided into two or more parts and each part is administered in different places of the subject. In a preferred embodiment, part of the dose is administered in three places.

 The present invention can be better understood from the following details of the experimental part. However, it should be apparent to any person skilled in the art that the methods and results discussed only illustrate the invention more fully described in the claims that follow the details of the experimental part.

The first series of experiments
Details of the experimental part.

Increased immunogenicity of GD3 conjugate vaccines;
Comparison of various carrier proteins and selection of GD3-KLH for further testing.

 Tumor-related gangliosides are known to be suitable targets for immune “attacks” against cancer, but they exhibit weak immunogenicity. Active immunization with them leads to a short-term immune response with a low titer of IgM antibodies. Covalent binding of weakly immunogenic antigens to immunogenic carrier proteins is an effective way to enhance the humoral immune response. GD3, the dominant ganglioside on malignant melanoma, was coupled to carrier proteins by two methods. It was attached using glucose oligosaccharide GD3, but this led to a loss of antigenicity and the formation of antibodies that could not react with GD3 or GD3 expressing melanoma cells. According to the second method, GD3 was modified by cleaving the double bond in the main chain of ceramide with ozone, introducing an aldehyde group and then combining this group with reductive amination with aminolysyl groups of proteins. Using this method, conjugates with synthetic polyantigenic peptides (MAPs) that detect repeats of the T cell epitope of malaria, outer membrane proteins (OMP) of Neisseria meningitidis, cationic bovine serum albumin (cB SA), cochlear lymphocytic hemocyanin (KLH) and polylysine were obtained. The antigenicity of the conjugates was confirmed by reactivity with various antibodies and immunogenicity was tested in mice. The antibody titer in immune serum was determined by ELISA and by immunostaining for purified glycosides in dot blots. The specificity of the serum reactivity was further analyzed by thin-layer immune chromatography using tumor tissue extracts. GD3 conjugate vaccines showed a significantly enhanced humoral immune response, especially vaccines with GD3-KLH conjugates. High titer IgM and IgG immune responses were induced. This method is suitable for other gangliosides; it can be suitable for the construction of ganglioside vaccines against various human cancers enriched with gangliosides.

 Materials and methods.

 Glycolipids. GM3, GM2 and GD1b and bovine brain extracts were presented by the Fidia Research Laboratory (Abano Terme, Italy). GD2 was obtained from GD1b by treatment with β-galactosidase (Cahan et al., 1982). GD3 (mel) was isolated from human melanoma tissue (Ritter et al., 1991). GD3 (bbm) (used to prepare the vaccine) and GT3 isolated from cow's milk buttermilk were kindly provided by Dr. R.K. Yu (Medical College of Virginia, Richmond, VA) (Ritter et al., 1990f). Disialyllactone (GD3 oligosaccharide) was isolated from bovine colostrum as previously described (Nicolai et al., 1978).

 Chemicals. Silica gel plates for high-resolution thin-layer chromatography (TLCHR) were obtained from E. Merck (Darmstadt, GRG); Sep-Pak C18 cassettes were obtained from Walters Associates (Milford, MA); disodium p-nitrophenyl phosphate and sodium cyanoborohydride were obtained from Sigma Chemical Cj. (St. Louis, MO); cyclophosphamide (cytoxan) was obtained from Mead Johnson (Syracuse, NY); the QS-21 containing saponin component Quil A was obtained from Cambridge Biotech (Wore ster, MA).

 Squirrels. Poly-L-lysine hydrobromide (viscometric molecular weight 3800) was purchased from Sigma, cochlear lymph hemocyanin (KLH) - y Calbiochem (LaJolla CA), cBSA immunomodulator - lmject Supercarrier - from Pierce (Rockfort, IL), outer membrane protein (OMP ) Niesseria Meningitidis was kindly provided by Dr. M. S. Blake (Rockefeller University New York, NY). The polyantigenic peptide (MAP) YAL-IV 294-1, containing 4 repeats of the T cell epitope of malaria, was donated by Dr. J.P. Tam (Rockefeller University New York, NY).

 Monoclonal antibodies. Anti-mouse rabbit immunoglobulins conjugated with horseradish peroxidase for ITLC and anti-mouse rabbit IgM and IgG conjugated with alkaline phosphatase for ELISA were obtained from Zymed (San Francisco, CA), anti-GD3 mAb R24 was generated (Houghton et a1., 1985) .

Serological tests. Enzyme-linked immunosorbent assay (ELISA) was performed as previously described (Livingston et a1., 1989). To control non-specific “adherence,” the immune serum was tested on plates that were treated similarly but to which no ganglioside was added, and the indicator was subtracted from the value obtained in the presence of ganglioside. The titer was defined as the largest dilution leading to a corrected absorption of 0.1 or higher. Immune staining of gangliosides with monoclonal antibodies or mouse serum was performed after separation on glass plates with silica gel for high-resolution thin-layer chromatography (TLCHR), as described above (Hamilton et a1., 1993). The plates were shown in solvent 1: a mixture of chloroform / methanol / water (25% CaCl ₂ ) with a ratio of 50:40: 10 (v / v) or solvent 2: a mixture of ethanol / n-butanol / pyridine / water / acetic acid with a ratio of 100 : 10: 10: 30: 3 (v / v) and was also visualized using a resorcinol / HCl reagent.

 Immunization. Six-week-old female BALB / cxC57BL76F1 mice (The Jackson Laboratory, Bar Harbor, ME) were injected intraperitoneally with cyclophosphamide (15 mg / kg) 3 days before the first immunization and were statistically assigned to the treatment groups. Groups of 4 or 5 mice were injected subcutaneously with three vaccines individually for 2 weeks, unless otherwise specified. Each vaccine contained 20 μg GD3 or 15 μg disialyllactose and 10 μg QS21 in a total volume of 0.1 ml PBS / mouse. Blood was collected from mice from the postorbital cavity 2 weeks before and 2 weeks after vaccination, unless otherwise specified.

Getting GD3 conjugate. GD3 (2 mg) was dissolved in 2 ml of methanol under the influence of ultrasound and cooled to -78 ^° C in an ethanol / dry ice bath. Ozone was obtained in an ozone generator (Del Industries, San Luis Obispo, CA) and passed through a sample for 30 minutes with vigorous stirring (Criegee, 1957, Wiegandt and Baschang, 1965). Excess ozone was displaced with nitrogen for 10 minutes. 100 μl S (CH ₃ ) ₂ (Pappas et al., 1966) was added, the sample was kept for 30 minutes at -78 ^° C, then 90 minutes at room temperature with vigorous stirring. The sample was dried over a stream of nitrogen and controlled by TLCHR. The long chain aldehyde was separated by adding 2 ml of n-hexane to a dry sample, followed by sonication for 5 minutes and centrifugation at 2000 g for 15 minutes. n-Hexane was carefully removed and discarded, and the sample was dried over a stream of nitrogen. The digested GD3 and native GD3 were separated by HPLC (Waters, System 501, Milford, VA) using a C18 reverse phase column (10 x 250 mm, Rainin Instruments, Kidge-field, NJ). Gangliosides were eluted with methanol, controlling for absorbance at 214 nm. Fractions were also analyzed by TLCHR. Fractions that contained GD3 digested were combined and evaporated at 37 ^° C using a rotary evaporator (Buchi, Switzerland). Obtained by cleavage of GD3, the carrier protein in PB S and 2 mg of sodium cyanoborohydride were incubated with gentle stirring for 48 hours at 37 ^° C. After 16 hours, another 1 mg of NaCNBH _{3 was} added. The progress of the interaction was controlled by TLCHR. In solvent 1 and solvent 2, the GD3 protein conjugates did not migrate and first appeared as a resorcinol-positive band. The mixture was dialyzed using a 1000 MWCO dialysis tube with three substitutions of each 4 L PBS at 4 ^° C. for 48 hours and passed through an extractigel (Pierce) detergent gel to finally remove unconjugated GD3. The samples were lyophilized and the protein and ganglioside contents in them were determined by Bioard protein analysis and by the determination of neuraminic acid according to the Svennerholm method (1957).

Disialyllactose was isolated from cow colostrum as previously described (Nicolai et a1., 1978). Carbohydrate was attached to the protein by reductive amination (Gray, 1974). 10 mg of disialyllactose was incubated with 2 mg of protein in 2 ml of PBS for 14 days at 37 ^° C. Initially, 2 mg of sodium cyanoborohydride was added and 1 mg of it was added every 3 days. The addition was monitored by TLCHR in solvent 2. The disialyllactose conjugates were purified by dialysis using a 1000 MWCO dialysis membrane followed by lyophilization. The content of protein and neuraminic acid was determined as described above. Disialyllactose was also conjugated to proteins according to the method described by Roy and Laferriere (1990). During this procedure, N-acryloylated glycopyranosylamine derivatives of the oligosaccharide are first formed, which are then conjugated by Michael reaction with the amino groups of the protein. Purification and determination of protein and neuraminic acid was performed as described above.

 IgG subclass definition. The IgG subclass was determined by ELISA using subclass-specific secondary monoclonal antibodies (MAbs). Secondary MAbs were used at the lowest dilution, in which reactivity to serum or negative control sera was not manifested. Goat anti-mouse IgG conjugated with alkaline phosphatase was used as the third antibody at a 1: 200 dilution.

FACS analysis of mouse antiserum. A single cell suspension of SK-ME L-28 melanoma cell line was obtained after treatment with 0.1% ethylenediaminetetraacetic acid (EDTA) in PBS and subsequent passage through a 26.5-gauge needle. Cells (3 × 10 ⁵ ) were incubated with 40 μl of diluted 1:20 serum before and after immunization for 30 minutes on ice. Cells were washed three times with 3% fetal calf serum in PBS. 30 μl of diluted (1: 50) and fluoresceinisothiocyanate-labeled goat anti-mouse IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was added as a secondary antibody and incubated on ice for 30 minutes. Cells were washed three times, as described above, and again suspended in 500 μl of 3% fetal calf serum in PBS and analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA).

Preparation and characterization of GD3 vaccines. Cow buttermilk GD3 was selectively cleaved at the C4-C5 double bond in the ceramide with ozone. Apparently, in methanol, methoxy peroxides are intermediate products that are readily reduced by dimethyl sulfite. As a result of this cleavage, a GD3 derivative with an aldehyde functional group in the position of the former double bond in the ceramide part was formed and the aldehyde in the long chain was eliminated (Fig. 1). Successfully cleaved GD3 migrated below native GD3 and, due to simultaneously cleaved unsaturated fatty acids, it appeared as a double band on TLCHR (see TLCHR, insert in Fig. 1). The densitometric determination of TLCHR showed a cleavage of more than 70% of GD3 isolated from cow buttermilk. Initial experiments with a prolonged period of ozone treatment did not change the ratio, indicating that 30% of GD3 from this source consists of analogues of sphinganin and phytosphingosine. It was found that the cleavage of GD3 at -78 ^° C with a reaction time of up to 1 hour, depending on the amount of GD3 used, is optimal. Cleaved GD3 was retained only in acidic or neutral phosphate buffers for up to 72 hours, but with an increased amount of by-product. Due to the β-elimination reaction, the release of the oligosaccharide portion of GD3 occurred in increasing amounts with increasing time, as described previously for the alkaline pH value (Wiegandt and Baseband, 1965). The carbohydrate portion isolated from GD3 did not migrate in solvent 1, but migrated along with disialyllactose isolated from bovine colostrum in solvent 2 used to separate oligosaccharides (not shown). The increased hydrophobicity of the cleaved GD3 compared to natural GD3 allowed it to be separated by HPLC on C18 reverse phase columns. The use of isocratic elution with methanol led to the formation of Schiff bases between the modified ganglioside and e-aminolysyl groups of proteins. They were reduced to form stable secondary amino groups between the ganglioside and the protein using sodium cyanoborohydride (Borch et a1., 1971). This reducing agent was selective and the aldehyde groups were not reduced in phosphate buffers with pH 6.5-7.5. The reaction was monitored by TLCHR and a change in the relationship between cleaved GD3 and the resorcinol-positive band that appeared initially was seen. This band indicated the formation of neoglycoconjugates. The reaction usually ended after incubation for 48 hours at 37 ^° C. Disialyllactose was easily removed by dialysis, an excess of cleaved GD3 was passed through a column to remove the detergent. The degree of combination was determined by determining sialic acid and protein. The mass ratio of GD3 and proteins in the conjugates depended on the availability of lysine groups in various proteins, it is shown in table 1. The average yield of GD3 conjugates and proteins was 30%.

 The carbohydrate portion of GD3, disialyllactose, was combined with proteins using two different techniques. Conjugation of disialyllactose was carried out by reductive amination, which led to the formation of glucose in the form with an open core, conjugated with proteins (Gray et a1., 1978). The technique, requiring a long period of incubation of oligosaccharides with proteins, allowed to obtain conjugates with a yield of less than 20%. The second oligosaccharide conjugation method (Roy and Laferriere 1990) led to the formation of a product with a closed glucose ring conjugated to a protein.

Serological reaction against GD3 after vaccination with vaccines containing GD3 protein conjugate
Serum before immunization did not show reactivity of IgM or IgG with GD3. Immunization with only 20 μg of GD3 or a mixture thereof with 10 μg of QS-21 adjuvant did not induce anti-GD3 antibodies (Table 1). Some groups of mice immunized with 20 μg of protein conjugated GD3 plus 10 μg of QS-21 showed an increased immune response against GD3. The GD3-poly-L-lysine conjugate, which is a high density GD3 epitope, induced a moderate titer IgM immune response (limit 1 / 20-1 / 320) and did not induce an IgG immune response. GD3 conjugated to the outer membrane proteins of Neisseria meningitidis (GD3-OM3) also induced a moderate IgM titer (limit 1 / 20-1 / 320) and a low IgG titer (limit 1 / 20-1 / 80). Only one mouse showed an IgM immune response with a high titer (1/1280) and a high IgG titer (1/2560) after vaccination with GD3-OMP, GD3 conjugated with cationized BSA (GD3-cBS A) showed a low IgM titer in the determination ELISA (limit 1 / 20-1 / 80) and a high titer of IgG (limit 1 / 20-1 / 2560). The synthetic MAP peptide containing the T cell epitope of malaria had 8 free amino groups at the N-terminal end and 4 groups were able to conjugate with GD3. The GD3-MAP conjugate induced a low IgM titer (limit 1 / 20-1 / 160) and only one mouse produced a low IgG titer 1/40. The GD3 conjugate with KLH (GD3-KLH) induced the strongest immune response (compared to other conjugates) with the highest IgM titer (limit 1 / 20-1 / 2560), as well as the highest IgG titer (limit 1 / 40-1 / 10240). Immunization with both types of conjugates of disialyllactose and protein induced only weak titer IgM, which was able to cross-react with GD3 ganglioside (limit 1 / 20-1 / 160), and did not induce a significant IgG immune response.

 The specificity of serum GD3 reactivity, as determined by immune thin layer chromatography.

 Immune thin layer chromatography (ITSX) allows you to test the GD3 antiserum for extracts of gangliosides from human body tissue and determine the specificity for gangliosides obtained from tumors. Examples of ITSX with extracts of human body tissues and high titers of IgM and IgG serum induced by immunization with GD3-KLH conjugate are shown in FIG. 3a and 3b. Serum was tested at a dilution of 1/150 for extracts of human brain gangliosides, neuroblastomas, human melanomas, and the GD3 immunogen (bbm) isolated from bovine colostrum. Reactivity on ITSC was compared with resorcinol-stained TLCHR, which showed the total ganglioside composition found in these tissues. The normal brain mainly contains GM1, GD1a, GD1b and GT1b, while the neuroblastoma extract contains in addition to the main gangliosides GD2 and GM2, and the melanoma extract contains mainly GM3 and GD3. The IgG antiserum showed specific reactivity only with GD3 in all three test tissue extracts (FIG. 3A) as control mAb R24. The IgM antiserum (FIG. 3), on the other hand, showed some cross-reactivity with structurally related gangliosides and sulfatide in the brain extract. Immune reactions induced by vaccination with other GD3 conjugates showed the same specific reactivity, but were weaker and more concentrated antiserum had to be used (not shown). High titer antisera identified by ELISA in mice immunized with GD3-cBSA had a high background on ITSX. The use of various blocking agents with these serums was unsuccessful. It was not possible to detect any specific reactivity with GD3 in the tissue extract.

 Determination of the specificity of GD3 reactive sera by staining in dot blots.

