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Philip O. Livingston - One of the best experts on this subject based on the ideXlab platform.

  • The known Immunologically active components of Astragalus account for only a small proportion of the Immunological Adjuvant activity when combined with conjugate vaccines.
    Planta Medica, 2010
    Co-Authors: Feng Hong, Govind Ragupathi, Wei-lie Xiao, Ping-chung Leung, K. Simon Yeung, Constantine George, Barrie R. Cassileth, Edward J. Kennelly, Philip O. Livingston
    Abstract:

    : The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an Immunological Adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this Adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an Immunological Adjuvant in future studies. Several flavonoids also had significant Adjuvant activity. However, when the activities of these known Immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the Immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to Adjuvant activity, or that the Adjuvant activity of Astragalus is greater than the activity of the sum of its parts.

  • consistent antibody response against ganglioside gd2 induced in patients with melanoma by a gd2 lactone keyhole limpet hemocyanin conjugate vaccine plus Immunological Adjuvant qs 21
    Clinical Cancer Research, 2003
    Co-Authors: Govind Ragupathi, John Gathuru, Susan E Krown, Chandra Hood, Linda Williams, Jedd D. Wolchok, Philip O. Livingston, Paul B Chapman, Roberta C Oldfield
    Abstract:

    Purpose: Melanomas, sarcomas, and neuroblastomas abundantly express the ganglioside GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. In addition, preclinical models have demonstrated the ability of a GD2-keyhole limpet hemocyanin (KLH) conjugate vaccine to induce antibodies that eliminate micrometastases. However, vaccination of patients with GD2-KLH has previously failed to induce a consistent relevant antibody response. We test here whether the use of GD2 lactone-KLH can overcome the low immunogenicity of GD2-KLH. Experimental Design: Eighteen patients with melanoma were vaccinated s.c. in the Adjuvant setting on weeks 0, 1, 2, 3, 10, and 24. Groups of 6 patients were entered at three dose levels (3, 10, or 30 μg) of GD2 lactone (GD2L) in vaccines containing GD2L-KLH plus the Immunological Adjuvant QS-21. Blood was drawn at regular intervals to assess the antibody response. Results: The vaccine was well tolerated. The majority of patients in all three dose levels produced anti-GD2 antibodies detectable by ELISA assay. Specificity for GD2 was also confirmed by immune thin-layer chromatography. Although there was no statistical difference in terms of titers between the three groups, patients at the 30-μg dose level had higher titers and longer lasting antibody responses overall by ELISA (median IgM/IgG peak titer 1:640/1:80) and generated the strongest cell surface reactivity by fluorescence-activated cell sorting (median IgM peak percentage positive cells/mean fluorescence intensity for pre- and postvaccination sera is 10%/63 and 70%/135). Patients vaccinated with the 30-μg GD2 dose also had the most potent complement dependent cytotoxicity using human complement, with 5 of 6 patients showing strong cell surface reactivity by fluorescence-activated cell sorting and >30% cytotoxicity by chromium release with a serum dilution of 1/100. Conclusions: GD2L-KLH conjugate vaccine plus Adjuvant QS-21 induces antibodies against GD2 that bind to the cell surface and induce complement-dependent cytotoxicity in the majority of patients with melanoma.

  • keyhole limpet hemocyanin conjugate vaccines against cancer the memorial sloan kettering experience
    Journal of Cancer Research and Clinical Oncology, 2001
    Co-Authors: Cristina Musselli, Philip O. Livingston, Govind Ragupathi
    Abstract:

