Govindaswami Ragupathi

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

Fully Synthetic Carbohydrate-Based Vaccines in Biochemically Relapsed Prostate Cancer: Clinical Trial Results With α-N-Acetylgalactosamine-O-Serine/Threonine Conjugate Vaccine

PURPOSE We report the synthesis of a mucin-related O-linked glycopeptide, alpha-N-acetylgalactosamine-O-serine/threonine (Tn), which is highly simplistic in its structure and can induce a relevant humoral response when given in a trimer or clustered (c) formation. We tested for an antitumor effect, in the form of a change in the posttreatment versus pretreatment prostate-specific antigen (PSA) slopes, that might serve as a surrogate for effectiveness of vaccines in delaying the time to radiographic progression. METHODS We compared the antibody response to immunization with two conjugates, Tn(c)-keyhole limpet hemocyanin (KLH) and Tn(c)-palmitic acid (PAM) with the saponin immunologic adjuvant QS21, in a phase I clinical trial in patients with biochemically relapsed prostate cancer. Patients received Tn(c)-KLH vaccine containing either 3, 7, or 15 microg of Tn(c) per vaccination. Ten patients received 100 microg of Tn(c)-PAM. QS21 was included in all vaccines. Five vaccinations were administered subcutaneously during 26 weeks with an additional booster vaccine at week 50. RESULTS Tn(c), when given with the carrier molecule KLH and QS21, stimulated the production of high-titer immunoglobulin M (IgM) and IgG antibodies. Inferior antibody responses were seen with T(c)-PAM. There was no evidence of enhanced immunogenicity with increasing doses of vaccine. An antitumor effect in the form of a decline in posttreatment versus pretreatment PSA slopes was also observed. CONCLUSION A safe synthetic conjugate vaccine in a trimer formation was developed that can break immunologic tolerance by inducing specific humoral responses. It seemed to affect the biochemical progression of the disease as determined by a change in PSA log slope.

Comparison of the effect of different immunological adjuvants on the antibody and T-cell response to immunization with MUC1-KLH and GD3-KLH conjugate cancer vaccines

While the importance of immunological adjuvants for optimal induction of antibody and T-cell responses against tumor antigens is clear, the relevant potency of different adjuvants is not clear. We have screened 19 different immunological adjuvants with KLH conjugate vaccines containing the two human cancer antigens (MUC1 peptide and GD3 ganglioside) in the mouse. ELISA assays for IgM and IgG antibody responses as well as proliferation and cytokine release (IFN-gamma and IL-4) for T-cell responses were performed. Six adjuvants stood out as being especially effective for induction of IgM and IgG antibodies against both MUC1 and GD3: QS-21, TiterMax, MoGM-CSF, MPL/DETOX and CpG ODN. Of these QS-21, MPL/DETOX and MoGM-CSF were uniformly effective at inducing potent proliferation and potent IFN-gamma and IL-4 responses against KLH while TiterMax and CpG ODN generated potent IFN-gamma responses but less potent proliferation or IL-4 release. Overall, as in our previous experience, QS-21 was the most effective adjuvant. There was no clear evidence for induction of T-cell immunity against either GD3 or MUC1 with any of the adjuvants. There was a strong correlation between the antibodies induced against MUC1 and GD3 with different immunological adjuvants and the strength of the IFN-gamma release against KLH. This suggests that the primary role of adjuvants in the context of these conjugate vaccines may be induction of higher levels of T-cell immunity against KLH, which then leads to higher levels antibody against the conjugated antigens.

Carbohydrate antigens as targets for active specific immunotherapy

Abstract Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewisy and globo-H (neutral glycolipids and glycoproteins), and Tn, TF and sTn (glycoproteins) are overexpressed in a variety of cancers. Antibodies against several of these carbohydrate antigens have been detected in sera from patients treated with cancer vaccines, and have been associated with a more favorable prognosis. Clinical responses have been reported after treatment with monoclonal antibodies against some of these antigens. Hence cell-surface carbohydrate antigens have been identified as suitable targets for immune attack by both active and passive immunotherapies. Different approaches have been adopted to induce immune responses against these carbohydrate antigens. These includes vaccination with whole or lysed tumor cells, purified or synthetic carbohydrates, immunogenic carbohydrate derivatives, or carbohydrates conjugated with immunogenic carriers and administered with immunological adjuvants. In the case of gangliosides, immunization with either whole tumor cells or cell lysates has only occasionally induced responses against carbohydrate antigens, and the antibodies were generally IgM antibodies of low titer. Compared with other methods of vaccination, conjugate vaccines have consistently induced the highest titer of IgM and IgG antibodies against gangliosides and other carbohydrate antigens. Preclinical and clinical studies with conjugate carbohydrate vaccines have induced IgM and IgG antibody responses capable of inducing complement-mediated cytotoxicity of tumor cells in vitro and associated with prolonged disease-free and overall survival in patients.