 The specificity of all high titers of IgM and IgG antisera (ELISA above 1/160) was studied using pure GM3, GD2, GD1b, GD3 and GT3 gangliosides isolated from bovine brain or buttermilk and GD3 isolated from human melanoma tissue. These structurally similar gangliosides were spotted on the nitrocellulose bands in equal amounts and treated with immune serum. Experiments with immunostaining on dot blots with sera obtained before and after immunization of mice with GD3-KLH and GD3-OMP conjugates are shown in FIG. 4. Serum did not show reactivity with these gangliosides. Sera obtained after immunization with GD3-KLH showed specific IgM and IgG reactivity with GD3 from bovine colostrum buttermilk (immunogen), as well as with GD3 isolated from human melanoma tissue. In some cases, cross-reactivity with GT3 was observed, and the reaction was also observed with a positive control R24 mAb (Houghton et al., 1985). High titer sera from mice immunized with GD3-cBSA conjugate showed only background reactivity, but did not show specific reactivity against any ganglioside (not shown). Dot blot reactivity induced by other GD3 conjugates were specific for GD3 (not shown). The results indicate that specific immune responses with high titers of IgM and IgG can be induced in mice with GD3-protein conjugates and that the strongest reactivity was induced with GD3-KLH conjugates. Apparently, the conjugation method protects important epitopes on the GD3 oligocaxapid chain and GD3 conjugates did not induce cross-reactivity with structurally related gangliosides.

 Cellular surface reactivity of immune serum as determined by FACS analysis.

 Mouse sera were tested for binding to cells of the FK-MEL-28 melanoma cell line, which is known to express cell surface GD3. A representative example of a FAC S assay using fluorescein isothiocyanate-labeled secondary goat anti-mouse antibodies is shown in FIG. 5. Serum was tested before and after immunization with GDS-KLH and QS-21. Before immunization, serum stained 8% of target cells; after immunization, 92%.

Here we describe an approach to creating vaccines for ganglioside conjugates with the aim of (1) establishing a combination reaction with proteins suitable for gangliosides of various tumors, (2) increasing the immunogenicity of GD3 as the main ganglioside associated with melanoma, and (3) determining the most effective carrier protein. Ganglioside conjugation should be carried out without affecting the immune dominant carbohydrate moiety. Modification of GD3 in its carbohydrate moiety, for example, the conversion of carboxy groups to amide groups, has been shown to increase the immunogenicity of synthetic antigens, but there was no significant cross-reactive humoral immune response with native GD3 (Ritter et al., 1990b). Therefore, this approach had in mind the combination of GD3 through its ceramide portion without affecting the carbohydrate portion. Ceramide, characteristic of all gangliosides, was digested with ozone at position 4 of the sphingosine base and a functional aldehyde group was introduced. The combination with proteins was carried out by reductive amination to form a stable amine group between the ganglioside and β-aminolysyl groups of proteins. Split
gangliosides by ozonolysis and subsequent conjugation have not yet been described; it was believed that the aldehyde intermediate of the gangliosides is unstable. Fragmentation is reported when double bond migration initiated by exposure to hydroxyl ions under alkaline conditions and β-elimination causes the release of the oligosaccharide moiety (Kanfer and Hakomori, 1983; Wiegandt and Baschang, 1965). It was found that the aldehyde functional group is quite stable at a neutral pH, Schiff bases with amino groups of proteins are easily formed, and β-elimination occurs to a small extent. A total yield of 30% was effective compared to that described for the conversion of gangliosides to lyso derivatives (Neuenhofer et a1., 1985). The aldehyde derivative GD3 no longer responded to immune thin layer chromatography (ITLC) with mAb R24. A similar phenomenon is described in connection with the reactivity of M2590 mAb with GD3, the reactivity depended on the acyl chain length (Itonori et al., 1989). Conversely, GD3 protein conjugates showed reactivity with mAb R24 in ITSX and Western blotting. This indicates that immune dominant epitopes in GD3 neoglycoconjugates have been restored.

 Once the conjugation method for the formation of the ganglioside vaccine was adopted, suitable carrier proteins had to be selected. Lowell et al., (1989) described an excellent vaccine system that induced an immune response with a high titer of antibodies by complexing bacterial carbohydrate and peptide antigens through a synthetic hydrophobic “foot” into the outer membrane proteins (OMP) of Neisseria meningitidis and was effective without additional adjuvant (Donnely, 1991). This system was directly suitable for gangliosides due to their amphipathic nature. In previous experiments, applicants absorbed gangliosides by hydrophobic interaction on these proteins and could induce immune responses with a high IgM titer (Livingston et al., 1993). Covalent attachment was used, but the GD3-OMP conjugate induced only random IgG responses, and the IgM response did not exceed test results without GD3 conjugation. Cationized BSA, which has been reported to be a strong immune modulator for protein antigens (Apple et a1., 1988), was able to increase the specific response to weakly immunogenic proteins after conjugation. GD3-CB6A conjugates induced only a moderate IgM response, but ELISA analysis showed a high titer of IgG antibodies. Further study of these high titer antisera by ITSX and dot blot immunostaining showed that the immune response was not specific for GD3. Another approach to vaccine preparation has been described by J. Tam et a1. (Tam, 1988; Tam and Lu, 1989), which proposed a polyantigenic peptide system (MAP). Based on an oligomeric branched lysine cortex, MAPs consist of 4 or 8 dendritic peptide processes containing B- and T-cell antigenic determinants. The immune response to peptides with the use of these constructs dramatically increased compared to peptides with only B- and T-cell antigenic determinants. When GD3 was attached to the terminal amino group of the MAP containing the T cell epitope of malaria, only a mild IgM immune response was detected and no IgG reaction against GD3 was detected. Although this approach is very effective for synthetic peptides, it seems to be better to use it for ganglioside vaccines. It was reported that it is possible to distinguish antibodies against GD3 gangliosides derived from tumor tissues and against normal tissues due to their different cell surface densities (Nores et a1., 1987). It was believed that the conjugate of GD3 with polylysine has a high density of GD3 epitopes combined on a single molecule. The immune response to GD3-polylysine was moderate, only the average IgM titer could be detected and there was no IgG immune response. Finally, mice immunized with cochlear lymph lymphocyte conjugated GD3, GD3-KLH, were able to generate immune responses with the highest titers of IgM and IgG, which were significantly higher than the titers generated by previous vaccines.

 It was found that these sera when tested by immunostaining on dot blots show high specificity for GD3 in human tissue extracts. Experiments on the IgM immune response versus time have shown results similar to those observed in previous studies (FIG. 2). Peak IgM titer was obtained after the third vaccination, when the vaccine was administered at two-week intervals. A rapidly decreasing immune response and continuous vaccination did not induce a significant increase in the humoral immune response. This is the first post showing the induction of an high IgG titer immune response with ganglioside vaccines. This immune response was significantly longer than the IgM response and was increased by continuous vaccination, but could not be compared with the exponential potentiation of the immune response, often observed with protein antigens. The subclass was mainly determined as IgGl; it was unclear whether the T cell dependent pathways of the vaccine with ganglioside conjugates were activated. Although the importance of T-cell assistance in B-cell maturation is undeniable, the regulation of the class of antibodies is controversial, and several reports have shown that isotope switching is possible by helper T-cells (Teale and Abraham, 1978). It was found that conjugates containing only the oligosaccharide part of GD3, did not react with R24 mAb and were not able to induce a significant immune response against GD3 ganglioside. Modification of glucose at the reducing end of the oligosaccharide chain during the conjugation process or the absence of part of the ceramide may affect the inherent presentation of the epitope and the detection of the immune system. Both methods used for conjugation were less effective and gave less yield. Inducing a specific immune response against tumor-associated gangliosides with less effective vaccines in patients who already had an immune response was associated with a better prognosis. Ganglioside conjugate vaccines have shown their ability to induce a long-lasting and specific IgG immune response in mice, suggesting that specifically the GD3-KLH conjugate may soon be suitable as a tumor vaccine for patients with melanoma.

 The second series of experiments.

Stage 1 Immunogolic Adjuvant Study
QS-21 in the body of patients with melanoma vaccinated with GM2 ganglioside covalently linked to KLH.

 Purpose: Determination of the optimal safe dose of the immunological adjuvant QS-21 for the induction of anti-GM2 antibodies.

 Premise.

 Patients with stage III JCC melanoma A have a relapse rate of two years and a mortality rate of three years 60-70% (Hilal et al., 1981; Eilber et al., 1976). Patients with stage IV melanoma who were not ill after surgery had a more ominous prognosis. There are no known treatments for changing this frequency and coefficient. The standard course of treatment for stage III melanoma after surgery is a thorough examination.

 It has been shown that some patients with melanoma have antibodies in the serum that react with highly conservative (evolutionarily stable) differentiating antigens of melanocytes. In some cases, it was noted that the presence of these antibodies was associated with an unexpectedly favorable course of the disease (Livingston et al., 1987). Since only a few patients have these antibodies in serum, attempts have been made to induce the formation of antibodies by immunizing these patients with melanoma vaccines containing the appropriate antigens. A whole cell vaccine was ineffective for this purpose (Livingston et a1., 1982). To receive the vaccine, it is now proposed to use purified antigens instead of whole melanoma cells. In recently completed studies, patients were vaccinated with BCG-GM2 and in 33 of 44 patients, the formation of short-lived IgM antibodies was detected (Livingston et al., 1989; Livingston, 1989), but the formation of IgG antibodies was rarely observed.

 There is a constant search for strong adjuvants or other ways to increase the immunogenicity of gangliosides, for example GM2, in particular to induce an IgG immune response. It was found that the most successful induction of the IgG immune response to gangliosides in mice is achieved by covalent attachment of gangliosides to the cochlear lymphocyte hemocyanin. The basis for this is the concept of split tolerance. A study of immunological tolerance and ways to overcome it showed that in various experimental systems, T-cell non-reactivity is faster induced and easier to maintain than B-cell non-reactivity (Romball et al., 1984; Weight, 1977). A content of circulating antigens suitable for maintaining T-cell tolerance is not able to maintain B-cell tolerance. Therefore, if T helper cells are provided (such as, for example, by strong other antigens, such as KLH covalently attached to the target immunogen), antibodies to tolerized T cell dependent antigens can be induced. This method has been successfully used to induce IgG antibodies against various carbohydrate antigens in experimental animals (Kundu et a1., 1980; Gray, 1978; Chang and Rittenderg, 1981; Longencker et a1., 1987) and recently against the polysaccharide antigen H. Influenza in children .

The molecular mass of KLH is very variable, but close to 2 • 10 ⁶ daltons. It was injected intradermally into patients by several researchers (Berd et a1., 1982) in an amount of 1 mg to induce delayed hypersensitivity (DTH). Pyrogen-free KLH obtained by Biomira Inc. (Edmonton, Canada), covalently coupled to GM2 at high epitope density (1000/1). Using these preparations in a mixture with immunological adjuvants, a high titer anti-GM2 IgG immune response was induced in mice.

 Of the immunological adjuvants tested in preclinical studies with vaccines of KLH conjugates, for example T-antigen-KLH, QS was the most effective. In most mice, the IgG antibody titer was 1/4000 and strong DTH was observed. When only T-KLH was administered, an average titer of 1/160 was observed and no DTH was detected. QS-21 is a carbohydrate extracted from the bark of the Quillaja saponaria Molina tree bark of South America. The monosaccharide composition, molecular weight, adjuvant effect and toxicity of a number of these saponins have been described (Kensil et a1., 1991). QS-21 was chosen due to its adjuvant ability and lack of toxicity. It has proven non-toxicity and high efficacy in increasing the immunogenicity of the FeLV subunit vaccine in cats (Marciani et a1.) And the recombinant HIV-1 vaccine in rhesus monkeys.

 In addition, it has been shown that some melanoma patients have suppressor cells that can interfere with immunization and that these cells can be inhibited by a low dose of cyclophosphamide (Livingston et al., 1987), with each patient receiving a low dose of cyclophosphamide before the first vaccination. It was found that this combined method increases the immunogenicity of glycolipids and other antigens in experimental animals and patients with melanoma (Livingston et al., 1987a, Livingston et al., 1989).

 Population to study.

 Patients with a high risk of stage III or IV malignant melanoma AJCC 2-8 months after surgical resection, microscopic pathology preparations of which were studied at the Memorial Hospital Department of Patology, in which no disease was clinically detected, are desirable. They must have a clinical effect state of 80 (Karnofsky) and an intended surgery (not including melanoma) after at least 5 years. Pregnant women, patients with an allergy to sea food, and patients with a creatine or bilirubin content higher than 2.0 are excluded. Patients may have previously been exposed to radiation, chemotherapy or immunotherapy (completed 8 weeks before vaccination).

 Examination of treatment.

 Patients must receive a physical examination at Memorial Hospital and an X-ray of the chest, a clinical blood test, a blood serum creatinine test and a blood function test and a liver function test within 3 weeks of treatment. Patients with an abnormal liver function test or chest x-ray results are acceptable unless further examination (e.g. CCT, tomograms) reveals melanoma.

 Getting the vaccine.

 Chemistry and cooking.

Drug substance
Title and Source
Correct name:
GM2-KLH, a tumor-associated synthetic glycoconjugate (S-TAG) - used for active specific immunotherapy.

 GM2-HSA, a tumor-associated synthetic glycoconjugate (S-TAG) - used to test the skin of patients undergoing specific specific immunotherapy with GM2-KLH conjugate.

Chemical Name:
11 ³ NeuAc-GgoSe ₃ Cer-hemocyanin snail lymph (KLH)
Lab Coding:
GM2-KLH series # 5
GM2-HSA Series # 1
Manufacturer: Biomira Inc. Research Center One, Edmonton Research and Development Park, 9411-20 Avenue Edmonton, Alberta T6N IE5 Canada
The materials used to obtain hapten GM2, see table. eleven
The materials used for conjugation, see table. 12.

Chemistry of manifestation
The data for GM2 and GM2-aldehyde, see table. thirteen.

The structure of GM2 and GM2 aldehyde was characterized by ¹ H NMR spectroscopy, thin layer chromatography (TCX), FAB mass spectroscopy and FT-IR spectroscopy.

Structural data
¹ H (DMSO-d ₆ : D ₂ ) δ: 9.48 (d, 1H, J = 2.0 Hz), 4.79 (d, 1H, J = 8.5 Hz, W-1), 4.26 (d, 1H, J = 8.0 Hz, P-1), 4.19 (d, 1H, J = 8.0 Hz, 1-1), 2.54 (dd, 1H, A-3e), 1.88 (s, 3H, Ac), 1.78 (s, 3H , Ac), 0.85 (t, 3H, J = 6.6 Hz, CH ₃ ).

FT-IR spectroscopy (KBr tablet, cm ^-1 ): 3439, 3420, 2952, 2923, 2851, 1634, 1070 (probably gem diol)
TCX: R _f = 0.5 (CHCl ₃ -CH ₃ OH-0.2% aqueous CaCl ₂ 5: 4: 1)
The data for KLH, GM2-KLH, HSA and GM2-HSA see table. 14.

 Snail lymph hemocyanin (KLH) is a large complex protein consisting of a number of subunits with a lower molecular weight. KLH is extracted from the mollusk lymph (Megathura crenulara) and purified. KLH, HSA and conjugates characterized Biomira Inc. gel filtration chromatography on sepharose CL-4B, isoelectric focusing (IEF) and colorimetric method using resorcinol hydrochloric acid (1) - see table 1, 7.

 1. L. Svennerholm, Biochimica et Biophysica Acta, 24, (1957), 604-611.

 2. Using the gel filtration method, whole molecules of KLH protein are eluted on CL-4B Sepharose.

The volume of the column, which indicated that the molecular weight of KLH is higher than 2 • 10 ⁶ . This value is consistent with the mass limit given in the literature for this protein.

 Technological scheme of obtaining GM2-KLH.