    Passively administered and actively induced antibodies have been associated with the eradication of circulating tumor cells and micrometastases in mice and humans. We have identified a series of cell surface carbohydrate and peptide antigens on melanomas, sarcomas, and cancer of the breast, prostate, ovary, and lung tissues. We found that breaking tolerance toward these tumor antigens was best achieved using vaccines containing antigens chemically conjugated to keyhole limpet hemocyanin (KLH) plus a potent Immunological Adjuvant (QS-21). To date, by using this approach to vaccination, antibodies have been induced in patients against glycolipid . antigens GM2, GD2, GD3, Fucosy1GM1, Globo H, and Lewis Y, and glycoprotein (mucin) antigens Tn, sialyl Tn, TF, and MUCL More recently, in a comparative study we investigated the T cell response induced by MUC1-KLH conjugates. Although a MUC1-specific T cell response was not consistently detected in any patient, the role of KLH in orienting the cytokine environment was crucial. We were able to confirm that KLH in these conjugate vaccines induces a Th1 T cell response as demonstrated by the high anti-KLH INF-7 secretion and the IgG1 and IgG3 subclasses of this high titer IgG antibodies induced. Clinical trials using KLH conjugated glycolipid and glycoprotein vaccines, are currently ongoing. These range from phase I/II single antigens trials with glycosilated MUC1, polysialic acid, synthetic Fucosyl GM1 and GD2, to phase II trials with a polyvalent vaccine containing six or seven antigens. Randomized phase 11 trials with polyvalent vaccines are planned for initiation in 2001–2002 in patients with ovarian, breast, and prostate cancer.

  • Effect of Immunological Adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates.
    Vaccine, 2000
    Co-Authors: Govind Ragupathi, Simone Cappello, Elliott Kagan, Philip O. Livingston
    Abstract:

    A year ago we described a comparison of 19 Immunological Adjuvants for their ability to augment antibody and T-cell responses against vaccines containing two cancer antigens, GD3 ganglioside and MUC1 peptide, covalently attached to keyhole limpet hemocyanin (KLH). As in our previous experience, the saponin fraction QS-21 was the most potent single Adjuvant but several other Adjuvants also had potent Adjuvant activity. Induction of an immune response against cancer antigens is generally difficult because these antigens are autoantigens. To get maximal benefit from the Adjuvant component of cancer vaccines we have now tested whether combinations of the optimal Adjuvants induced an improved immune response compared to QS-21 alone. Since over the intervening year a new semi-synthetic saponin Adjuvant (GPI-0100) containing the dodecylamide derivative of hydrolyzed naturally-occurring saponins had become available, this was tested as well. Twelve different Adjuvant combinations and GPI-0100 were compared for their ability to augment (1) antibody responses against GD3 and MUC1 and (2) T-cell responses against GD3, MUC1 and KLH. GPI-0100 and five Adjuvant combinations were superior to QS-21 alone for induction of IgM and IgG antibodies against MUC1 and/or GD3: QS-21 plus bacterial nucleotide CpG, QS-21 plus monophosphoryl lipid A (MPL), QS-21 plus non-ionic block copolymer CRL-1005, QS-21 plus Titermax and Titermax plus CpG. Antibody responses were documented both by ELISA against purified antigens and by FACS for cell surface reactivity. There was no evidence for T-cell immunity against GD3 or MUC1. The antibody responses against GD3 and MUC1 were, however, strongly correlated with IFN-gamma release and DTH against KLH. These results demonstrate that combinations of Immunological Adjuvants are able to augment antibody and T-cell responses to these conjugates beyond that attainable with QS-21 alone, and again confirm the absolute necessity of potent Adjuvants or Adjuvant combinations for optimal immunogenicity with conjugate vaccines.

  • Immunization of ovarian cancer patients with a synthetic Lewisy-protein conjugate vaccine : A phase 1 trial
    International Journal of Cancer, 2000
    Co-Authors: Paul Sabbatini, Samuel J. Danishefsky, Maria Spassova, Andrzej Zatorski, David Spriggs, Philip O. Livingston, William G Bornmann, Valery Kudryashov, Carol Aghajanian
    Abstract:

    As the initial step in developing carbohydrate-based vaccines for the treatment of ovarian cancer patients in an Adjuvant setting, 25 patients were immunized with a Lewisy pentasaccharide (Ley)-keyhole limpet hemocyanin (KLH)-conjugate vaccine together with the Immunological Adjuvant QS-21. Four different doses of the vaccine, containing 3, 10, 30, and 60 μg of carbohydrate were administered s.c. at 0, 1, 2, 3, 7, and 19 weeks to groups of 6 patients. Sera taken from the patients at regular intervals were assayed by ELISA for reactivity with naturally occurring forms of Ley (Ley-ceramide and Ley mucin) and by flow cytometry and a complement-dependent cytoxicity assay for reactivity with Ley-expressing tumor cells. The majority of the patients (16/24) produced anti-Ley antibodies as assessed by ELISA, and a proportion of these had strong anti-tumor cell reactivity as assessed by flow cytometry and complement-dependent cytotoxicity. One serum, analyzed in detail, was shown to react with glycolipids but not with glycoproteins or mucins expressed by ovarian cancer cell line OVCAR-3. The vaccine was well tolerated and no gastrointestinal, hematologic, renal, or hepatic toxicity related to the vaccine was observed. On the basis of this study, Ley-KLH should be a suitable component for a polyvalent vaccine under consideration for the therapy of epithelial cancers. Int. J. Cancer 87:79–85, 2000. © 2000 Wiley-Liss, Inc.

Govind Ragupathi - One of the best experts on this subject based on the ideXlab platform.

  • a phase i study of unimolecular pentavalent globo h gm2 stn tf tn immunization of patients with epithelial ovarian fallopian tube or peritoneal cancer in first remission
    Cancers, 2016
    Co-Authors: Roisin E Ocearbhaill, Govind Ragupathi, Maria Spassova, Jianglong Zhu, Qian Wan, Svetlana Mironov, Guangbin Yang, Alexia Iasonos, Sara Kravetz, Ouathek Ouerfelli
    Abstract:

    We conducted a phase I study in ovarian cancer patients to evaluate the safety and immunogenicity of a synthetic unimolecular pentavalent carbohydrate vaccine (Globo-H, GM2, sTn, TF, and Tn) supported on a peptide backbone, conjugated to keyhole limpet haemocyanin (KLH), and mixed with Immunological Adjuvant QS-21. Twenty-four advanced-stage, poor-risk, first-remission ovarian cancer patients were enrolled from January 2011–Septermber 2013. Three dose levels were planned (25, 50, 100 mcg) with three cohorts of six patients each, with an additional 6-patient expansion cohort at the MTD. ELISA serologic IgM and IgG responses for each antigen was defined as positive response if antibody titers were ≥1:80 over the respective patient’s pre-vaccination serum. The study would be considered positive if at least four of 12 patients treated at the MTD showed immune responses for at least three of the five antigens. Twenty-four patients (median age, 54 years [range, 36–68]) were included in the safety analysis. Histology was high-grade serous in 22 patients (92%); 18 had stage III and six stage IV disease. The vaccine was well-tolerated at all doses, with no DLTs. At the highest treated dose, IgG and/or IgM responses were recorded against ≥3 antigens in 9/12 patients (75%), ≥4 in 7/12 (58%), and 5 in 3/12 (25%). With a median follow-up of 19 months (range, 2–39), 20 patients (83%) recurred and six (25%) died. The unimolecular pentavalent vaccine construct was shown to be safe and immunogenic. Such a construct greatly simplifies regulatory requirements and manufacturing, facilitates scalability, and provides adaptability.

  • The known Immunologically active components of Astragalus account for only a small proportion of the Immunological Adjuvant activity when combined with conjugate vaccines.
    Planta Medica, 2010
    Co-Authors: Feng Hong, Govind Ragupathi, Wei-lie Xiao, Ping-chung Leung, K. Simon Yeung, Constantine George, Barrie R. Cassileth, Edward J. Kennelly, Philip O. Livingston
    Abstract:

    : The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an Immunological Adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this Adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an Immunological Adjuvant in future studies. Several flavonoids also had significant Adjuvant activity. However, when the activities of these known Immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the Immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to Adjuvant activity, or that the Adjuvant activity of Astragalus is greater than the activity of the sum of its parts.