Carbohydrate vaccines that induce antibodies against cancer. 2. Previous experience and future plans

In conclusionThe primary function of antibodies is the elimination of circulating viral or bacterial pathogens from the blood-stream, lymphatics and interstitial spaces, and so, once induced, antibodies should be ideally suited for eliminating tumor cells and micrometastases from these spaces as well. Natural or tumor-induced and vaccine-induced antibodies against human cancer-associated antigens have been correlated with an improved clinical outcome. In the mouse, passive administration of monoclonal antibodies against cell-surface antigens 1–4 days after tumor challenge, and active induction of antibodies with vaccines, has resulted in prolonged survival or complete protection from tumor growth. This is a setting similar to the adjuvant setting in humans. Carbohydrates are the most abundant antigens at the cell surface of cancer cells, where they play important roles in cell-cell interactions, proliferation and the metastatic process. They have been shown to be excellent targets for immune attack by antibodies against human cancers, especially in the adjuvant setting. Vaccines containing these carbohydrate antigens covalently attached to immunogenic carrier proteins, such as KLH, plus potent immunological adjuvants, such as QS-21, effectively induce antibodies against these antigens in patients, which can result in complement-mediated lysis of antigen-positive tumor cells. Phase III trials with KLH conjugate vaccines have been initiated in the adjuvant setting against two carbohydrate antigens, the ganglioside GM2 and the blood-group-related antigen sTn. As the immunogenicity of additional vaccines is confirmed in small pilot trials, trials with polyvalent vaccines against two to five different antigens tailored for particular cancer types are planned.

Induction of antibodies against GD3 ganglioside in melanoma patients by vaccination with GD3-lactone-KLH conjugate plus immunological adjuvant QS-21.

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.

Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates

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.

On the power of chemical synthesis: Immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines

Synthetic carbohydrate cancer vaccines have been shown to stimulate antibody-based immune responses in both preclinical and clinical settings. The antibodies have been observed to react in vitro with the corresponding natural carbohydrate antigens expressed on the surface of tumor cells, and are able to mediate complement-dependent and/or antibody-dependent cell-mediated cytotoxicity. Furthermore, these vaccines have proven to be safe when administered to cancer patients. Until recently, only monovalent antigen constructs had been prepared and evaluated. Advances in total synthesis have now enabled the preparation of multivalent vaccine constructs, which contain several different tumor-associated carbohydrate antigens. Such constructs could, in principle, serve as superior mimics of cell surface antigens and, hence, as potent cancer vaccines. Here we report preclinical ELISA-based evaluation of a TF–Ley–Tn bearing construct (compound 3) with native mucin glycopeptide architecture and a Globo-H–Ley–Tn glycopeptide (compound 4) with a nonnative structure. Mice were immunized with one or the other of these constructs as free glycopeptides or as keyhole lymphet hemocyanin conjugates. Either QS-21 or the related GPI-0100 were coadministered as adjuvants. Both keyhole lymphet hemocyanin conjugates induced IgM and IgG antibodies against each carbohydrate antigen, however, the mucin-based TF–Ley–Tn construct was shown to be less antigenic than the unnatural Globo-H–Ley–Tn construct. The adjuvants, although related, proved significantly different, in that GPI-0100 consistently induced higher titers of antibodies than QS-21. The presence of multiple glycans in these constructs did not appear to suppress the response against any of the constituent antigens. Compound 4, the more antigenic of the two constructs, was also examined by fluorescence activated cell sorter analysis. Significantly, from these studies it was shown that antibodies stimulated in response to compound 4 reacted with tumor cells known to selectively express the individual antigens. The results demonstrate that single vaccine constructs bearing several different carbohydrate antigens have the potential to stimulate a multifaceted immune response.

A FULLY SYNTHETIC GLOBO H CARBOHYDRATE VACCINE INDUCES A FOCUSED HUMORAL RESPONSE IN PROSTATE CANCER PATIENTS : A PROOF OF PRINCIPLE

Human trials on the globo H carbohydrate vaccine (see picture, KLH=the carrier protein keyhole limpet hemocyanin) show that it produces strong IgM, and in some cases IgG, responses in patients with progressive and recurrent prostate cancer. Furthermore, these antibodies not only recognize synthetic antigens, but also globo H-positive tumors in biopsy extracts and tumor tissues.

Reevaluation of the cellular immune response in breast cancer patients vaccinated with MUC1.

Conjugation of antigens to a carrier protein like keyhole limpet hemocyanin (KLH) has proven effective in clinical trials for inducing antibodies against selected tumor antigens. The impact of this approach on T‐cell immunity has not been previously tested. We utilized peripheral blood mononuclear cells (PBMC) obtained at leukapheresis from 6 breast cancer patients vaccinated 4 times each with a 106‐amino acid‐long MUC1 peptide conjugated with KLH plus immune adjuvant QS‐21. Proliferation after 6 days of in vitro culture and an interferon gamma ELISPOT assay with and without 6 days of in vitro sensitization with the immunizing antigen were used. Parallel experiments employed the use of the cytokine IL2. Our results indicate that despite a high response to KLH in all patients with precursor frequencies as high as 1/120 peripheral blood lymphocytes and augmentation of proliferation in excess of 200‐fold after vaccination, the T‐cell response against MUC1 peptide was minimal and inconsistent. The strength and consistency of the vaccine‐induced T‐cell response against KLH in these patients excludes general immune incompetence and assay insensitivity or inconsistency as explanations for the weak and inconsistent response against MUC1. We conclude that for any report of augmented T‐cell responses against MUC1 to be convincing, one or more postimmunization blood samples will be needed to demonstrate augmented MUC1‐specific immunity consistently on multiple occasions. Assuming this criteria, convincing induction of T‐cell immunity against MUC1 by vaccination has yet to be described.