Stage 1 - cleaning GM2:
GM2 (FIDIA) passing for viral test
↓
Silica gel column chromatography
1. chloroform-methanol, 65:35
2. chloroform-methanol-water, 65: 35: 4
↓
To conduct QC tests, we used:
1. TCX
2. ¹ H NMR spectroscopy
Stage 2 - cleaning KLH:
KLH was dissolved in PBS, pH 7.5 (about 3 mg / ml)
↓
Centrifuged
↓
A sample of dissolved KLH was passed through a Sepharose column to determine molecular weight
↓
Diafiltered with sequential use of the following buffers:
1. PBS, pH 7.5
2. Tris-HCl, EDTA, pH 7.75
3. Tris-HCI, EDTA, 0.5% DOC, pH 7.75
4. Tris-HCI, EDTA, pH 7.75
5. PBS, pH 7.5
Sterile pyrogen-free PBS was added until a volume of 75 ml was established.
↓
Centrifuged
↓
Sterile filtered
↓
Biorad protein analysis
↓
A KLH sample was passed through a Sepharose column to determine molecular weight
↓
Set the concentration to 10 mg / ml with PBS buffer, pH 7.5
Aliquots of KLH were placed in serum tubes
↓
and frozen at -25 ^o +5 ^o C
↓
In the process of testing used:
1. Isoelectric Focusing (IEF)
2. Test for pyrogen lysate Limulus amebocyte (LAL)
↓
Stage 3 - synthesis of GM2-aldehyde (compound # 2):
GM2 (compound # 1)
↓
(1) O ₃ , methanol
↓
(2) CH ₃ S CH ₃
GM2-aldehyde (compound # 2, may be heme diol)
In the tests conducted at home, they used:
1. TLC
↓
Stage 4 - conjugation of hapten GM2 with KLH:
↓
Thawed sterile, pyrogen-free KLH
↓
KLH was added to the hapten in a ratio of 4: 1 (mass / mass)
↓
Incubated at room temperature with shaking for 3 minutes
↓
NaBH ₃ CN was added to the hapten / KLH mixture at a ratio of 1: 1 (w / w) to hapten
↓
The reaction mixture was carefully stirred at room temperature overnight and then at 40 ^o C for 4 days
↓
Stage 5 - diafiltration of the conjugate:
The conjugate was diafiltered using
1. PBS, pH 7.5
2. Tris / EDTA, pH 7.75
3. Tris / EDTA / 0.05% DOC, pH 7.75
4. Tris / EDTA, pH 7.5
5. PBS, pH 7.5
The conjugate was aseptically removed from the Amicon titration apparatus
↓
Centrifuged
↓
The conjugate was sterile filtered.
↓
In the QC test process, the following were used:
1. Biorad protein analysis
2. Sepharose gel filtration
3. Isoelectric Focusing (IEF)
↓
Conjugate concentration was aseptically set to 1 mg / ml
↓
The conjugate was distributed in 1 ml of sterile, pyrogen-free serum and frozen at -20 ^o +5 ^o C
↓
Final QC Test:
1. Enzyme-linked immunosorbent assay with affinity chromatography (EIA)
2. Test for pyrogen LAL
3. Biorad protein analysis
4. Analysis using resorcinol-HCl
5. Test for rabbit pyrogen
6. General security test
7. Sterility test
8. Test for impurities for cyanide
The method of obtaining conjugates GM2-KLH
The preparation of the semi-synthetic glycoconjugate GM2-KLH for ASI and the semi-synthetic glycoconjugate GM2-HSA for skin testing are carried out in 5 stages:
1. Purification of the resulting GM2 (cow source) (compound # 1).

 2. Purification of cochlear lymphocytic hemocyanin (KLH).

 3. Synthesis of GM2-aldehyde (compound # 2).

 4. Conjugation of hapten GM2 with KLH.

 5. Diafiltration of the conjugate.

Stage 1: Purification of GM2 (compound # 1):
Name: Ganglioside GM2
Abbreviation: II ³ NeuAc-GgOse ₃ Cer
A GM2 ganglioside sample (cow source), source material provided by FIDIA, is sent for viral testing (8CFR protocol). All glass products are washed with distilled acetone and then with distilled ethanol and then kept at 130 ^° C. for 18 hours before use. A silica gel column (Michel-Miller S 795-10) (30.5 g, 60H silica gel, Art 7736, E. Merck) was filled at 5.3 psi (SSI Model 300Lo pump) using a mixture of chloroform and methanol (65:35 ) as a solvent. GM2 (200 mg) is administered as a concentrated solution of a mixture of chloroform and methanol (65: 35). Elution is carried out with this solvent and then with a mixture of chloroform, methanol and water (65: 35: 4). Fractions were analyzed by TLC (R _f 0.6, chloroform-methanol-0.2% aqueous CaCl ₂ , 5: 4: 1 mixture). The fractions containing GM2 were combined and evaporated to give a creamy white amorphous solid.

A method for testing this material (compound 1) involves ¹ H NMR spectroscopy and thin layer chromatography (TLC) to confirm the identity and purity of the ganglioside. Test results must meet the specifications specified in the chemistry of new developments. If this material is found to be unclean, then this purification is repeated.

 Stage 2: Obtaining a sterile, pyrogen-free snail lymphocyte hemocyanin (KLH).

Getting KLH:
This entire procedure is carried out inside a Class 100 biohazard chamber. Snail lymph hemocyanin (KLH) obtained from Calbiochem is dissolved in 100 ml of sterile pyrogen-free phosphate buffered saline (PBSP, pH 7.5). This solution was incubated at 2 ^° - 6 ^° C for 18 hours to dissolve KLH in the solution. The solution is then centrifuged at 200 rpm for 30 minutes. The supernatant was taken and sample was passed through a CL-4B Sepharose gel column to determine the molecular weight of untreated KLH.

 Before dialysis, KLH, the Amicon ultrafiltration unit with stirring, is sterilized and pyrogen free by washing it 4 times with sterile water for injection (WFI) first and then filling it with 95% ethanol and keeping the ethanol in it under stirring for 2 hours. The installation is again rinsed with WFI water, then autoclaved.

The supernatant containing KLH was poured into a sterile, pyrogen-free Amicon diafiltration unit with a YM 30 filter (a material with a molecular weight of 30,000 was separated). The total volume of KLH is then adjusted to 350 ml sequentially with the following sterile, pyrogen-free or low pyrogen-free buffers:
1. 1 complete replacement of PBS with pH 7.5 (sterile, pyrogen-free)
2. 3 complete replacements for Tris-HCl, EDTA, pH 7.67 (sterile, low pyrogen content)
3. 2 complete replacements for Tris-HCl, EDTA, pH 7.75 with 0.5% deoxycholic acid (DOC) (sterile, low pyrogen content)
4. 4 complete replacements for Tris-HCl, EDTA, pH 7.75 (sterile, low pyrogen content)
5. 3 complete replacements of PBS with pH 7.5 (sterile, pyrogen-free).

 Each buffer change consisted of reducing the Amicon unit volume to 50 ml or lower and then adding buffer until a volume of 350 ml was reached.

Sterile, pyrogen-free PBS is prepared using chemicals that are heated at 180-185 ^° C. for 4.5 hours. The chemicals are added to sterile water for injection (WFI) and mixed in a sterile, pyrogen-free container. Chemicals or other buffers must not be heated to depyrogenate them, since they melt at such extreme temperatures, so these buffers are obtained in sterile WFI water in sterile, pyrogen-free containers and sterilely filtered through a 0.22 μm filter. The pH of PBS and Tris-HCl buffers is adjusted to the required value using a sterile, pyrogen-free 2 sodium hydroxide solution.

 The DOC in Tris-E TA buffer, pH 7.75, serves to decompose pyrogens into their smaller subunits that pass through the filter, while the KLH protein is retained on the Amicon filter (8, 9).

 The KLH solution was aseptically removed from the Amicon unit and centrifuged again at 2000 rpm for 30 minutes. The solution is then transferred to a sterile, pyrogen-free graduated cylinder and the final volume is set at 75 ml using a sterile, pyrogen-free PBS with a pH of 7.5.

 The supernatant is then sterile filtered through a 0.22 μm filter, fixing the protein with a lower molecular weight. A KLH sample was passed through a CL-4B Sepharose column to determine if the molecular weight of KLH had changed as a result of processing it with buffers (in particular, DOC) compared to the results of column chromatography of untreated KLH. The molecular weight should not vary significantly. An aliquot of KLH was selected and Biorad protein analysis was performed using KLH for a standard curve. The final volume of the KLH solution is aseptically set using sterile, pyrogen-free PBS to obtain a final protein concentration of 10 mg / ml.

 LAL testing is performed to determine the pyrogen content in purified KLH. The pyrogen content should be below 10 EU / mg for KLH, which is used for the conjugation procedure.

 Isoelectric focusing (IEF) is performed to control the purity and identity of KLH. Past batches of KLH were used in parallel as standards.

Aliquots of 10 ml KLH solution are dispensed into sterile, pyrogen-free 30 ml serum tubes and closed with sterile, pyrogen-free tubes. KLH is then frozen at -20 ^° ± 5 ^° C until the hapten conjugation time.

Stage 3: Synthesis of GM2-aldehyde (compound # 2, heme diol)
All glass products were washed with distilled methanol and dried (130 ^° C.) for 18 hours before use. A solution of purified ganglioside is prepared. GM2 (compound # 1) (40 mg) in distilled methanol (10 ml) was stirred at -15 ^° C (cooling with dry ice in ethanol) and ozone gas was passed through the solution for 7 minutes (Orec 03V10-0 ozonizer). Then, a stream of argon is passed through the solution under the control of the TLC reaction (chloroform-methanol-0.2% aqueous solution of CaCl ₂ , 5: 4: 1). The solvents are then removed under reduced pressure and the resulting material is dissolved in distilled methanol. Methyl sulfide (200 ml) was added to this solution, and the reaction mixture was stirred at room temperature for 1 hour. The solvents are then removed and the residue triturated with diethyl ether (4 x 25 ml). The resulting white solid (compound # 2) was dried in vacuo for 15 minutes to remove the remaining solvent and then used directly in the subsequent conjugation step.

Due to the unstable nature of the obtained aldehyde (β-elimination), compound 2 is identified, as usually only TLC. TLC usually indicates the presence of a small amount of sphinganin or phytosphingosine analogue (has the same R _f as compound # 1) and a small amount of reducing sugar (R _f 0.32).

Stage 4: Conjugation of the hapten GM2 with KLH (or HSA)
All operations are carried out in a Class 100 biohazard chamber.

 Two ampoules, each containing 10 ml of frozen sterile pyrogen-free KLH (10 mg / ml), are thawed to room temperature immediately before use.

 The aseptically measured amount (16 ml) of KLH protein is added to a bottle containing lyophilized hapten GM2 and a magnetic stirring bar. The solution was gently mixed for 3 minutes at room temperature to transfer the entire hapten into the solution.

Sodium cyanoborohydride (NaBH ₃ CN) (40 mg) was added to the hapten / KLH solution and the flask was sealed with a stopper equipped with a sterile filter needle. The solution was gently shaken and then incubated overnight at room temperature. Then the solution is incubated at 40 ^o C for 40 days.

Stage 5: Diafiltration of glycoconjugates (GM2-KLH)
The contents of the hapten / KLH reaction vial are aseptically transferred to a sterile, pyrogen-free Amicon filter unit with a YM-30 filter. Filtered nitrogen is passed to establish an operating pressure in the Amicon chamber of 1.125 at. The conjugate is then diafiltered using sequentially the following sterile, pyrogen-free or low pyrogen-containing buffers:
1. 2 complete replacements of PBS with pH 7.5 (sterile, pyrogen-free)
2. 2 complete replacements for Tris-HCI, EDTA with pH 7.75 (sterile, low pyrogen content)
3. 2 complete Tris-HCI replacements with pH 7.75 and 0.5% deoxycholic acid (DOC) (sterile, low pyrogen)
4. 4 complete replacements of Tris-HCI with pH 7.75 (sterile, low pyrogen content)
5. 3 complete replacements of PBS with pH 7.5 (sterile, pyrogen-free).

 The glycoconjugate is then aseptically removed from the filtration unit and centrifuged for 30 minutes at a speed of 2000 rpm. The supernatant is then sterile filtered using a 0.22 mm filter fixing the lower proteins.

A glycoconjugate sample is obtained and the following QC tests are performed:
1. Gel filtration through sepharose
2. Isoelectric Focusing (IEF)
3. Protein analysis according to the Biorad method
Based on the results of the protein analysis, the final volume of the glycoconjugate is determined using a sterile, pyrogen-free buffer with a pH of 7.5 at such a level that a protein concentration of 1 mg / ml is reached.

The final glycoconjugate is then dispensed in 0.5 ml portions in a Class 100 bio-safe chamber with a 0.1 ml deflection volume into sterile, pyrogen-free, 1 ml transparent serum borosilicate tubes with red rubber stoppers and frozen at -20 ^o C In the process of filling the tubes, the air inside the filled space is controlled by exposing two plates with blood agar to the action of air near the working space inside the chamber for at least 30 minutes. These plates are then transferred to an incubator (37 ^° C.) and incubated for 1-2 days. The plates are then examined for the presence of bacterial or fungal colonies.

The tubes are placed inside the chamber with a label indicating the product name, batch number and number of tubes. The camera is then sealed and a label with the same information is placed on the outside of the camera. The camera is quarantined in the refrigerator for 1-2 days before labeling can be performed. After the last QC tests are done, tagging is required. The product is tagged by personnel, then tagging is checked by the Quality Control department or Regulatory Affairs department. The final product document is then signed by the Director of Regulatory Affairs and Vice President and COO Immunotherapeutics division. The product is then freed and stored in the refrigerator for the "freed" product at -20 ^o C.

Each series of GM2-KLH and GM2-HSA conjugates is subjected to the following tests for ultimate quality control:
1. Enzyme-linked immunosorbent assay with affinity chromatography (EIA)
2. Test for LAL-pyrogen
3. Protein analysis according to the Biorad method
4. Resorcinol-HCl-carbohydrate analysis
5. Test for rabbit pyrogen
6. General security test
7. Sterility test
8. Testing for impurities for cyanide.

 GM2-KLH gets Biomira Inc. (Edmonton, Alberta), it is used under IND U.S. Food and Drug Administration. The molar ratio of GM2 to KLH is 800/1 (real 200-1400). The conjugate is fed at a concentration of 0.57 mg per 0.5 ml of phosphate buffered saline (PBS). This amounts to approximately 70 μg of GM2 ganglioside and 500 μg of KLH per 0.5 ml of PBS. On the day of vaccination for the initial 5 immunizations, 0.5 ml of the vaccine should be placed in individual syringes and delivered to the clinic for administration. This dose, containing 70 μg GM2, as shown in previous studies with GM2 and various adjuvants, is an effective dose. The last (sixth) immunization should contain half of this dose, 35 μg GM2 and 250 μg KLH.

QS-21 extracted Cambridge Bioscience Inc. (Worcester, MA) from the bark of the Quillaja saponaria Molina tree bark by chromatography on silica and chromatography with reverse phase, as described previously (16). The purified conjugate GM2-KLH and QS-21 are tested for sterility using standard methods in a bacteriological laboratory to control pyrogenicity for rabbits and safety for rabbits and mice. They are aliquoted and stored at a temperature of -15 ^o to -25 ^o C. On the day of vaccination, 570 μg GM2-KLH (or 285 μg for the sixth vaccination) is mixed with QS-21, injected into an individual syringe, labeled and delivered to the clinic .

 4 doses should be used: 10, 50, 100 and 200 μg, each of which is diluted to a total volume of 0.25 ml of PBS. The first group of 3 patients should receive 6 vaccines containing 10 μg QS-21, the next 3 patients - 50 μg QS-21, the next 3 patients - 100 μg QS-21 and then 3 patients - 200 μg QS-21. No patient should proceed to the next dose until all 3 patients who have received the previous dose receive at least two vaccinations. If toxicity is not observed at a dose of 200 mcg and if the immunological reactivity to the GM2 antigen and KLH does not leave the rectilinear region within 50-200 mcg, then a dose of 400 mcg can be administered to 3 additional patients. After determining the safe and maximally immunogenic dose, 6 additional patients should be immunized with the same dose to better determine the humoral immune response.

 IND for GM2-KLH plus QS-21 owns MSKCC.

 Vaccination.

Three to five days before the first immunization, 200 mg / m ² cyclophosphamide IV is administered. Applicants have successfully used this dose and regimen in past vaccination studies. Four vaccinations were then given subcutaneously at intervals of two weeks, starting 2-30 weeks after surgical resection of the entire known lesion. Two additional vaccinations were carried out at two-month intervals.

 Rating.

 Serological reaction: The main endpoint of this test is the serological reaction. Blood of the peripheral system (30 ml) should be taken immediately before each vaccination and 2 and 5 weeks after the fourth, fifth and sixth vaccinations. Therefore, blood can be taken over a 3-month interval, while a detectable amount of GM2 antibodies is maintained. Serum obtained 2 weeks after the fourth, fifth, and sixth vaccination in all patients should be ELISA tested for antibodies against GM2 and related gangliosides. Patients with a titer of 1/80 and above, as determined by ELISA or immune thin-layer chromatography for various glycolipids that respond specifically, are considered to have a serological reaction (serological respondents). Additional serum from serological respondents can be tested to better determine the humoral immune response to vaccination. An additional 60 ml of peripheral blood could be obtained 2 weeks after the fourth vaccination to study the immune response against GM2 at the clonal level if the applicants had evidence of the induction of a high titer of anti-GM2 IgG antibodies. The supernatants from EBV-transformed lymphoblasts and then the resulting hybridomas should be used for this purpose.