  • a bivalent conjugate vaccine in the treatment of biochemically relapsed prostate cancer a study of glycosylated muc 2 klh and globo h klh conjugate vaccines given with the new semi synthetic saponin Immunological Adjuvant gpi 0100 or qs 21
    Vaccine, 2005
    Co-Authors: Susan F. Slovin, Shemeeakah Powell, Matthew P Jefferson, Andrew Wilton, Celina Fernandez, Meghan Diani, Govind Ragupathi, Maria Spassova, Celso A. Reis, Henrick Clausen
    Abstract:

    GPI-0100 is a semi-synthetic saponin with modifications designed to augment stability and diminish toxicity. Two batches of GPI-0100 (the second with higher purity) were tested with doses ranging between 100 and 5000 μg in groups of five treated prostate cancer patients who had no evidence of disease except for rising PSA levels. GPI-0100 was mixed with a bivalent vaccine containing the glycolipid Globo H and the glycosylated mucin MUC2 conjugated to keyhole limpet hemocyanin (KLH). All doses were well tolerated and antibody titers against Globo H and MUC-2 escalated with the increasing dose levels. At the 5000 μg dose level in this patient population, toxicity remained minimal with only occasional grade II local toxicity at vaccination sites and occasional sporadic grade I elevations in ALT. Compared with a subsequent trial with the same bivalent vaccine plus QS-21 at the maximal tolerated dose of 100 μg, the 5000 μg dose of GPI-0100 produced comparable antibody titers.

  • Vaccination of Small Cell Lung Cancer Patients with Polysialic Acid or N-Propionylated Polysialic Acid Conjugated to Keyhole Limpet Hemocyanin
    Clinical cancer research : an official journal of the American Association for Cancer Research, 2004
    Co-Authors: Lee M. Krug, Govind Ragupathi, Chandra Hood, Harold J. Jennings, Mark G. Kris, Zhongwu Guo, Vincent A. Miller, Barbara Pizzo, Leslie B. Tyson
    Abstract:

    Purpose: Long chain polysialic acid (polySA) is a side chain on embryonal neural cell adhesion molecules that, in the adult, is largely restricted to small cell lung cancer (SCLC). Long chains of polySA are also expressed on group B meningococcus. In this clinical trial, we aimed to elicit an immune response against polysialic acid to target clinically inapparent residual disease in patients with SCLC who had successfully completed initial therapy. Experimental Design: Patients were vaccinated with either 30 μg unmodified polySA or N -propionylated-polySA (NP-polySA), conjugated to keyhole limpet hemocyanin (KLH) and mixed with 100 μg of Immunological Adjuvant QS-21 at weeks 1, 2, 3, 4, 8, and 16. Results: Of the 5 evaluable patients vaccinated with unmodified polySA, only 1 mounted an IgM antibody response to polySA. On the other hand, all 6 of the patients vaccinated with NP-polySA produced IgM antibodies to NP-polySA and these cross-reacted with unmodified polySA in all but 1 case. IgG antibodies to NP-polySA were observed in 5 of the patients, but these did not cross-react with polySA. The presence of IgM antibodies reactive with SCLC cell lines was confirmed in this group by flow cytometry. Complement-dependent lysis of tumor cells could not be demonstrated. However, postimmunization sera induced significant bactericidal activity against group B meningococcus when combined with rabbit complement. Conclusions: Vaccination with NP-polySA-KLH, but not polySA-KLH, resulted in a consistent high titer antibody response. We are now conducting a de-escalation dosing study with NP-polySA-KLH to better assess the immunogenicity, toxicities, and optimal dose of this vaccine. We plan to incorporate this vaccine as a component of a polyvalent vaccine with GM2, fucosylated GM1, and Globo H to target SCLC.

  • consistent antibody response against ganglioside gd2 induced in patients with melanoma by a gd2 lactone keyhole limpet hemocyanin conjugate vaccine plus Immunological Adjuvant qs 21
    Clinical Cancer Research, 2003
    Co-Authors: Govind Ragupathi, John Gathuru, Susan E Krown, Chandra Hood, Linda Williams, Jedd D. Wolchok, Philip O. Livingston, Paul B Chapman, Roberta C Oldfield
    Abstract:

    Purpose: Melanomas, sarcomas, and neuroblastomas abundantly express the ganglioside GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. In addition, preclinical models have demonstrated the ability of a GD2-keyhole limpet hemocyanin (KLH) conjugate vaccine to induce antibodies that eliminate micrometastases. However, vaccination of patients with GD2-KLH has previously failed to induce a consistent relevant antibody response. We test here whether the use of GD2 lactone-KLH can overcome the low immunogenicity of GD2-KLH. Experimental Design: Eighteen patients with melanoma were vaccinated s.c. in the Adjuvant setting on weeks 0, 1, 2, 3, 10, and 24. Groups of 6 patients were entered at three dose levels (3, 10, or 30 μg) of GD2 lactone (GD2L) in vaccines containing GD2L-KLH plus the Immunological Adjuvant QS-21. Blood was drawn at regular intervals to assess the antibody response. Results: The vaccine was well tolerated. The majority of patients in all three dose levels produced anti-GD2 antibodies detectable by ELISA assay. Specificity for GD2 was also confirmed by immune thin-layer chromatography. Although there was no statistical difference in terms of titers between the three groups, patients at the 30-μg dose level had higher titers and longer lasting antibody responses overall by ELISA (median IgM/IgG peak titer 1:640/1:80) and generated the strongest cell surface reactivity by fluorescence-activated cell sorting (median IgM peak percentage positive cells/mean fluorescence intensity for pre- and postvaccination sera is 10%/63 and 70%/135). Patients vaccinated with the 30-μg GD2 dose also had the most potent complement dependent cytotoxicity using human complement, with 5 of 6 patients showing strong cell surface reactivity by fluorescence-activated cell sorting and >30% cytotoxicity by chromium release with a serum dilution of 1/100. Conclusions: GD2L-KLH conjugate vaccine plus Adjuvant QS-21 induces antibodies against GD2 that bind to the cell surface and induce complement-dependent cytotoxicity in the majority of patients with melanoma.

Sucharita Adluri - One of the best experts on this subject based on the ideXlab platform.

  • induction of antibodies against gd3 ganglioside in melanoma patients by vaccination with gd3 lactone klh conjugate plus Immunological Adjuvant qs 21
    International Journal of Cancer, 2000
    Co-Authors: Michael L. Meyers, Sucharita Adluri, Lisa Howard, Cristina Musselli, Philip O. Livingston
    Abstract:

    The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the Immunological Adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 μg of ganglioside plus 100 μg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40–1/1,280 in all patients and IgG titers of 1/160–1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28. Int. J. Cancer 85:659–666, 2000. © 2000 Wiley-Liss, Inc.

  • induction of antibodies against gd3 ganglioside in melanoma patients by vaccination with gd3 lactone klh conjugate plus Immunological Adjuvant qs 21
    International Journal of Cancer, 2000
    Co-Authors: Govind Ragupathi, Sucharita Adluri, Lisa Howard, Michael L. Meyers, Cristina Musselli, Philip O. Livingston
    Abstract:

    The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the Immunological Adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 microg of ganglioside plus 100 microg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40-1/1,280 in all patients and IgG titers of 1/160-1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28.

  • specificity analysis of sera from breast cancer patients vaccinated with muc1 klh plus qs 21
    British Journal of Cancer, 1999
    Co-Authors: Sucharita Adluri, Vinod Ramnath, Shengle Zhang, Teresa Gilewski, Philip O. Livingston
    Abstract:

    The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH). Six breast cancer patients immunized with this conjugate plus the Immunological Adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the β turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells. © 1999 Cancer Research Campaign

  • Tumor cell reactivity mediated by IgM antibodies in sera from melanoma patients vaccinated with GM2 ganglioside covalently linked to KLH is increased by IgG antibodies
    Cancer Immunology Immunotherapy, 1997
    Co-Authors: P. Livingston, Sucharita Adluri, Friedhelm Helling, Shengle Zhang, Govind Ragupathi, Linda Graeber, Martin Fleisher
    Abstract:

     Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials. A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the Immunological Adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials. For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients. With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge. Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells. Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells. Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations. Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction. However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone. In addition, ADCC was induced in all seven patients. The results were the same whether the sera were obtained after 2 months or 2 years of immunizations. These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies.

