Synthesis of selected LeY and KH-1 analogues: a medicinal chemistry approach to vaccine optimization.

[structure: see text] As part of our ongoing anticancer vaccine program, we recently found that antibodies generated in response to the KH-1-KLH construct recognized not only KH-1 antigen but also the Lewis Y (Le(y)) antigen as well, with antibody titer levels much higher than those observed after immunization with individual Le(y)-KLH vaccine constructs. In an attempt to explore the structure-antigenic relationship of these carbohydrate epitopes, several analogues of both KH-1 and Le(y) were synthesized. A convergent synthetic approach to the analogues was designed on the basis of well-established glycal methodology, employing a minimum number of building blocks to generate competent antigens with high stereoselectivity and reasonable yield.

Fully synthetic carbohydrate-based vaccines in biochemically relapsed prostate cancer: clinical trial results with alpha-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.

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-Le(y)-Tn bearing construct (compound 3) with native mucin glycopeptide architecture and a Globo-H-Le(y)-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-Le(y)-Tn construct was shown to be less antigenic than the unnatural Globo-H-Le(y)-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.

Constructing an adenocarcinoma vaccine: immunization of mice with synthetic KH-1 nonasaccharide stimulates anti-KH-1 and anti-Le(y) antibodies.

There is mounting evidence to suggest that immunization-based strategies can be used to mobilize the human immune system against specific carbohydrate antigens displayed on the surface of cancer cells. Following isolation and identification, such antigens can be administered as conjugate vaccines. The tumor-associated carbohydrate antigen KH-1 is 1 such antigen and may serve as a potential target for immunization against adenocarcinoma. However, a serious impediment to the application of a vaccine-based approach involving this antigen is that its availability from natural sources is severely limited. In order to overcome this limitation, we have developed an efficient total synthesis of this complex glycolipid. We have extended our synthesis to reach a structurally related analog in which the ceramide portion of KH-1 is replaced with an allyl substituent. These synthetic advances have led to the preparation of 2 potential vaccine constructs, each based on the conjugation of the KH-1 nonasaccharide and the carrier protein keyhole limpet hemocyanin (KLH). In 1 construct (KH-1-Et-KLH), the nonasaccharide is conjugated to KLH via a simple ethyl linkage, while in the other (KH-1-MMCCH-KLH), conjugation is mediated by a 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) cross-linker. We report here the immunological properties of these 2 constructs. Mice were immunized with either of the 2 KH-1-KLH vaccine candidates or the KH-1 ceramide, along with the immunological adjuvant QS-21. Immunization with the ceramide served as a negative control and, as expected, failed to stimulate the production of antibodies against the KH-1 glycolipid. The construct in which the KH-1 nonasaccharide is linked to KLH via a simple alkyl chain stimulated significant quantities of IgM antibodies, whereas the construct linked to KLH by MMCCH induced high titers of both IgM and IgG antibodies. Inhibition data demonstrated that antibodies generated in response to immunization with the KH-1-KLH constructs recognize not only the KH-1 antigen but also the Lewis(y) (Le(y)) antigen, which, from a structural perspective, is similar to the 4 residues located at the non-reducing end of the KH-1 nonasaccharide. Thus, the KH-1-KLH constructs elicit an immune response that successfully targets 2 adenocarcinoma markers. As assessed by FACS analysis, the antibodies raised were strongly reactive with the KH-1/Le(y) positive cell line MCF-7 but not with KH-1 and Le(y) negative melanoma cell lines. Based on the results of our study, a KH-1-KLH plus QS-21 vaccine is being prepared for clinical evaluation.

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.

Comparison of antibody titers after immunization with monovalent or tetravalent KLH conjugate vaccines.

Antigens such as ganglioside GD3, neutral glycolipid Lewis(y) (Le(y)) and mucins MUC1 and MUC2 are over-expressed on the cell surface of many tumors. We have shown previously that conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) and the use of immunological adjuvant QS-21 is the optimal approach for inducing high titer IgM and IgG antibodies. These antibodies are able to bind with natural antigens on the tumor cell surface and mediate complement dependent cytotoxicity and/or antibody dependent cell mediated cytotoxicity. Immunization of patients with monovalent vaccines containing these and a variety of other antigens have demonstrated both the consistent immunogenicity and the safety of these vaccines. Now, in preparation for the use of polyvalent conjugate vaccines in the clinic, we have addressed for the first time with conjugate vaccines against cancer antigens several questions in the pre-clinical setting, including whether immunogenicity of the individual components is decreased in the polyvalent vaccine and issues relating to vaccine formulation and administration. We have immunized groups of mice with GD3-KLH, Le(y)-KLH, MUC1-KLH and MUC2-KLH conjugates and QS-21 separately or mixed and administered at one or four sites. High titer IgM and IgG antibodies were induced against each of the four antigens whether administered singly in separate mice, at separate sites in the same mice, or mixed and administered at a single site or at four sites, or administered subcutaneously (s.c.) or intraperitoneally (i.p.). These antibodies reacted specifically with the respective antigens and tumor cells expressing these antigens. There was no evidence of suppression of the antibody response against any one of the antigens by the presence of the other conjugates in the vaccine. Immunogenicity of the four individual antigens conjugated to KLH and QS-21 is not affected by mixing the four together and administering them at a single subcutaneous site.

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.