 Slow-type hypersensitivity: testing of the skin for delayed-type hypersensitivity against KLH, GM2 and GM2 attached to human serum albumin should be performed during the fifth immunization. Reactions with an induction of more than 5 mm in 48 hours should be considered positive, but in the case of GM2, further testing of the skin with various gangliosides should be carried out.

 Clinical period: Patients should be examined at Memorial Hospital during their fourth or sixth vaccination, chest x-ray and fluoroscopic profile should be performed during the sixth vaccination. Additional and subsequent monitoring should be carried out by their oncologists.

 Criterion for stopping immunization.

 Local relapse, which is usually treated with local therapy (surgery or injection at the site of injury), does not serve as a reason for stopping immunization if the patient is freed from the disease. Immunization should be discontinued if the patient develops a relapse disease that requires systemic treatment or radiation therapy.

 Statistical discussions.

 This study phase 1 deals primarily with the assessment of toxicity and IgG immune response against GM2 and KLH after vaccination. Three patients should be vaccinated 4 times with each time increasing dose of QS-21. If the IgG immune response continues to increase with each dose increase, then the highest dose can be selected, causing no more than systemic toxicity of 1 degree or local toxicity of 2 degrees. If toxicity is not observed, then administration of the fifth dose should be tested. Based on a study of mice, it is assumed that the IgG immune response after the second or third dose will be in the form of a peak or plateau. Once a safe and immunogenic dose limit has been established, 3-6 additional patients should be immunized with the dose that turned out to be the most immunogenic, and the next higher and lower doses, to be sure of the degree of toxicity and immunogenicity of these doses.

 Risk.

GM2-KLH: 100 patients who were given vaccines containing 200 μg GM2 did not observe any toxicity due to GM2. KLH was used in doses of 1 mg to immunize and test the immune status in 50 patients without toxicity (15). 18 patients were immunized with vaccines containing GM2-KLH. One patient experienced pain and soreness when touching at the vaccination site for 48 hours, attributed to GM2-KLH, probably delayed-type hypersensitivity to KLH. No dose reduction was required. The only suspected side effect of GM2-KLH was inflammation of the vaccination sites for several days. If there is a fever with a temperature above 38 ^o C or strong local toxicity, which limits the normal activity, then the dose of GM2-KLH in the following vaccines can be reduced by factor 3.

Saponins have not been used in humans. They are known to be hemolytic, but the QS-21 fraction was chosen because it did not exhibit obvious toxicity at such high doses as 125 μg per mouse, cat, or rhesus monkey. When counting on m ² or kg, this dose is hundreds of times higher than the maximum dose proposed here.

 Autoimmune or hypersensitivity reactions to vaccine components or skin test antigens are theoretical, but operate within the scope of the possibility.

 Toxicity criterion: Toxicity should be graded according to the Common Toxicity Criteria developed by the National Cancer Institute (NCl). These criteria are found in documents with the IRB.

 The importance of learning.

 To increase the immune response to cancer, one can try two main methods of non-specific immunopotentiation, which form the main part of the past and present achievements of cancer immunotherapy, and specific immunization, which was not really appreciated in the treatment of cancer, but significantly contributed to the fight against infectious diseases. This is the knowledge of a microbial antigen, which allows the development of successful specific immunization against infections. On the other hand, the lack of knowledge of human cancer antigens has hindered the proper investigation of specific immunization in the context of cancer using vaccines with a specific cancer-related antigenicity and demonstration of their immunogenicity in cancer patients. Recent progress in detecting surface antigens of melanoma cells now provides an opportunity to investigate specific immunization.

 Results.

 GM2-KLH was used as an antigen in a phase 1 study with QS-21. Patients with stage III and IV AJCC melanoma who did not have this disease after surgical resection of the entire known affected area were immunized with 500 μg GM2-KLH (ratio (GM2 / KLH was 200-1400 / 1, GM2 dose 70 μg) plus 10, 50 , 100 or 200 μg of QS-21 in groups of 3 patients each. All patients received a total of 6 immunizations. No toxicity was observed associated with a dose of 10 or 50 μg of QS-21. The dose of 100 μg was also quite tolerant, but each of 3 patients who were given this dose had, at least after the first of 6 vaccinations, tangible swelling and pain on pressing for 24-48 hours and 2 of these patients had this phenomenon after 2 of 6 immunizations. No systemic side effects were detected. 3 patients who were given 200 mcg experienced pain on pressing and pain in each vaccination. injection sites for 2-10 days and mild fever and flu symptoms, including mild headache and diffuse pain, within 8-24 hours after most of the vaccinations. Serological responses against GM2 and KLH for these 12 patients are summarized at the bottom of Table 2. Although toxicity with a dose of 200 μg was not really maximally tolerable, it was significant and there was no evidence that it was associated with increased immunogenicity compared with a dose of 100 μg . In the usual way, an additional 9 people were immunized, and 3 of them received a dose of 50, 100 or 200 μg to confirm these toxicity and immunogenicity results. IgM and IgG titers of 6 patients receiving GM2-KLH plus 100 or 200 μg QS-21 are shown in FIG. 6. It can be seen that GM2-KLH plus QS-21 (100 μg) is a shock immunogenic vaccine, far superior to the GM2 / BCG or GM2-KLH plus DETOX or BCG vaccine both in titer and duration of IgM and IgG antibodies.

 Having shown that the GM2-KLH and GD3-KLH plus QS-21 vaccines are significantly more immunogenic than previous ganglioside / BCG vaccines, the immunogenicity of GD2, GD3 and GD3-lactone in these vaccines of new conjugates should be tested in a clinical setting. The basis for the selection of these gangliosides as immunogens in melanoma is the quantitative analysis of 20 melanoma biopsies using thin-layer immune chromatography to quantify the content of these gangliosides on melanoma and various normal tissues.

 6-12 patients with melanoma should be vaccinated with each of these ganglioside conjugates that have been obtained and covalently linked to KLH. Conjugates that detect immunogenicity in these prospective studies should then be combined and tested in an additional group of 12 patients as an entry into a large multicenter study aimed at determining the effect of persistently immunogenic multivalent melanoma ganglioside vaccine on relapse rate and survival.

 The peak titer of antibodies against ganglioside GM2 after immunization with GM2-KLH plus various immunogenic adjuvants are given in table. 2.

Third series of experiments
The cell surface gangliosides GM2, GD2 or GD3 are often overexpressed in malignant melanoma. Applicants had previously discovered that immunization of patients with GM2 melanoma and BCG induced an IgM immune response in most patients and that patients with a high titer of antibodies to GM2 showed increased survival. As usual with carbohydrate antigens (which are T-independent), the IgM immune response was short-lived and the IgG immune response was rare. To increase immunogenicity, applicants conjugated GM2 covalently with cochlear lymphocytic hemocyanin (KLH). GM2-KLH vaccine was administered to melanoma patients with one of three adjuvants: BCG, DETOX, or QS-21. A more effective vaccine was GM2-KLH with QS-21. She induced a much higher titer and a longer IgM immune response to GM2 and a persistent IgG response (IgGI and IgG3 isotype). She also induced the highest titer of antibodies against KLH. The results confirmed that the GM2-KLH plus QS-21 conjugate vaccine caused significant T-cell assistance. Since no significant toxicity was detected, the method using such a vaccine is attractive for increasing the immunogenicity of other gangliosides, for example GD2 and GD3, and for determining the effect of ganglioside antibodies on the development of melanoma. In addition, the discovery that QS-21 significantly enhances the immunogenicity of GM2-KLH suggests that such an increase may be valid for other conjugate vaccines, many of which are usually used without adjuvant.

 Introduction

One of the changes that occur in the process of malignant transformation is the altered nature of the expression of cell surface gangliosides in some types of cancer, including malignant melanoma (1). In normal melanocytes, the main ganglioside is GM3. The content of other gangliosides, which include GD3, GM2, GD1a and GT1b, is less than 10% of the total number of gangliosides (2). In malignant melanoma, activation of glycosylating enzymes leads to increased expression of GD3, GD2, GM2 and 9-0-acetyl-GD3 (3, 4). These overexpressed gangliosides are attractive targets for immunotherapy, including active immunization with ganglioside vaccines. In a number of studies involving immunization with GM2 vaccines for patients with malignant melanoma, applicants found that vaccination (after a low dose of cyclophosphamide and with BCG as adjuvant) induces the formation of IgM antibodies on GM2 in most patients (5) and that the disease-free period and survival is rising
in patients producing a high titer of antibodies to GM2 (6, 7). However, the induced immune response to GM2 has the characteristics of a T-independent immune response (mainly IgM antibodies, short duration, unstable IgG immune response, lack of a secondary immune response) and other melanoma gangliosides, GD3 and GD2, are not immunogenic when administered with the same image (8). Since suitable epitopes are carbohydrates, applicants have explored approaches to enhancing immunogenicity based on the successful development of carbohydrate vaccines for bacterial infections. Applicants have found that in mice, the immunogenicity of GD3 is markedly enhanced by covalent binding to cochlear lymphocytic hemocyanin and that mice immunized with GD3-KLH conjugate and QS-21 adjuvant have a high titer IgM immune response, followed by a strong, long-lasting IgG immune answer (9). Applicants have now begun to test conjugated ganglioside vaccines in a clinical setting and report here the first results of immunization of patients with malignant melanoma GM2-KLH conjugate vaccine.

 Materials and methods.

Patients
Patients with stage III malignant melanoma were considered suitable if all metastatic lesions were removed within the last 4 months. None of the patients had undergone prior chemotherapy or radiation therapy.

Receiving and administration of a vaccine
GM2-KLH vaccine: GM2-KLH conjugate was obtained by Biomira Inc. (Edmonton, Alberta) as previously described for the GD3-KLH conjugate vaccine (9). Briefly, the conjugation procedure included ozone cleavage of the double bond of the ceramide part of GM2, the introduction of the aldehyde group and conjugation with reductive amination with the KLH aminolysyl groups. The molar ratio of GM2 / KLH was approximately 800/1, and the vaccine was administered with a concentration of conjugate of 0.57 mg in 0.5 ml of normal saline. This amount was the dose for one patient and contained 70 μg GM2 and 500 μg KLH. Each group of 6 patients received GM2-KLH conjugate without adjuvant, GM2-KLH with DETOX, GM2-KLH with BCG and GM2-KLH with QS-21.

Four vaccinations were given intradermally, in extreme cases, with direct lymphatic drainage at 2-week intervals and the next additional vaccination at 8-week intervals. 4-6 days before the first vaccination, 200 mg / m ² cyclophosphamide was administered intravenously to all patients (Cytoxan, Mead Johnson and Co., Evansville, N).

 Immunological adjuvants: DETOX was prepared and presented by Ribi Immunochem Research Inc. (Hamilton, MT) as a lyophilized oil emulsion. It consisted of cell wall cages (CWS) of Galmette-Guorin bacillus and monophosphoryl lipid A (MPLA) from salmonella minnesota R595. On the day of vaccination, 0.25 ml of DETOX (250 μg CWS and 25 μg MPLA) were mixed with GM2-KLH. The vaccine (final volume 0.75 ml) was mixed by swirling for 2-3 minutes and administered to patients for 15 minutes. BCG was acquired from Bionetics Research Inc. (Rockville, MD). On the day of vaccination, 107 viable units of BCG in 0.1 ml of normal saline were added to the GM2-KLH vaccine in each individual syringe (total volume 0.6 ml). The contents were mixed and administered to the patient for 15 minutes. Adjuvant QS-21 (homogeneous saponin isolated from Quillaja saponaria Molina bark and purified) (10, 11) was kindly provided by Cambridge Biotech. Inc. (Worcester, MA). 100 μg or 200 μg QS-21 was diluted with 0.25 ml of normal saline and mixed with GM2-KLH. The vaccine (final volume 0.75 ml) was mixed by swirling for 2-3 minutes and was administered for 15 minutes.

Gangliosides
Cow brain GM2 and GD1b were donated to us by the Fidia Research Laboratory (Abano Terme, Italy). Cow brain GM3, GM1, and GD1a were purchased from Sigma Chemical Co (St. Louis, Mo.). Asialo-GM2 was obtained by treating GM2 with 0.1 M trifluoroacetic acid at 100 ^° C. for 1 hour, followed by separation on a C18 Sep-Pak reverse phase column (Waters, Milford MA). GD2 was obtained from GD1b by treatment with β-galactosidase (12). GD3 isolated from cow buttermilk was kindly provided by Dr.RKYu (Medical Colledge of Virginia, Richmond, VA).

Reagents and monoclonal antibodies
Silica gel plates for high-resolution thin-layer chromatography (TLCHR) were obtained from E. Merck (Darmstadt, Germany). 4-Chloro-1-naphthol and the disodium salt of p-nitrophenyl phosphate were obtained from Sigma. For enzyme-linked immunosorbent assays (ELISA), alkaline phosphatase conjugated goat anti-human IgM (Kierkgaard and Perry Labs, Gaithersburg, MD) and purified murine anti-human IgG (Southern Biotech, Birmingham, AL) and then conjugated to alkaline phosphatase Biochemical ) Horseradish peroxidase-conjugated goat anti-human IgM or IgG purchased from TAGO (Burlingame, CA) was used for immunoassay with dot blot staining and immuno thin layer chromatography (HTLC) Rabbit anti-mouse immunoglobulin, conjugated with peroxidase, x and rabbit anti-mouse IgM and IgG conjugated with alkaline phosphatase for ELISA were used with control monoclonal mouse antibodies, obtained from Zymed (San Francisco, CA). Anti-GM2 mAb 696 was obtained from Kyowa Hakko Kogyo (Tokyo, Japan) and anti-GM3 mAb R24 was generated (13).

Serological tests
ELISA was performed as described previously (6). To control non-specific “adherence,” immune serum was also tested on plates that were treated identically but to which ganglioside was not added, and the reading was subtracted from the value obtained in the presence of ganglioside. The titer was defined as the highest dilution giving a corrected absorption of 0.1 or higher. Immunostaining of gangliosides with monoclonal antibodies or human serum was carried out after staining on nitrocellulose bands (14) or separation on glass plates with silica gel for TLCHR, as described previously (3). The plates were developed in a mixture of chloroform / methanol / water (0.25% CaCl ₂ ) with a ratio of 50:40:10 (v / v) and the gangliosides were visualized by staining with resorcinol / HCl reagent or monoclonal antibodies.

IgG subclass definition
The IgG subclass was determined by ELISA using subclass-specific secondary murine anti-human mAbs IgG1, IgG2, IgG3 and IgG4. Used secondary mAb from various sources (table 4). Alkaline phosphatase conjugated to goat anti-mouse IgG (Southern Biotech, Birmingham, AL) was used as the third antibody at a 1: 200 dilution.

Complement mediated cytotoxicity test
A check for complement mediated cytotoxicity was performed by assay with ⁵¹ Cr release for 4 hours. Cells from the GM2-positive melanoma cell line SK-MEL-173 served as target cells, 2x106 cells were labeled with 100 µ CiNa ₂ ⁵¹ CrO ₄ (New England Nuclear, Boston, MA) in 10% FCS RPMI for I hour at 37 ^o C in a CO ₂ incubator. Cells were washed twice and 104 cells / well in a 96-well round-bottom plate (Corning New York, NY) were labeled and incubated with 1: 5 diluted serum before and after vaccination or only with medium for 1 hour at 37 ^° C in CO ₂ incubator. Cells were washed and incubated with human complement (Sigma) at a dilution of 1: 4 for 4 hours at 37 ^° C. The plates were centrifuged at 500 g for 5 minutes and an aliquot of 125 μl of each well supernatant was taken to determine ⁵¹ Cr released. All assays were repeated three times, they included control wells for maximum isolation in 1% NP-40 (Sigma) and for spontaneous isolation in the absence of complement.

Результаты
Введение вакцины и побочные действия
24 пациента иммунизировали вакциной GM2-KLH. Группы из 6 пациентов получали GM2-KLH без иммунологического адъюванта или с DETOX, BCG или QS-21 Ни локальной, ни системной токсичности не обнаружили после введения только GM2-KLH. Вакцины, содержащие DETOX, вызывали образование узелковых утолщений в местах вакцинации у 4 из 6 пациентов, не исчезающих в течение 2-10 недель. У 4 пациентов вакцинация вызывала эритемы и индурацию 3-10 см, но только с минимальной болезненностью. У одного пациента образовалась эритема и индурация 25 см после одной иммунизации и у второго пациента была слабая лихорадка и недомогание в течение 72 часов после первой иммунизации. Для этого пациента дозу DETOX для следующей иммунизации снизили до 50 мкг CWS и 5 мкг MPLA. BCG вызывал локальное воспаление и появление струпьев в одном и том же месте у всех пациентов, которые залечивались через 2-12 недель. Когда это имело место, дозу BCG снижали с 1х107 жизнеспособных единиц до конечной дозы 3х106 единиц у 4 пациентов и 1х106 единиц у одного пациента. Шестой пациент имел туберкулезное воздействие и положительный PPD-тест. Его поэтому начали вакцинировать с дозы 1х106 единиц, которую в конце снизили до 1х105 единиц. QS-21 индуцировал слабую локальную эритему и болезненность при дотрагивании в течение 24-48 часов у всех пациентов при дозе 100 мкг. Доза QS-21 вызывала локальную болезненность и воспаление, длящиеся 2-10 дней после всех иммунизаций, а также симптомы гриппа, включая небольшую лихорадку (ниже 38^oC) головную боль и миалгию в течение 8-24 часов после 3 из 18 иммунизаций. Не наблюдались нейрологические нарушения или другие побочные действия.The percentage of specific lysis was calculated as follows:

results
Vaccine administration and side effects
24 patients were immunized with GM2-KLH vaccine. Groups of 6 patients received GM2-KLH without an immunological adjuvant or with DETOX, BCG, or QS-21. Neither local nor systemic toxicity was found after administration of GM2-KLH only. Vaccines containing DETOX caused the formation of nodular thickenings at the vaccination sites in 4 of 6 patients who did not disappear within 2-10 weeks. In 4 patients, vaccination caused erythema and induction of 3-10 cm, but only with minimal pain. One patient had erythema and an induction of 25 cm after one immunization, and the second patient had mild fever and malaise within 72 hours after the first immunization. For this patient, the dose of DETOX for the next immunization was reduced to 50 μg CWS and 5 μg MPLA. BCG caused local inflammation and scabs at the same place in all patients who healed after 2-12 weeks. When this was the case, the dose of BCG was reduced from 1x107 viable units to a final dose of 3x106 units in 4 patients and 1x106 units in one patient. The sixth patient had a tuberculosis effect and a positive PPD test. Therefore, they began to vaccinate him with a dose of 1x106 units, which at the end was reduced to 1x105 units. QS-21 induced weak local erythema and tenderness when touched for 24-48 hours in all patients at a dose of 100 mcg. The dose of QS-21 caused local pain and inflammation lasting 2-10 days after all immunizations, as well as flu symptoms, including a slight fever (below 38 ^o C), headache and myalgia for 8-24 hours after 3 of 18 immunizations. No neurological abnormalities or other side effects were observed.

Humoral immune response to GM2-KLH conjugate vaccines
Prior to vaccination, no IgG antibodies against GM2 were found and IgM antibodies were found in only three patients with a titer of 1:80 in two and 1: 320 in one. The remaining 21 patients did not detect GM2 reactivity prior to vaccination. The results of serum analyzes before and after immunization with ELISA and immunostaining of dot blots are summarized in Table 3. The IgM antibody titers after immunization with GM2-KLH or GM2-KLH and BCG or GM2-KLH and DETOX were similar (average titer 1: 80-1: 160). In contrast, 5 or 6 patients immunized with GM2-KLH and QS-21 had an IgM antibody titer of 1: 1280 or higher, significantly higher than titers in other groups or in patients previously immunized with unconjugated GM2 and BCG vaccines (6). In addition, immunization with GM2-KLH and QS-21 induced a persistent IgG response at first in all patients; only 2 of the other 18 patients receiving GM2-KLH vaccines produced comparable IgG titers.

 Successive anti-GM2 IgM and IgG antibody titers in patients receiving GM2-KLH and QS-21 are shown in FIG. 6. Peak IgM titers were detected after the third or fourth vaccination and remained high in most patients for at least 20 weeks. Repeated immunizations at weeks 14 and 22 did not further increase IgM titers. IgG titers of 1: 160 or higher were detected 2 weeks after the fourth vaccination in five of six patients. The titers dropped to 1:40 or less, but quickly rose again after re-vaccination to the previous level (average 1: 160) and remained at that level for more than 11 weeks. The second re-vaccination in most cases did not have a noticeable effect on antibody titer. Thus, the response to revaccination showed only one of two characteristics of the classical secondary immune response. The immune response showed up more quickly, but the antibody titer did not rise above the titer after the initial immunization.

 Antibodies to KLH in serum were not determined prior to vaccination. After vaccination, the serum of all patients showed reactivity with KLH, as shown in Table 3. The highest titer of IgG antibodies was after administration with QS-21, significantly higher than in all other groups, including the next best group of patients vaccinated with GM2-KLH and BCG (p below 0.005). In the QS-21 group, there was no correlation between the intensity of the immune response to GM2 and the immune response to KLH.

The specificity analysis of antibodies against GM2
The specificity of antibodies against gangliosides detected in patient sera before and after immunization was determined by immunostaining of dot blots using ganglioside standards GM3, Asialo-GM2, GM2, GM1, GD2, GD3, GD1a and GD1b (Fig. 7). IgM and IgG antibodies before immunization in most patients showed weak reactivity with asialo-GM2, some patients also had IgM antibodies against GM1 and GD1b. The reactivity of antibodies with these gangliosides did not change after immunization. The only vaccine-induced changes were strong reactivity with GM2 and weak reactivity with GD2. Dot blot immune staining was rated as 0, 1+, 2+, and 3+ in intensity. Reactivity of 3+ IgM antibodies against GM2 was detected in the serum of five of six patients immunized with GM2-KLH and QS-21, in one of six patients immunized with GM2-KLH and BCG, and in two of six patients immunized with GM2-KLH without adjuvant or GM2-KLH and DETOX. The reactivity of 3+ IgG antibodies was found in five of six patients immunized with GM2-KLH and QS-21, and was not found in any of the patients in other groups.

Serum after vaccination of patients immunized with GM2-KLH and QS-21 was also analyzed by immuno thin layer chromatography (Fig. 8A) to determine the reactivity with GM2 and other gangliosides of the melanoma tissue extract. Most patients' sera showed strong IgM and IgG reactivity with GM2 isolated from bovine brain or melanoma. Reactivity was also shown by a slowly migrating band in an extract of melanoma, presumably GD2, when compared with the value of R _f purified standard GD2 (not shown).

 To confirm the GD2-specific reactivity of IgG antibodies, patient N2 serum after vaccination was pre-incubated with GM2 or GD2 before immunostaining (Fig. 8B). Reactivity with GM2 and GD2 in the melanoma ganglioside extract was completely inhibited by preincubation with GD2. On the other hand, preliminary incubation of the same serum with GD2 led to the inhibition of only GD2 reactivity and did not change the reactivity with GM2. These results suggest the presence of two antibody populations, one reacting only with GM2, the other reacting with GM2 and GD2.

Determination of IgG antibody subclass
High IgG titers from six patients immunized with GM2-KLH and QS-21 were analyzed by ELISA using an IgG subclass-specific secondary antibody panel. The results are summarized in table 4. IgG antibodies of all six sera were subclasses of IgG1 and IgG3.

Complement mediated cytotoxicity
The function of the anti-GM2 antibody effector in the serum of patients vaccinated with GM2-KLH and QS-21 (1: 5 dilution) was determined by complement-mediated cytotoxicity assays. As shown in table 5, the serum after vaccination of all three patients lysed CM2-positive SK-MEL-173 melanoma cells in the presence of human complement. Serum prior to vaccination did not show cytotoxicity when complement was added. Serum after vaccination was not cytotoxic to GM2-negative melanoma cells or without complement addition. Undoubtedly, these are only preliminary results, and a more detailed study of the functions of the cell surface binding effector and the cytotoxicity of vaccine-induced antibodies and their subclasses is currently underway.

 Discussion.

 In a number of studies of patients with malignant melanoma, it was objectively necessary to design vaccines that are effective in inducing the production of antibodies against three gangliosides, often overexpressed in melanoma -GM2, GD2 and CD3. The initial approach to this was to vaccinate patients with unconjugated gangliosides adsorbed by BCG. In this way, we were able to induce the production of antibodies against GM2 (5, 6), but not against GD2 or GD3. Antibodies against GM2 induced by GM2-BCG vaccines were mainly of the IgM class, the immune response was short-term and re-immunization led again to a short period of production of IgM antibodies, similar to the period of the primary immune response - all characteristics of a T-cell-independent immune response are well known when studying other carbohydrate antigens. However, vaccine-induced antibody production against GD2 in patients with stage III melanoma after surgery has been associated with increased survival. This observation suggests that melanoma gangliosides are suitable candidates for vaccine design and that vaccines for melanoma gangliosides with increased immunogenicity can lead to better clinical results. Since suitable epitopes of melanoma gangliosides are carbohydrates, it is useful to consider studies that were designed to increase the immunogenicity of other carbohydrate vaccines, namely vaccines against certain bacterial infections.

 The main difference between the immune response of carbohydrate antigens and protein antigens is that it does not depend on the thymus. The concept that carbohydrate antigens are thymus-independent (TI) is based on the observation that neonatally thymectomized mice as well as thymic mice exhibit unaffected humoral immune responses to bacterial polysaccharides (15). B cells that respond to TI antigens have several characteristics. They appear later in ontogenesis, are long-living, and do not require T cells for activation, at least in vivo. Although T cells are required for B cells to respond to TI antigens in vitro, the nature of the action of T cells is not well understood and is clearly different from NHC-restricted T-cell assistance in the T-dependent immune response to protein antibodies. Although T cells are not necessary for an in vivo immune response to TI antigens, antibody titer is higher when T cells are present, which confirms a general increase in T cell activity, but again by unknown mechanisms (16).

 In attempts to increase the immunogenicity of carbohydrate antigens, a large number of approaches have been investigated. These included chemical modification (17), administration with adjuvants, non-covalent complexation with proteins, covalent attachment to immunogenic protein carriers (18) and replacement of the carbohydrate epitope with a protein replica or peptides synthesized de novo (so-called mimitopes), (19) or anti-idiotype antibodies (20). Most of these approaches lead to increased T-cell assistance for a carbohydrate-specific immune response. Although each approach has shown promise in initial experimentation, the covalent attachment of carbohydrate antigens to immunogenic E-dependent carrier proteins, which was first proposed for haptens (21) and then for saccharides (22), is the concept that has been dealt with most vigorously, leading to the creation of vaccines, which, as shown in some examples, are most effective in recent clinical trials.

 Excellent examples of these are vaccines for the conjugation of H. influenzae type b polysaccharide (Hib) proteins. The four vaccines that have been developed over the past decade differ in carbohydrate components, protein carriers, and linkers between carbohydrate and protein (23-27). In comparative studies in children, conjugate vaccines induced a significantly stronger immune response than the unconjugated polysaccharide phosphoribosyl / ribiphosphate (PRP) Hib vaccine (28). The most interesting observations are that newborns who were first immunized with HbOC (oligosaccharide / non-toxic diphtheria toxin) or PRP-OMPC (Neisseria meningitidis type B outer membrane protein complex) and were later treated with a non-conjugated PRP vaccine showed an anamnestic IgG immune response, even if they were treated at the age at which they did not respond to primary immunization with the unconjugated vaccine (29, 30). T cells are now involved, but it is still far from clear how they interact with Hib PRP-reactive B cells. The fact that increased immunogenicity and T-dependence require covalent binding between PRP and protein suggests that the proximity between protein and PRP should not be compromised at least in the early phase of antigen processing. When the isotope and biological activity of antibodies induced by Hib PRP and Hib PRP conjugates is the same, it turns out that B cells that correspond to the conjugate-induced T cell signal are quantitatively identical to cells that only Hib PRP bind.

 Drawing on the significant experience gained in developing carbohydrate vaccines for bacterial infections, the applicants have explored similar approaches over the past few years to attempts to increase the immunogenicity of melanoma gangliosides. Chemical modification of GD3, leading to the formation of amide, lactone or gangliosidol or O-acetylation, allows the preparation of derivatives that are very effective in inducing antibody formation in patients with melanoma. However, antibodies induced by these derivatives of GD3 do not cross-react with GD3 (31,32). Anti-idiotypic BEC-2 antibodies mimicking GD3 was obtained by immunizing mice with R24 monoclonal antibodies that recognize GD3. Rabbits immunized with BEC-2 produced antibodies against GD3 (33). Initial studies of the immunogenicity of BEC-2 in humans are currently underway.

On conjugate vaccines, the initial studies with GD3 in mice dealt with three questions - the development of a conjugation method, the selection of a carrier protein and the choice of adjuvant (7). The optimal conjugation technique included ozone cleavage of the GD3 double bond in the ceramide chain, the introduction of the aldehyde group and the combination of reductive amination with protein aminolysis groups. It was found that of the five tested carriers, poly-L-lysine, cochlear lymph lymphocyte hemocyanin (KLH), cationized bovine serum albumin, the Neisseria meningitidis outer membrane protein complex (OMRS), and polyantigenic peptide constructs containing 4 repeats of T-branched malaria epitope on branched polylysine cortex, KLH is the most effective. Non-covalent GD3 / KLH complexes were non-immunogenic. The best adjuvant was QS-21, a homogeneous saponin fraction isolated from the bark of Quillaja saponaria Molina and purified. Characteristics of the humoral immune response to immunization with GD3-KLH and QS-21 conjugate included
a) a high initial titer of IgM antibodies,
b) a rapid secondary increase in the titer of IgM antibodies after re-immunization,
c) maintaining the titer of IgM antibodies after re-immunization for up to 10 weeks and
d) persistent production of high titer IgG antibodies in parallel with the production of IgM antibodies, with the exception of an initial delay of two weeks.

 These findings are now reproduced in patients (people) with melanoma immunization with another ganglioside conjugate vaccine, GM2-KLH, using the same conjugation technique. It turns out, as with studies in mice, QS-21 is a significantly more effective adjuvant than DETOX or BCG, with acceptable toxicity.

 The anti-GM2 immune response has many characteristics of a T cell dependent response. It was long-term, and antibodies of the IgGl and IgG3 subclass (usually associated with a T-cell-dependent immune response) were induced. As shown with Hib PRP vaccines, these isotypes were the same as those occasionally induced with weak titers of unconjugated GM2 / BCG vaccines. The lack of a clear repeat response in the long-term high titer IgM and IgG immune response after vaccination within three or five months after the initial series can be explained by the fact that patients were immunized initially at two-week intervals. In a classic experiment showing a secondary immune response to protein antigens, a second injection of antigen is performed 4 weeks after the first. The content of antibodies after the first immunization is higher for one or two weeks after injection, then decreases to very low values until the second injection of antigen after four weeks. In the immunization schedule chosen by the applicants, the initial immune response did not decrease, but increased stepwise in response to the first four vaccinations at two-week intervals, anticipating the secondary immune response, which is more dramatic in the classical experiment. In contrast to the immune response to most protein antigens, the IgM immune response was long-term and the IgM antibody titer remained higher than that of IgG antibodies, even after repeated booster immunizations, as is typical for carbohydrate antigens. Therefore, the immune response against gangliosides that contain a relatively short oligosaccharide chain linked to the lipid chain and which are autoantigens shows much in common with the immune response against Hib-PRP and other bacterial carbohydrates.

 The development of a CM2 conjugate vaccine will make it possible to determine whether a higher titer of anti-GM2 IgM and IgG antibodies, maintained for longer periods, is more effective in delaying the recurrence of melanoma than lower titers of mainly IgM antibodies present for shorter periods , in patients immunized with unconjugated GM2. In addition, applicants can now test whether conjugation with immunogenic protein carriers provides the immunogenicity of GD2 and GD3 to the main gangliosides that do not induce an immune response in patients with melanoma when given as unconjugated vaccines. If this can be done, the construction and testing of polyvalent melanoma ganglioside vaccines could be an attractive next step.

Fourth series of experiments
Elevated doses of the QS-21 saponin fraction were administered as an immunological adjuvant in a phase 1 study at a constant dose of melanoma ganglioside GM2 covalently attached to cochlear lymphocytic hemocyanin (KLH). Twenty-eight patients with stage III or IV AJCC melanoma who did not have the disease after surgery received six subcutaneous vaccinations over a period of more than 5 months. Local and systemic reactions were as with the introduction of QS-21. Doses of 100 mcg or less induced mild local pain and inflammation at the vaccination sites, persisting for 2-4 days, and a rare, short-term mild fever and malaise, but without disability. A dose of 200 μg induced mild fever and malaise after 30% vaccination and local reactions of 20 cm in diameter appeared in all patients, which led to a limited effect of the injected limbs for 5-10 days. The titers of IgM and IgG antibodies against GM2 and IgG antibodies against KLH were higher at doses of QS-21 of 100 and 200 μg. Antibodies against QS-21 were not detected. This study establishes a 100 μg QS-21 dose as the optimal, well-tolerated dose for inducing antibodies against GM2 and KLH in melanoma patients.

 Introduction

 Applicants and other employees have melanoma patients vaccinated with a series of vaccines of whole melanoma cells selected for expression of various glycoprotein and ganglioside antigens (1, 2). The ganglioside most commonly recognized by antibodies in serum after immunization was GM2 ganglioside. GM2 ganglioside is a differentiating antigen overexpressed on the surface of human malignant melanoma cells. It was found that injection of GM2 alone is not immunogenic, therefore GM2 vaccines containing various immunological adjuvants were tested (3, 4). Immunization with purified GM2 ganglioside adhered to Bacillus Calmette-Guerin (BCG) induced the production of anti-GM2 IgM antibodies in most melanoma patients, and antibody producing patients had a longer disease-free period and better survival compared to patients who did not produce antibodies (4, 5). In a randomized study with 122 melanoma patients who did not have the disease after surgery, the majority of patients (86%) who received the GM2 / BcG vaccine produced antibodies (6). Patients who produced anti-GM2 antibodies had a significantly longer disease-free period and higher overall survival than patients with a negative antibody test. A comparison of the two research directions shows that patients receiving the GM2 / BCG vaccine had an 18% longer interval without disease and an 11% longer survival compared to the BCG control group, although the difference was not statistically significant. The immune response was short-lived, mainly with moderate titer IgM production. Similar approaches with vaccines of other melanoma gangliosides GD2, GD3 and 9-0-acetyl-GD3 / BCG in patients caused only random immune responses with a low titer (7). The need for a more effective vaccine was obvious.

 Landsteiner's classical experiments with hapten-carrier conjugates have been successfully used to vaccinate children against various antigens, including bacterial capsular polysaccharides (8). Based on this, several different carbohydrate tumor antigens were conjugated with cochlear lymph hemocyanin (KLH) and showed that they provoke an IgM and IgG immune response with a higher titer than previously observed titers when using unconjugated vaccines (9-11). Applicants tested the immunogenicity of GD3 coupled to various carrier molecules in mice and determined KLH as the optimal carrier (12). The GM2-KLH conjugate vaccine was tested separately or in a mixture with immunological adjuvants BCG and DETOX in melanoma patients, but the immune response is not significantly different from the response induced by GM2-BCG vaccines, only moderate titer IgM antibodies were induced (13). A stronger immunological adjuvant was needed.

QS-21 is a saponin fraction purified almost to homogeneity and obtained from the bark of Quillaja saponaria Molina; it was chosen due to its high adjuvant effect and low toxicity (14, 15). Saponins were used as immunogenic adjuvants in various kits (a review is given). QS-21 was used to enhance the immune response against E. coli polysaccharide (16) and the humoral immune response and T-cell response against HIV-1 sheath protein (17), ovalbumin (18) and feline leukemia virus (19). Cytotoxic T cells were induced against HIV-infected cells and ovalbumin-transfected cells by immunization with protein / QS-21 mixtures (17, 18). Applicants tested various new immunological adjuvants with various carbohydrate antigen-KLH vaccines and showed that immunological adjuvants QS-21 and SAF _m most effectively increase IgM and IgG antibody titers (9). The experiments focused on the use of QS-21, because it was the least toxic and was acceptable for testing with conjugate vaccines. GD3-ganglioside-KLH plus QS-21 vaccines in mice induced persistent high titer IgM and IgG immune responses (12), providing the basis for testing this approach in melanoma patients.

 Described here is the result of a phase 1 study using a constant dose of GM2-KLH plus an increasing dose of QS-21. Since purified QS-21 and partially purified saponins, such as Quil A, were not previously used in humans, the goal was to determine the maximum tolerated dose suitable for repeated administration to an outpatient and the optimal dose to enhance the immune response against GM2.

 Materials and methods.

Gangliosides
Cow brain GM2 and GD1b were donated by the Gidia Research Laboratory (Abano Terme, Italy). Cow brain GM3, GM1 and GD1a were purchased from Sigma Chemical Co. (St. Louis, MO). GD2 was obtained from GD1b by treatment with β-galactosidase (20). GD3 was isolated from cow buttermilk and was kindly provided by Dr.RKYu (Medical Colledge of Virginia, Richmond, VA).

Reagents and monoclonal antibodies
Silica gel plates for TLCHR were obtained from E. Merck (Darm stadt, Germany); 4-chloro-1-naphthol, the disodium salt of p-nitrophenyl phosphate was obtained from Sigma. Goat anti-human IgM conjugated with alkaline phosphatase (Kierkegaarg and Perry Labs, Gaithersburg, MD) and mouse anti-human LgG, purified thereafter with goat anti-mouse IgG conjugated with alkaline phosphatase (Southern Biotech), were used for ELISA. Horseradish peroxidase-conjugated goat anti-human IgM or IgG purchased from TAGO (Burlingame, Calif.) Was used for immune staining of dot blots and immune thin layer chromatography. Conjugates of rabbit anti-mouse immunoglobulins with horseradish peroxidase for ITSX and rabbit anti-mouse IgM and IgG conjugated with alkaline phosphatase for ELISA were used with control monoclonal antibodies and were obtained from Zymed (San Francisco, CA). anti-GM2 mAb 696 was obtained from Kyowa Hakko Kogyo (Tokyo, Japan) and anti-GD3 mAb R24 was generated (21).

Patients
Melanoma patients with stage AJCC III or IV disease were considered appropriate if all symptoms of metastatic disease were eliminated within the last 8 months. None of the patients had received chemotherapy or radiation therapy before. Patients were examined for toxicity at each vaccination. In addition, patients were instructed to measure local reactions and temperature. These results were recorded and then viewed during the next clinical visit. Four patients (one for each of the 4 doses) received only 4 immunizations, since another treatment was necessary due to the progress of the disease.

Preparation and administration of vaccines
GM2-KLH conjugate was obtained by Biomira Inc. (Edmonton, Alberta). The molar ratio of GM2 / KLH was approximately 800/1. The conjugate was administered in an amount of 0.57 mg per 0.5 ml of normal saline. This dose is for one administration to the patient, it contains 79 μg GM2, 500 μg KLH and 0.5 ml of normal saline. An adjuvant QS-21 containing a saponin component isolated from the bark of the Quillaja saponaria Molina tree was introduced by Cambridge Biotech. Corp. (Worcester, MA). The purity of QS-21 was determined to be greater than 98% analytical reversible-phase HPLC. Then, 50, 100 or 200 μg of QS-21 was diluted with 0.25 ml of normal saline and mixed with GM2-KLH. The vaccine (final volume 0.75 ml) was vortexed for 2-3 minutes and administered for 15 minutes. Four vaccinations were performed subcutaneously at two-week intervals and then two vaccinations at eight-week intervals. First, 12 patients were vaccinated, 3 for each of the 4 doses of QS-21. Then 1 additional patient received a dose of 10 μg and 3 additional patients received 3 higher doses. 200 mg / m ² cyclophosphamide (Cytoxan, Mead Johnson and Co., Evansville, IN) was administered IV to all patients 3-6 days before the first vaccination.

Serological and delayed-type hypersensitivity (DTH) analyzes
Enzyme-linked immunosorbent assay (ELISA) was performed as previously described for gangliosides (5). For ELISA assays, QS-21, QS-21: ethylenediamine (prepared by attaching ethylenediamine to the carboxy group of glucuronic acid QS-21) was applied to plates coated with glutaraldehyde Immulon 4 (Dynatech Labs, Chanlilly, VA). To control non-specific “adherence,” immune sera were also tested on plates that were treated identically but to which ganglioside-KLH or QS-21 was not added, and the indicator was subtracted from the value obtained in the presence of antigen. The titer was defined as the largest dilution leading to a corrected absorption of 0.1 or higher. Immune staining of gangliosides with monoclonal antibodies and human blood serum was performed after staining on nitrocellulose strips, as described previously (22). Patients' skin was tested for GM2, GM2-HSA and KLH during the fifth vaccination. 25 μg of each of them was diluted with 0.05 ml of PBS and was administered intradermally. The results were interpreted as described previously (5).

 Results.

Toxicity
The number of vaccines administered containing only GM2-KLH or conjugate in combination with different doses of QS-21, and the local and systemic reactions associated with these vaccinations are shown in Table 6. Whereas GM2-KLH or this conjugate with 10 μg QS-21 sometimes caused weak local erythema and induction; increased doses of QS-21 led to an increase in the frequency and severity of local reactions. At doses of 10 and 50 μg, this was associated with mild soreness at the pressing site and erythema and induction of 2-4 cm for 24-48 hours (toxicity above degree 1 was not observed). At the highest doses, these reactions became more visible with the largest reaction in the amount of 8-10 cm and in some cases (at a dose of 200 μg) reached 20 cm in diameter. These reactions usually lasted 2-4 days, in one case 7 days at a dose of 100 mcg, and usually 7 days, in one case 10 days at a dose of 200 mcg. In no case were analgesics stronger than tylenol required, and patients at doses of 10, 50 and 100 μg continued to engage in their labor activities. The activity of the vaccinated limbs was limited to 5-8 days after the introduction of mainly vaccines containing 200 μg, but all evidence of local reactions disappeared within two weeks before the next examination. No ulceration and drainage or subcutaneous nodule enlargement were detected (as was found with most other adjuvants). Although occasional short-term mild manifestations of fever or myalgia were observed after the administration of vaccines containing a lower 3 dose of QS-21, approximately a third of the 200 μg QS-21 vaccinations were associated with these symptoms. In general, systemic symptoms were most noticeable after re-immunization. For all patients from the 200 μg QS-21 group, the dose of QS-21 was reduced to 100 μg or 50 μg for the third and fourth immunizations due to increased local and systemic reactions (side effects of immunization with these reduced doses are shown in Table 6 for 100 μg doses and 50 mcg). These doses were well tolerated and, therefore, for all 5 patients (in one patient the disease progressed and was excluded from the protocol after 4 immunizations), the dose was increased again to 200 μg for the fifth immunization. Two out of five fifths of immunizations were associated with fever and flu-like symptoms. One of them also caused local 20 cm erythema and induction. The dose of QS-21 for the sixth vaccination was reduced to 100 μg for this patient and as a result there was no development of systemic symptoms and the local reaction was weak (3-4 cm erythema and induction). None of the four remaining immunizations using 200 μg QS-21 caused systemic symptoms. Although the general systemic symptoms were very mild, local erythema, induction and soreness at a dose of 200 mcg were noticeable enough so that the applicants did not choose higher doses of QS-21 to continue vaccination.

Humoral immune response against GM2 after vaccination (see table 7)
Immunization with GM2-KLH alone resulted in antibody titers similar to those in previous studies with vaccines containing GM2 adhered to the surface of BCG. Five out of 6 patients produced IgM antibodies, only one produced IgG antibodies (low titer). The titer of IgM antibodies increased with increasing doses of QS-21. The reciprocal average titer of IgM antibodies after immunization with GM2-KLH alone was 80, after administration of the conjugate with 10 or 50 μg QS-21 was 480 and 240 (respectively), and after 100 and 200 μg QS-21 was 1280. The titer of IgG antibodies increased progressively with increasing doses from 0 in patients receiving only GM2-KLH to 10, 60, 200 and 640 in patients receiving 10, 50, 100 and 200 μg of QS-21, respectively. A noticeable IgM antibody content and detectable IgG antibody content (ELISA and dot-blot immune staining) were detected in each patient receiving GM2-KLH plus QS-21. Sequential titers of IgM and IgG antibodies for groups of six patients receiving 100 and 200 μg of QS-21 are shown in FIG. 9. In general, the immune responses were exactly the same in the two groups, although IgG titers (ELISA) were slightly higher at a dose of 200 μg. Immune staining of dot blots against GM2 confirmed the specificity for GM2 and the similarity of reactions at doses of 100 and 200 μg (see table 7 and Fig. 10). The average IgM response of dot-blots against GM2 increased from 2+ in patients receiving only GM2-KLH, to values between 2+ and 3+ in patients receiving 10 or 50 μg QS-21, and up to 3+ in patients receiving 100 and 200 μg QS-21. IgG reactions increased from 0 in patients not receiving QS-21 to 2+ in patients receiving 10 or 50 μg QS-21, and to 3+ in patients receiving 100 or 200 μg QS-21.

The specificity analysis of the humoral immune response against GM2
Immune staining of dot blots with sera obtained before and after immunization in two representative patients of each of the 5 groups of immunized patients is shown in FIG. 10. Prior to immunization, IgM reactivity against GM1 was detected in the sera of many patients, as applicants described previously, but reactivity was detected against GM2 or other gangliosides. After immunization, GM1 reactivity was not detected, but IgM and IgG antibodies against GM2 were detected in all patients. As previously described by applicants (13), these GM2-induced antibodies sometimes show cross-reactivity with GM2 (patient 1, dose 100 μg).

Serological responses against KLH and QS-21
As shown in table 7, reactivity against KLH was not detected before immunization and this reactivity progressively increased with increasing doses of QS-21. While doses of QS-21 of 50, 100, and 200 μg induced significantly higher titers of anti-KLH IgG antibodies compared with a dose of 0 or 10 μg, there was no significant difference between serological titers induced at doses of 50, 100, and 200 μg . Anti-QS-21 antibody titers were determined by ELISA after four immunizations. No significant increase in antibodies against QS-21 was observed in serum samples before and after immunization (data not shown).

 DTH responses against GM2 and KLH.

 Neither erythema nor induction was detected at the skin testing sites for GM2 or GM2-KLH. Noticeable erythematous reactions were detected after 24 and 48 hours in most places of skin testing for KLH. These reactions were the strongest in patients who received a dose of 200 mcg (average diameters 4.0 to 6.0 cm). They were associated only with minimal induction, which suggests a combination of DTH and antibody-mediated reactions, which makes accurate quantification of the DTH response (by patients at home) difficult.

Other clinical studies
Using the same dose of vaccine as described above (Biomira. Inc.), six patients with advanced disease were treated. An additional six patients with advanced disease were again given a 1/10 dose of GM2-KLH with 100 μg QS-21. Antibody titers in both groups were lower than in patients with an early stage of the disease, but a dose of GM2 of 7 μg was found to be as effective in inducing titers of IgM and IgG antibodies as a dose of 70 μg.

 In another study, 6 patients with an early stage of the disease were given the GM2-KLH construct. IgG antibody titers were comparable to those after administration of the Biomira construct, although IgM antibody titers were sometimes lower. However, all patients produced antibodies after immunization and the vaccine was well tolerated.

 Further study will include 18 patients receiving GM2-KLH vaccines, initially weekly (the indicated scheme is preferred for studying mice). Patients were randomized, with some not receiving a preliminary low dose of cyclophosphamide and others previously receiving a low dose of cyclophosphamide. At that time, 10 patients were studied and it turned out that there was no difference between patients receiving cyclophosphamide and patients not receiving phosphamide, and the antibody titer according to the new scheme was at the same time preferable to titers in previous studies with the same conjugate.

 Discussion.

 This was a phase 1 study designed to identify the maximum tolerant dose (MTD) of QS-21 for use in outpatient kits, and the dose of QS-21, which provides the strongest immunopotentiating effect. Probable MTD was set as 200 mcg QS-21 for vaccination. Despite the weak manifestation of fever and malaise and strong local inflammatory reactions at a dose of 200 μg, MTD cannot be considered in the context of a high dose of IL2 or combination chemotherapy, which is used for hospitalized patients with progressive melanoma (23, 24), they were important in the context of outpatient treatment cancer patients in a set of adjuvants and may be acceptable for immunization against infectious diseases of normal recipients. A dose of 100 μg, however, caused only two cases of mild fever in 44 injections and local inflammatory reactions that did not interfere with daily activities and were usually limited to 2-4 days. This dose was quite tolerant for a group of patients in a clinical study. There was a clear increase in local and systemic toxicity with increasing doses from 100 μg to 200 μg, which made us indecisive for a further increase in dose. While fever and malaise at the highest dose were similar to fever and malaise, sometimes manifested by other immunological adjuvants, for example BCG and DETOX (15.5), the local reactions were completely different. A dose of 200 μg QS-21 when injected subcutaneously caused a erythema or induction with a diameter of 10-20 cm that was hot and wet when touched. This local reaction spreads more than the reaction usually caused by doses of DETOX or BCG, inducing comparative systemic symptoms. An unexpected characteristic of these reactions was that after a few days (or at most after 10 days), these reactions were completely weakened and it was not obvious that the vaccination was carried out at this place. None of the patients showed ulcer, drainage, or nodule formation at the injection site.

The second unexpected discovery of this study was that QS-21, at any dose used, elicited a qualitatively different immune response to GM2 ganglioside. Even at a dose of 10 mcg
all patients produced IgG antibodies that were detectable by immunostaining for anti-GM2 dot blots. GM2-KLH vaccines or these vaccines with optimal doses of BCG, or DETOX, or GM2 adhering to the surface of BCG, the Minnesota Salmonella mutant R595 or proteosomes only rarely elicited more than one measurable IgG response among 6 immunized patients (2, 4, 5 ) Anti-GM2 IgM and IgG antibody titers continued to increase with increasing doses of QS-21, but as it turned out, reached a plateau between doses of 100 and 200 μg. The anti-KLH IgG antibody titer grew similarly with increasing doses of QS-21. Peak IgG titers were significantly higher at doses of 50, 100 and 200 μg than at doses of 0 and 10 μg, but the titers at three higher doses did not differ significantly from each other. These results indicate that doses of 100 and 200 μg of QS-21 induce an optimal immune response against GM2 and that a dose of 100 μg is more tolerant. First of all, the answers to testing erythematic skin against KLH were unexpected in patients immunized with GM2-KLH or GM2-KLH plus various doses of QS-21. They can be represented by a combination of antibody-mediated arthus-like and DTH responses. It is also possible that the lowest dose of KLH in skin tests compared with those used in other studies (26) (25 μg as opposed to 100 μg) was insufficient to produce a strong, measurable DTH reaction, or that the epitope density of the ganglioside is high GM2 on KLH in vaccines does not allow for the normal processing and presentation of antigens necessary to induce a classic DTH response against KLH.

 Despite the widespread use of QS-21 and other saponin-containing adjuvants in experimental animals and in veterinary practice (14-19), the mechanism of action remains unknown. From these studies, some conclusions can be drawn on the mechanism of action of QS-21. The absence of palpable nodules at the injection sites confirms that there is no depot effect with the formation of granulomas. Despite the effectiveness of QS-21 as an adjuvant, it appears to be a weak immunogen. Even after repeated immunizations, measurable immune responses were not obtained. Therefore, unlike other adjuvants, for example Parvum C, BCG and Freund's complete adjuvant, the immunogenicity of QS-21 cannot contribute to its adjuvant effect. Mild fever and malaise at a dose of 200 μg, and erythema and induction at the injection site confirm that cytokine is released. Intradermal injection of IL-2 led to very similar reactions, which are characteristic of classic DTH responses during biopsies (27, 28). Partial switching of anti-GM2 antibodies to an IgG-immune response involves the induction of T-cell assistance, possibly as a result of cytokine induction.

 Applicants have recently shown superiority of KLH over other carriers tested to increase the immunogenicity of gangliosides (12), and here applicants have shown an optimal dose of QS-21 to further enhance this effect. As applicants prepare for a large phase III ganglioside vaccine study in patients with stage III AJCC melanoma, there are a number of variables that remain to be tested. Applicants are currently conducting a preclinical study with this vaccine (ganglioside-KLH plus QS-21) plus the use of some other immunological adjuvants. In addition, clinical trials are planned with different immunization schedules and different doses of GM2-KLH. Unexpected discoveries in the phase 1 study described here are 1) the lack of evidence of granuloma formation at any dose of QS-21 and 2) that at a completely tolerated dose of 100 μg, the serological response against GM2 was strikingly quantitatively and qualitatively superior to any response using previously tested GM2 vaccines .

 The fifth series of experiments.

 Introduction

 The incidence of malignant melanoma has increased rapidly in the last 10 years. In 1992, over 32,000 cases of progressive melanoma and 6,700 deaths from melanoma were reported in the United States (1). After adequate resection of primary melanoma, the five-year survival rate varies from more than 95% for patients with primary tumors with a thickness of not more than 0.75 mm to 50% for patients with a thickness of primary tumors of more than 4.0. mm (2). Patients with regional lymph node metastases (stage III AJ CC) have a five-year survival rate of 25-35% after elective or therapeutic dissection (3). It was shown that no additional therapeutic measures reduce the rate of relapse and do not increase survival after surgery in these patients. A large number of agents were tested in these complementary treatment studies (reviewed in references 4, 5), including chemotherapy, non-specific immunomodulators, interferons, and various types of melanoma vaccines. For melanoma vaccines, the challenge was to develop methods that can control the immunogenicity of vaccines by the strength and specificity of the generated immune response. Since humoral immune responses to vaccines can be quantified and analyzed with an accuracy that is only now possible with cellular immune responses, the applicants have focused on melanoma antigens that induce humoral immunity. Of these serologically defined antigens, gangliosides, in particular GM2, have been identified as attractive targets for active immunization (5-8). Applicants have recently shown that immunization with purified GM2 adhering to BCG after preliminary administration of a low dose of Cy led to the induction of IgM antibodies in a large percentage of melanoma patients (8, 9). The immune response to GM2 showed a T-cell-independent result, i.e., a short duration, the absence of an IgG response and the absence of a secondary immune response during repeated vaccinations. The induced antibodies were cytotoxic to GM2-expressing melanoma cells in the presence of human complement, and patients who produced anti-GM2 antibodies after immunization had a significantly longer interval without disease and greater survival than patients without such immunization (9). The purpose of this study was to confirm the beneficial effects of producing vaccine-induced anti-GM2 antibodies in a randomized controlled trial.

 Materials and methods.

Patients
Patients with pathologically documented melanoma metastases limited to regional cutaneous and lymph nodes (AJCC stage III) were suitable 2–13 weeks after complete resection of skin metastases or regional lymph nodes. Other eligibility criteria include: age more than 15 years; health status according to Karnofsky not less than 90; serum creatinine less than 2.5 mg / dl, serum bilirubin less than 2.5 mg / dl and the absence of chemotherapy or radiation therapy for a period of 8 weeks before vaccination. Pregnant women were excluded. All patients were examined and their suitability was determined within two weeks before randomization. Subsequent clinical medical observation was performed by the patient’s initial oncologist.

Randomization and follow-up
Informed consent was obtained from all patients and was distributed according to the number of positive lymph nodes (1, 2-4, not more than 5), the presence of an enduring disease, and the interval between surgery and vaccination (2-6 weeks, 7-13 weeks). Patients were randomized for GM2 / BCG vaccination or only BCG vaccination by a computer (to ensure a reasonably equal number of patients in the two study groups). None of the patients and medical staff were informed of the type of vaccine administered until two weeks after the last vaccination. Information about the subsequent medical examination of all patients was received within one week every 4-6 months by phone from their initial doctors. Relapse data, documented radiographically or pathologically, was used to determine the interval without disease. Since the two non-specific components of the current vaccination approach, BCG and low-dose cyclophosphamide may have demonstrated antitumor activity (10, 11, 12), they were considered more suitable than the absence of such treatment for the control purpose.

GM2 / BCG vaccine
The GM2 ganglioside used to prepare the vaccines was obtained from two sources, the brain tissue of cats with Tay-Sachs disease (transmitted in these cats as an autosomal recessive trait) and GM1, obtained from the brain of domestic dairy cattle and purchased from Sureico, Beliefonte, PA . Tay-Sachs cats brain sections (provided by Dr. Mario Ratazzi, Mt. Sinai Hospital, New York, NY) were extracted with chloroform / methanol and the extract was peracylated, chromatographed on florsilil to remove phospholipids, deacetylated, dialyzed and chromatographed on DEAE columns -sephadex (13). After dialysis of GM2, the main fraction was separated by preparative thin layer chromatography. GM2 was obtained from bovine brain GM1 by terminal galactose cleavage with β-galactosidase, as previously described (14). GM2 from two sources were indistinguishable by thin layer chromatography (TLC) and immune TLC using murine monoclonal antibodies recognizing GM2 (13). Each portion of GKR had a purity higher than 98% as determined by thin layer chromatography and densitometric scanning. Servings were tested using routine sterility and safety tests on Guinean pigs and mice.

Purified GM2 was dried and stored at 4 ^o C. 10 ⁷ viable units (or 3 • 10 ⁶ for use in patients with a positive PPD test) BCG strain Tice (University of Illinois, Chicago, IL) was suspended in distilled water by ultrasound together with 200 μg dried and purified GM2. The suspension was lyophilized and stored at -80 ^° C. The residue was again suspended in 0.5 ml of phosphate buffered saline (PBS) shortly before the vaccine was administered. Under these conditions, GM2 was found to adhere to BCG, probably due to the formation of hydrophobic bonds, as reported earlier by applicants (8). BCG suspended in PBS for use in the control group. All patients in GM2 / BCG-rpynne received 200 μg GM2 per vaccination. These vaccines and the vaccination protocol were approved by the Memorial Hospital Institutional Review Board and were used under the IND US Food and Drug Administration. Patients initially received GM2 from the brain of cats. Then an incremental portion of GM2 derived from GM1 was used, since GM2 from the brain was no longer available. All patients received vaccines by intradermal injection in 6-10 places of the limb with intact lymphatic drainage, and this administration was repeated twice at two-week intervals, using different limbs each time. Repeated immunizations were performed two and five months after the initial vaccination series.

Cy introduction
A single dose of 200 mg / m ² Cy (Cytoxan, Mead Johnson and Co., Evansville, IN) was administered intravenously to all patients 5-7 days before the first and fourth vaccine injections.

Ganglioside reagents
GD2 was obtained by treatment of GD1b with β-galactosidase (GWJourdian, University of Michigan, Ann Arbor, MI) according to published methods (14). GM1 bought from Supelco (Bellefonte, PA). GD3 was donated to Fidia Research Laboratories (Abano Terme, Italy) and GDib was purchased from the same source. GM3 was isolated from canine red blood cells and purified (13). GM2 for dot blot immunostaining (Fig. 11) was obtained from GM1 by treatment with β-galactosidase or extracted from human melanoma biopsy samples as described previously (13).

Serological methods
Serum was obtained during each vaccination, 2-6 weeks after the introduction of vaccines 3, 4 and 5 and 3 months after the introduction of vaccine 5. ELISA for antibodies against GM2 or BCG (9) was performed with patient sera and rabbit anti-human IgM or anti-human IgM - "second" antibodies, or protein A conjugated to an alkaline protease (Kirkegaard and Perry Labs, Gaithersburg, MD). Values obtained with normal sera from donors without GM2 reactivity were subtracted from values obtained with patient sera. Antibody titer was determined as the highest dilution of serum, at which a corrected absorption of more than 0.10 was obtained, as previously described (9). Dot blot immune staining was performed as previously described (8, 9) and was divided into degrees 0, 1+, 2+ and 3+, as shown in FIG. 11. Serum was classified as positive if GM2 reactivity was 2+ or 3+ on dot blots with an ELISA titer of at least 1/20 or 1+ on dot blots with an ELISA titer of at least 1/80. Analyzes of complement-dependent cytotoxicity were performed as previously described with normal human serum (diluted 1/3) as a source of complement and visual quantification of non-viable cells by Giemsa staining (8, 15).

Skin Testing for Delayed Hypersensitivity (DTH)
Single-layer liposomes were obtained from egg phosphatidylcholine and passed through a 0.1 micron filter ten times. On the day of skin testing (2 weeks after the fourth vaccination), 1.2 mg of phosphatidylcholine liposomes and 25 μg GM2 were mixed in PBS and sonicated in a Bronson 1200 ultrasonic disintegrator with a water bath (Shelton, CT). Skin tests were performed with a) 25 μg GM2 and b) 25 μg / 1.5 mg GM2 / liposomes after 48 hours, as described previously (16, 17).

 Results.

Patient Characteristics
A total of 123 patients were registered between March 10, 1987 and March 17, 1989. Upon examination, it was found that one patient had an AJCC stage II disease (did not have lymph nodes) and therefore was not included in the analysis. All (122) eligible patients, not including vaccines, were randomized to the analysis. Two patients (one in each group) decided not to participate in the study after the first immunization, because they found that it was too difficult to overcome the uncertainties of experimental therapy and randomization. One patient in the BCG group received only two immunizations when they found that the disease had progressed and the immunization was stopped. All other patients received at least an initial series of three immunizations and remained in the protocol until the study was completed or before the disease progressed. One patient in the BCG group accidentally received GM2 / BCG in the fifth vaccination (he produced antibodies against GM2 and remained healthy). The average follow-up time is five years and three months with a minimum follow-up time of four years and three months after surgery before randomization.

 The characteristics of 122 randomized patients are shown in Table 8. The single most important prognostic indicator for patients with stage III melanoma is the number of positive lymph nodes (3). Other factors associated with poor prognosis include head and neck or torso as the main site and persistent melanoma (3). GM2 / BCG and all BCG groups were comparable in their prognostic indicators.

Side effects
Initial administration of Cy sometimes caused nausea or malaise for 2-24 hours, but was generally well tolerated. An intradermal injection of BCG resulted in inflammation and scab formation with drainage in all patients who received three or more immunizations. To maintain the inflammatory response at a suitable level, the dose of BCG was reduced by factor 3 when scab formation or drainage was first detected. Therefore, the average dose of BCG at the time of the last immunization was 3 • 10 ⁶ viable units in both study groups. A dose of 10 ⁷ units was supported only in 10% of patients; for some patients, a further dose reduction to 10 ⁶ units was required. GM2 did not affect the inflammatory response to BCG and did not cause additional side effects.

Serological response against GM2
Successive titers of anti-GM2 IgM antibodies before and after GM2 / BCG vaccination are shown in FIG. 10 for the first five patients (1987) and the last five patients (1989) who received all series of GM2 / BCG immunizations. As in the previous study (9), the immune response was predominantly IgM-type, an increase in titer was often observed after each immunization, which was more likely to be a repeated primary immune response than a secondary immune response. Sera before immunization and after immunization (peak titers) of all patients were also tested by immunostaining of dot blots to confirm the specificity of antibodies detected by ELISA. The results of the immunostaining of dot blots with 10 sera shown in FIG. 10 are shown in FIG. 11. Random reactivity against GM1 was detected with a low titer in serum prior to immunization, as described previously (8, 9). Vaccine-induced reactivity was limited by GM2 reactivity; GM3, GD2, GD1b, and GD3 reactivity were not detected. Antibodies against GM2 were able to mediate complement dependent cytotoxicity in tests for GM2-expressing tumor cells with human complement (table 9). No changes in this characteristic of immune responses and specificity were observed in the patient for a period of two years. The results of the serological analysis for all patients are summarized in Table 10. Seven of 64 patients in the BCG group were shown to have anti-GM2 antibodies defined as ELISA (average titer 1/40) and immunostaining on dot blots (average intensity 2+). Antibodies were previously present in 5 cases and were first detected after BCG vaccination in 2 cases. Anti-GM2 antibody titers increased 4 times or more after BCG immunization in 3 out of 5 patients with anti-GM2 antibodies before BCG vaccination. In contrast, only one in 58 patients who received GM2 / BCG showed GM2 reactivity from two tests prior to immunization and 50 of 58 patients showed antibodies against GM2 (average titer ELISA 1/160) in their serum after immunization, as evidenced by immunostaining dot blots (average titer 3+). The total serum IgM response against GM2 (2+ or 3+ on dot blots and an ELISA titer of at least 1/20 or 1+ on dot blots and an ELISA titer of at least 1/80) in the two groups was 11% for only BCG and 86% for GM2 / BCG. IgG antibodies were not detected in the serum of any patient prior to immunization and were not induced in any patient by BCG immunization (table 10). GM2 / BCG vaccination induced a positive IgG immune response in eight patients (mean ELISA titer 1/80, mean dot blot response 2+). These responses were short-term (average duration of 8 weeks) and usually did not increase with additional immunization. IgG reactivity in all cases was limited by reactivity with GM2 (data not shown). All 8 patients also had IgM antibodies, the titer of which was higher and the duration of their presence was longer than that of IgG antibodies.

DTH response to GM2
Skin tests for DTH against GM2 and other antigens were performed after an initial series of three vaccinations. All patients who received three or more immunizations showed strong DTH responses against BCG. Eight patients (four patients vaccinated with BCG and four patients vaccinated with GM2 / BCG) showed positive DTH responses to GM2 and GM2 liposomes (used to maintain GM2 at the site of skin testing) as an induction of 10 to 33 mm. Further testing showed that these 8 patients had similar reactivity with liposomes without gangliosides and with other gangliosides. Thus, in any patient there was no evidence of GM2-specific DTH.

Serological response against BCG
The sera of all patients were tested for reactivity (ELISA) of IgG against 10 ³ viable organisms / well BCG dried on ELISA plates as described for gangliosides. Reactivity was not detected prior to vaccination. After vaccination, the sera showed anti-BCG antibody titers of 1 / 40-1 / 80 in both study groups, regardless of whether the patients produced anti-GM2 antibodies or not.

Correlation of the humoral immune response and immunization with a disease-free period and survival
Comparison of the disease period and overall survival for all patients producing anti-GM2 antibodies, documented by two analyzes (ELISA titer of at least 1/180 and dot blot staining of 1+ or ELISA titer of at least 1/20 and dot blot staining of at least 2+ ), with such indicators of patients who did not produce anti-GM2 antibodies, is shown in FIG. 12. A significant difference is visible both for the period without the disease and for overall survival (p = 0.004 and 0.02, respectively, log test). Of the six patients who produced antibodies against GM2 before immunization (five received BCG and one received GM2 / BCG), only one (the patient in the BCG group) had a recurrent disease. This confirms that the natural production of antibodies against GM2 in patients with melanoma is associated with a favorable course of the process. Conversely, of the eight patients who did not produce anti-GM2 antibodies after immunization with GM2 / BCG, six had recurrent melanoma and died. This confirms that the lack of antibody production against GM2 after vaccination indicates an unfavorable prognosis. The average titers of antibodies against BCG in six patients producing natural GM2 antibodies were the same as the titers in eight patients with negative responses to antibodies against GM2 in the part vaccinated with GM2 / BCG. This indicates that these patients did not differ in their ability to provide a serological response to an unrelated antigen.

 When six patients producing antibodies against GM2, before randomization (six in the BCG part and one in the GM2 / BCG part) were excluded from the analysis, the disease-free period in the GM2 / BCG group was significantly longer than in the BCG group ( p = 0.02, log test), and a tendency to increase overall survival was also observed in the GM2 / BCG group (Fig. 13). The curves turned into a plateau for a time of 40-52 months after surgery, the difference of 23% and 14% was for the period without disease and overall survival, respectively. The small number of patients producing IgG antibodies that react with GM2 after immunization does not make it possible to conclude from the relative contribution of IgM and IgG antibodies against GM2. Five out of eight patients who tested positive for anti-GM2 IgG antibodies remained healthy over the same time period.

 A comparison of two immunized groups that are randomized is shown in FIG. 14. The curves form a plateau at a time after surgery of 40 to 52 months, a difference of 18% for the period without disease and 11% for overall survival was in favor of GM2 / BCG vaccination. These differences were not statistically significant. The degrees of the disease-free period (30%) and overall survival (46%) after 51 months (the minimum period of the subsequent medical examination) in the BCG group were similar to the degrees that the applicants had previously observed in patients randomized for BCG administration or control purposes (28) . As shown in FIG. 14, the beneficial effect of immunization was more evident when two immunized groups were classified by the number of positive nodes (1 versus 2 or more). Immunization had less impact on the disease-free period in patients with only one positive node. In contrast, the disease-free period in patients with two or more positive nodes receiving GM2 / BCG was significantly longer than in patients immunized with BCG only (p = 0.02). A similar trend was observed for survival (p = 0.08, log test).

 Discussion.

 The applicants made significant efforts to develop ganglioside vaccines that induce high titers of serum antibodies (18, 19), because 1) gangliosides are the main components of the surface of melanoma cells (14, 20-25), 2) human monoclonal antibodies with specificity for gangliosides can to isolate with relatively high frequency in patients with melanoma (14, 25-28) and 3) human tumor cells expressing ganglioside antigens can be lysed with antibodies against gangliosides in the presence of human complement eka (13, 29). It has been shown that ganglioside GM2, in particular, is an immunogen for humans and vaccines containing purified GM2 and BCG as an adjuvant cause the formation of antibodies against GM2 in melanoma patients who were previously given a low dose of cyclophosphamide (8, 9). The study presented here was intended to answer two questions: does vaccination with the GM2 / BCG vaccine induce the production of antibodies against GM2 in a high percentage of melanoma patients and does the induction of antibodies against GM2 change the course of the disease in patients with stage III melanoma after complete resection of the entire identified tumor.

 Regarding the induction of antibodies against GM2, immunization with the GM2 / BCG vaccine was clearly effective in the majority of patients. Of the 58 patients who received the GM2 / BCG vaccine, 50 produced antibodies against GM2, detected by both ELISA and immunostaining on dot blots. As in early experiments (9), the induced antibodies against GM2 were mainly IgM-class; IgG antibodies were detected less frequently. The correlation between the production of antibodies against GM2 and a more favorable clinical course (9) was also confirmed and expanded here for a more homogeneous group of patients without stage III AJCC melanoma. Patients producing antibodies against GM2 (natural production or production induced by the vaccine) had a significantly longer period without disease and overall survival than patients who did not show a humoral response. The initial presence of antibodies against GM2 (before vaccination) was found in six patients in this series and was associated with a particularly favorable prognosis. Since the coverage of patients with spontaneous production of antibodies against GM2 was the same (10%) for the general group and patients with melanoma (8, 9). their relationship with the development and growth of melanoma remains unknown. It is known that the titer of previously present anti-GM2 antibodies did not change during the nine-month follow-up period, whereas the titer of vaccine-induced anti-GM2 antibodies was usually able to be detected only within 8-12 months after immunization. This observation increases the likelihood that consistent antibody production may be important for a favorable clinical outcome, and this indicates the need for the development of GM2 vaccines that produce long-term immune responses against GM2. In contrast to vaccinated patients who developed anti-GM2 antibody production, 8 patients who did not produce anti-GM2 antibodies after immunization with GM2 / BCG showed particularly rapid progression of the disease, with a relapse usually found before the entire series of five immunizations was given. Other prognostic factors were no less favorable for these patients and their immune response against BCG was not less strong than the response in patients with a positive immune response against GM2. This indicates that general immunosuppression is not the main mechanism.

 The association between the production of anti-GM2 antibodies and improved prognosis has also been confirmed in studies of the antigen designated OFA-1 (31), a cell surface antigenic system expressed by many melanomas. Several lines of evidence indicate that the reactivity of antibodies against OFA is mainly directed against GM2 ganglioside (30, 25). Patients with elevated anti-OFA-1 IgG titers, having them before immunization or after immunization with irradiated melanoma cells, had increased survival.

 Although the present study reinforces the evidence that anti-GM2 antibody production is associated with a favorable prognosis in patients with melanoma, it also illustrates the problems that arise with randomized vaccine trials. Pre-production (before immunization) of antibodies against GM2 in 5 patients in the control group as opposed to 1 patient in GM2 / BCG-rpynne, the lack of an immune response against GM2 in 8 vaccinated patients in GM2 / BCG-rpynne helped to smooth out the difference between the two randomized vaccinated groups before the point at which the therapeutic result in the GM2 / BCG group was negligible. Obviously, patients with a preliminary presence of antibodies against GM2 should be excluded or at least allocated to separate groups in future studies. An equally important task is the need to increase the immunogenicity of vaccines. In this regard, the applicants investigated approaches that were successfully developed in the context of carbohydrate vaccines against bacterial infections (review in 32), including chemical modification of gangliosides to produce closely related congeners (33, 34), conjugation of ganglioside with immunogenic carrier proteins (35) and the use of stronger adjuvants (36). Applicants have shown that this approach was most effective for conjugating GM2 with cochlear lymph hemocyanin (KLH) and administering GM2-KLH conjugate with QS-21 (Saponaria Quillaja saponin extract) as an adjuvant. In a search study in patients with melanoma, the administration of this vaccine led to the detection of titers of anti-GM2 IgM antibodies significantly higher than those after GM2 / BCG and, more importantly, the persistent production of anti-GM2 antibodies belonging to the IgG class, thus apparently transforming the T-independent immune response into a T-dependent immune response (37).

 An additional approach to improving the clinical efficacy of the GM2 / BCG vaccine described herein should be to introduce additional immunogenic melanoma gangliosides into the vaccine. Mouse experiments show that conjugation with KLH and the use of QS-21 as an adjuvant also increase the immunogenicity of GD3 (35), the most highly expressed ganglioside by melanoma, which has, so far, shown very weak immunogenicity in patients with melanoma. If these statements could be confirmed in a study of people, the applicants could have the basis for the construction of a polyvalent ganglioside conjugate vaccine that includes several major melanoma gangliosides, thereby circumventing the heterogeneity of ganglioside expression found in human melanomas. Applicants believe that the results of the study described here confirm further studies of this approach.

Claims (34)

 1. A composition for stimulating or increasing the production of antibodies that recognize a ganglioside, comprising a) a conjugate i) a ganglioside derivative comprising an unchanged oligosaccharide portion and an altered ceramide portion, ii) an immunogenic protein-based carrier, b) a saponin derived from Quillfria saponaria bark Moline, and c) a pharmaceutically acceptable carrier, wherein the relative amounts of conjugate and saponin are effective to stimulate or increase antibody production in a subject; moreover, the protein-based immunogenic carrier is obtained from a protein selected from the group consisting of a malarial T-cell epitope, an external membrane protein Neisseria meningitidis, cationic bovine serum albumin, Keyhole Limpet hemocyanin or its derivative, polylysine and human serum albumin, characterized in that in said conjugate, the ganglioside derivative is conjugated to a protein-based immunogenic carrier via a ceramide carbon of a ganglioside derivative linked to a protein-based immunogenic carrier.  2. The composition according to claim 1, characterized in that the ceramide part comprises a sphingosine base and in the said conjugate the ganglioside derivative is conjugated to the protein-based immunogenic carrier via the C-4 carbon of the sphingosphine base of the ceramide part of the ganglioside derivative bound to the protein-based immunogenic carrier.  3. The composition of claim 1, wherein the protein-based immunogenic carrier comprises an ε-aminolysyl group and, in said conjugate, the ganglioside derivative is conjugated to a protein-based immunogenic carrier via the ε-aminolysyl group of the immunogenic protein carrier.  4. The composition according to claim 1, characterized in that the ceramide portion comprises a sphingosine base, and the protein-based immunogenic carrier comprises an ε-aminolysyl group and, in said conjugate, the ganglioside derivative is conjugated to the protein-based immunogenic carrier via a C-4 carbon of the sphingosine base of the ceramide parts of a ganglioside derivative with ε-aminolysyl group of an immunogenic protein carrier.  5. The composition according to any one of claims 1 to 4, characterized in that the saponin obtained from the bark of Quillaria saponaria Molina is QS-21.  6. A composition for stimulating or increasing the production of antibodies that recognize a ganglioside, comprising a) a conjugate i) an oligosaccharide portion of the ganglioside with ii) cochlear lymph hemocyanin b) saponin derived from Quillaria saponaria molina bark, and c) a pharmaceutically acceptable carrier, wherein amounts of conjugate and saponin are effective to stimulate or increase antibody production in a subject.  7. The composition according to claim 6, characterized in that the saponin obtained from the bark of Quillaria saponaria Molina is QS-21.  8. The composition according to any one of claims 1 to 5, characterized in that the ganglioside derivative is conjugated with cochlear lymphocytic hemocyanin.  9. A composition according to any one of claims 1 to 5 and 8, characterized in that the ganglioside derivative is a ganglioside derivative selected from the group consisting of GM2, GM3, GD2, GD3, GD3 lactone, O-acetyl GD3 and GT3.  10. The composition according to claim 9, characterized in that the ganglioside derivative is a derivative of GM2 or GD2.  11. The composition of claim 10, wherein the ganglioside derivative is a GM2 derivative.  12. The composition of claim 10, wherein the ganglioside derivative is a derivative of GD2.  13. The composition according to PP. 1-12, characterized in that it comprises a conjugate in an amount of from about 1 μg to about 200 μg.  14. The composition according to p. 13, characterized in that it comprises a conjugate in an amount of from about 50 μg to about 90 μg.  15. The composition according to 14, characterized in that the amount of conjugate is about 70 μg.  16. The composition according to p. 13, characterized in that it comprises a conjugate in an amount of from about 1 μg to about 10 μg.  17. The composition according to p. 16, characterized in that the amount of conjugate is about 7 μg.  18. The composition according to any one of claims 1 to 17, characterized in that it comprises saponin in an amount of from about 10 μg to about 200 μg.  19. The composition according to p. 18, characterized in that the amount of saponin is about 100 μg.  20. The composition according to p. 18, characterized in that the amount of saponin is about 200 μg.  21. The composition according to any one of claims 1 to 5 and 8-20, characterized in that the molar ratio of the ganglioside derivative to the immunogenic protein carrier is in the range from about 200 to about 1400.  22. The composition according to claim 6 or 7, characterized in that the molar ratio of oligosaccharide to hemocyanin of the lymph of the cochlea is in the range from about 200 to about 1400.  23. A method of stimulating or increasing the production of antibodies that recognize a ganglioside in a subject, which comprises administering to the subject an effective amount of a composition that comprises a) a conjugate i) of a ganglioside derivative comprising an unchanged oligosaccharide moiety and an altered ceramide moiety, with ii) a protein-based immunogenic carrier b) saponin derived from the bark of Quillaria saponaria molina; c) a pharmaceutically acceptable carrier, wherein the relative amounts of conjugate and saponin are effective to stimulate or increasing antibody production in a subject; moreover, the protein-based immunogenic carrier is obtained from a protein selected from the group consisting of a malarial T-cell epitope, external membrane protein Neisseria meningitidis, cationic bovine serum albumin, cochlear lymphocyte hemocyanin or its derivative, polylysine and human serum albumin, characterized in that said conjugate, the ganglioside derivative is conjugated to a protein-based immunogenic carrier via a ceramide carbon of the ganglioside derivative bound to the immunogenic protein carrier.  24. A method for preventing or treating cancer in a subject, which comprises administering to the subject an effective amount of a composition for preventing or treating cancer, which comprises a) a ganglioside derivative conjugate, which comprises an unchanged oligosaccharide moiety and an altered ceramide moiety, with ii) an immunogenic carrier protein, b) saponin derived from the bark of Quillaria saponaria Molina c) a pharmaceutically acceptable carrier, while the relative amounts of conjugate and saponin are effective to stimulate or increase oi producing antibodies in a subject; moreover, the protein-based immunogenic carrier is obtained from a protein selected from the group consisting of a malarial T-cell epitope, an external membrane protein Neisseria meningitidis, cationic bovine serum albumin, cochlear lymphocyte hemocyanin or a derivative thereof, polylysine and human serum albumin; characterized in that in said conjugate, the ganglioside derivative is conjugated to a protein-based immunogenic carrier via a ceramide carbon of the ganglioside derivative bound to the immunogenic protein carrier.  25. The method according to item 23 or 24, characterized in that the ceramide part comprises a sphingosine base and wherein in the said conjugate the ganglioside derivative is conjugated to a protein-based immunogenic carrier via the C-4 carbon of the sphingosphine base of the ceramide part of the ganglioside derivative associated with the immunogenic protein carrier .  26. The method according to item 23 or 24, wherein the protein-based immunogenic carrier comprises an ε-aminolysyl group and wherein in the said conjugate the ganglioside derivative is conjugated to the immunogenic protein carrier via the ε-aminolysyl group of the immunogenic protein carrier.  27. The method according to item 23 or 24, characterized in that the ceramide part comprises a sphingosine base, and the protein-based immunogenic carrier comprises an ε-aminolysyl group, and where in the said conjugate the ganglioside derivative is conjugated to the immunogenic protein carrier via C-4 carbon of the sphingosine base the ceramide portion of the ganglioside derivative bound to the ε-aminolysyl group of the immunogenic protein carrier.  28. The method according to paragraph 24, wherein the cancer is cancer of epithelial origin.  29. The method according to paragraph 24, wherein the cancer is a cancer of neuroectodermal origin.  30. The method according to p. 29, characterized in that the cancer of neuroectodermal origin is melanoma.  31. The method according to item 23 or 24, characterized in that the introduction is carried out in two or more places.  32. The method according to p, characterized in that the introduction is carried out in three places.  33. A method of stimulating or increasing antibody production in a subject, comprising administering to the subject an effective amount of a composition comprising a) a conjugate i) an oligosaccharide portion of the ganglioside with ii) cochlear lymph hemocyanin, b) saponin derived from Quillaria saponaria Molina bark, c) a pharmaceutically acceptable carrier while the relative amounts of conjugate and saponin are effective to stimulate or increase antibody production in a subject.  34. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a composition for preventing or treating cancer, comprising a) conjugate i) oligosaccharide portion of ginglioside with ii) cochlear lymphocytic hemocyanin, b) saponin derived from Quillaria saponaria molina bark and c ) a pharmaceutically acceptable carrier, wherein the relative amounts of conjugate and saponin are effective to stimulate or increase antibody production in a subject.

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