  • gm2 klh conjugate vaccine increased immunogenicity in melanoma patients after administration with Immunological Adjuvant qs 21
    Cancer Research, 1995
    Co-Authors: Friedhelm Helling, Sucharita Adluri, Shengle Zhang, Ann Shang, Michele Calves, Michael B Longenecker, R. Rao Koganty, Herbert F. Oettgen, Philip O. Livingston
    Abstract:

    Abstract The cell surface gangliosides GM2, GD2, and GD3 are often overexpressed in malignant melanoma. We have shown previously that immunization of melanoma patients with GM2 and Bacillus Calmette-Guerin induced an IgM antibody response in most patients and that patients with high titer GM2 antibodies showed increased survival. As is commonly seen with carbohydrate antigens (which are T independent), the IgM response was short lived, and an IgG response was rarely observed. To increase immunogenicity, we conjugated GM2 covalently with keyhole limpet hemocyanin (KLH). GM2-KLH vaccine was given to melanoma patients alone or with one of the three Adjuvants: Bacillus Calmette-Guerin, DE-TOX, or QS-21. The most effective vaccine was GM2-KLH with QS-21. It induced a much higher titer, a longer-lasting IgM GM2 antibody response, and a consistent IgG response (isotype IgG1 and IgG3). It also induced the highest titer anti-KLH response. The results suggest that the conjugate GM2-KLH plus QS-21 vaccine elicited significant T-cell help. Because there was no serious toxicity, this vaccine approach is attractive for augmenting the immunogenicity of other gangliosides, such as GD2 and GD3, and to determine the effects of ganglioside antibodies on the course of melanoma. In addition, the finding that QS-21 significantly increased the immunogenicity of GM2-KLH suggests that it may do the same for other conjugate vaccines, many of which are currently used without Adjuvant.

Cristina Musselli - One of the best experts on this subject based on the ideXlab platform.

  • keyhole limpet hemocyanin conjugate vaccines against cancer the memorial sloan kettering experience
    Journal of Cancer Research and Clinical Oncology, 2001
    Co-Authors: Cristina Musselli, Philip O. Livingston, Govind Ragupathi
    Abstract:

    Passively administered and actively induced antibodies have been associated with the eradication of circulating tumor cells and micrometastases in mice and humans. We have identified a series of cell surface carbohydrate and peptide antigens on melanomas, sarcomas, and cancer of the breast, prostate, ovary, and lung tissues. We found that breaking tolerance toward these tumor antigens was best achieved using vaccines containing antigens chemically conjugated to keyhole limpet hemocyanin (KLH) plus a potent Immunological Adjuvant (QS-21). To date, by using this approach to vaccination, antibodies have been induced in patients against glycolipid . antigens GM2, GD2, GD3, Fucosy1GM1, Globo H, and Lewis Y, and glycoprotein (mucin) antigens Tn, sialyl Tn, TF, and MUCL More recently, in a comparative study we investigated the T cell response induced by MUC1-KLH conjugates. Although a MUC1-specific T cell response was not consistently detected in any patient, the role of KLH in orienting the cytokine environment was crucial. We were able to confirm that KLH in these conjugate vaccines induces a Th1 T cell response as demonstrated by the high anti-KLH INF-7 secretion and the IgG1 and IgG3 subclasses of this high titer IgG antibodies induced. Clinical trials using KLH conjugated glycolipid and glycoprotein vaccines, are currently ongoing. These range from phase I/II single antigens trials with glycosilated MUC1, polysialic acid, synthetic Fucosyl GM1 and GD2, to phase II trials with a polyvalent vaccine containing six or seven antigens. Randomized phase 11 trials with polyvalent vaccines are planned for initiation in 2001–2002 in patients with ovarian, breast, and prostate cancer.

  • induction of antibodies against gd3 ganglioside in melanoma patients by vaccination with gd3 lactone klh conjugate plus Immunological Adjuvant qs 21
    International Journal of Cancer, 2000
    Co-Authors: Michael L. Meyers, Sucharita Adluri, Lisa Howard, Cristina Musselli, Philip O. Livingston
    Abstract:

    The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the Immunological Adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 μg of ganglioside plus 100 μg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40–1/1,280 in all patients and IgG titers of 1/160–1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28. Int. J. Cancer 85:659–666, 2000. © 2000 Wiley-Liss, Inc.

  • induction of antibodies against gd3 ganglioside in melanoma patients by vaccination with gd3 lactone klh conjugate plus Immunological Adjuvant qs 21
    International Journal of Cancer, 2000
    Co-Authors: Govind Ragupathi, Sucharita Adluri, Lisa Howard, Michael L. Meyers, Cristina Musselli, Philip O. Livingston
    Abstract:

    The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the Immunological Adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 microg of ganglioside plus 100 microg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40-1/1,280 in all patients and IgG titers of 1/160-1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28.

  • Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells
    Cancer Immunology Immunotherapy, 1999
    Co-Authors: Sarah Cappello, Cristina Musselli, Fred-thomas Brezicka, Philip O. Livingston, Govind Ragupathi
    Abstract:

    Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus Immunological Adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 Adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC.

Shengle Zhang - One of the best experts on this subject based on the ideXlab platform.

  • specificity analysis of sera from breast cancer patients vaccinated with muc1 klh plus qs 21
    British Journal of Cancer, 1999
    Co-Authors: Sucharita Adluri, Vinod Ramnath, Shengle Zhang, Teresa Gilewski, Philip O. Livingston
    Abstract:

    The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH). Six breast cancer patients immunized with this conjugate plus the Immunological Adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the β turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells. © 1999 Cancer Research Campaign

  • Tumor cell reactivity mediated by IgM antibodies in sera from melanoma patients vaccinated with GM2 ganglioside covalently linked to KLH is increased by IgG antibodies
    Cancer Immunology Immunotherapy, 1997
    Co-Authors: P. Livingston, Sucharita Adluri, Friedhelm Helling, Shengle Zhang, Govind Ragupathi, Linda Graeber, Martin Fleisher
    Abstract:

     Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials. A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the Immunological Adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials. For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients. With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge. Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells. Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells. Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations. Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction. However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone. In addition, ADCC was induced in all seven patients. The results were the same whether the sera were obtained after 2 months or 2 years of immunizations. These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies.

  • gm2 klh conjugate vaccine increased immunogenicity in melanoma patients after administration with Immunological Adjuvant qs 21
    Cancer Research, 1995
    Co-Authors: Friedhelm Helling, Sucharita Adluri, Shengle Zhang, Ann Shang, Michele Calves, Michael B Longenecker, R. Rao Koganty, Herbert F. Oettgen, Philip O. Livingston
    Abstract:

    Abstract The cell surface gangliosides GM2, GD2, and GD3 are often overexpressed in malignant melanoma. We have shown previously that immunization of melanoma patients with GM2 and Bacillus Calmette-Guerin induced an IgM antibody response in most patients and that patients with high titer GM2 antibodies showed increased survival. As is commonly seen with carbohydrate antigens (which are T independent), the IgM response was short lived, and an IgG response was rarely observed. To increase immunogenicity, we conjugated GM2 covalently with keyhole limpet hemocyanin (KLH). GM2-KLH vaccine was given to melanoma patients alone or with one of the three Adjuvants: Bacillus Calmette-Guerin, DE-TOX, or QS-21. The most effective vaccine was GM2-KLH with QS-21. It induced a much higher titer, a longer-lasting IgM GM2 antibody response, and a consistent IgG response (isotype IgG1 and IgG3). It also induced the highest titer anti-KLH response. The results suggest that the conjugate GM2-KLH plus QS-21 vaccine elicited significant T-cell help. Because there was no serious toxicity, this vaccine approach is attractive for augmenting the immunogenicity of other gangliosides, such as GD2 and GD3, and to determine the effects of ganglioside antibodies on the course of melanoma. In addition, the finding that QS-21 significantly increased the immunogenicity of GM2-KLH suggests that it may do the same for other conjugate vaccines, many of which are currently used without Adjuvant.

  • Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients
    Cancer Immunology Immunotherapy, 1995
    Co-Authors: Sucharita Adluri, Shengle Zhang, Kenneth O. Lloyd, Fiedhelm Helling, Steven H. Itzkowitz, Shigenobu Ogata, Philip O. Livingston
    Abstract:

    Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without Adjuvant, TF-KLH plus the Immunological Adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the Immunological Adjuvant QS-21, and the serological response was monitored. Enzyme- linked immunosorbent assay (ELISA), dot-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus Immunological Adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins