Carbohydrate antigens as targets for active specific immunotherapy

 Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewis^y 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. Received: 6 August 1996 / Accepted: 20 September 1996     

Role of tumor-associated gangliosides in cancer progression

Neuroectodermic tumors can mostly be characterized by the presence of tumor-associated glycosphingolipid antigens, such as gangliosides, defined by monoclonal antibodies. Recently, cumulative evidence indicates that gangliosides modify the biological effects of several trophic factors, in vitro and in vivo, as well as the mitogenic signaling cascade that these factors generate. The functional roles of gangliosides in tumor progression can be revisited: (i) ganglioside antigens on the cell surface, or shed from the cells, act as immunosuppressors, as typically observed for the suppression of cytotoxic T cells and dendritic cells, (ii) certain gangliosides, such as GD3 or GM2, promote tumor-associated angiogenesis, (iii) gangliosides strongly regulate cell adhesion/motility and thus initiate tumor metastasis, (iv) ganglioside antigens are directly connected with transducer molecules in microdomains to initiate adhesion coupled with signaling, and (v) ganglioside antigens and their catabolites are modulators of signal transduction through interaction with tyrosine kinases associated with growth factor receptors or other protein kinases. Given the potential importance of these sialylated gangliosides and their modulating biological behavior in vivo, further studies on the role of gangliosides are warranted.     

A Systems Immunology Approach to the Host-Tumor Interaction: Large-Scale Patterns of Natural Autoantibodies Distinguish Healthy and Tumor-Bearing Mice

Traditionally, immunology has considered a meaningful antibody response to be marked by large amounts of high-affinity antibodies reactive with the specific inciting antigen; the detection of small amounts of low-affinity antibodies binding to seemingly unrelated antigens has been considered to be beneath the threshold of immunological meaning. A systems-biology approach to immunology, however, suggests that large-scale patterns in the antibody repertoire might also reflect the functional state of the immune system. To investigate such global patterns of antibodies, we have used an antigen-microarray device combined with informatic analysis. Here we asked whether antibody-repertoire patterns might reflect the state of an implanted tumor. We studied the serum antibodies of inbred C57BL/6 mice before and after implantation of syngeneic 3LL tumor cells of either metastatic or non-metastatic clones. We analyzed patterns of IgG and IgM autoantibodies binding to over 300 self-antigens arrayed on slides using support vector machines and genetic algorithm techniques. We now report that antibody patterns, but not single antibodies, were informative: 1) mice, even before tumor implantation, manifest both individual and common patterns of low-titer natural autoantibodies; 2) the patterns of these autoantibodies respond to the growth of the tumor cells, and can distinguish between metastatic and non-metastatic tumor clones; and 3) curative tumor resection induces dynamic changes in these low-titer autoantibody patterns. The informative patterns included autoantibodies binding to self-molecules not known to be tumor-associated antigens (including insulin, DNA, myosin, fibrinogen) as well as to known tumor-associated antigens (including p53, cytokeratin, carbonic anhydrases, tyrosinase). Thus, low-titer autoantibodies that are not the direct products of tumor-specific immunization can still generate an immune biomarker of the body-tumor interaction. System-wide profiling of autoantibody repertoires can be informative.     

The reverse proteomics for identification of tumor antigens

The identification of tumor antigens is essential for the development of anticancer therapeutic vaccines and clinical diagnosis of cancer. SEREX (serological analysis of recombinant cDNA expression libraries) has been used to identify such tumor antigens by screening sera of patients with cDNA expression libraries. SEREX-defined antigens provide markers for the diagnosis of cancers. Potential diagnostic values of these SEREX-defined antigens have been evaluated. SEREX is also a powerful method for the development of anticancer therapeutics. The development of anticancer vaccines requires that tumor antigens can elicit antigen-specific antibodies or T lymphocytes. More than 2000 antigens have been discovered by SEREX. Peptides derived from some of these antigens have been evaluated in clinical trials. This review provides information on the application of SEREX for identification of tumor-associated antigens (TAA) for the development of cancer diagnostics and anticancer therapeutics.     

Dendritic cells for specific cancer immunotherapy

The characterization of tumor-associated antigens recognized by human T lymphocytes in a major histocompatibility complex (MHC)-restricted fashion has opened new possibilities for immunotherapeutic approaches to the treatment of human cancers. Dendritic cells (DC) are professional antigen presenting cells that are well suited to activate T cells toward various antigens, such as tumor-associated antigens, due to their potent costimulatory activity. The availability of large numbers of DC, generated either from hematopoietic progenitor cells or monocytes in vitro or isolated from peripheral blood, has profoundly changed pre-clinical research as well as the clinical evaluation of these cells. Accordingly, appropriately pulsed or transfected DC may be used for vaccination in the field of infectious diseases or tumor immunotherapy to induce antigen-specific T cell responses. These observations led to pilot clinical trials of DC vaccination for patients with cancer in order to investigate the feasibility, safety, as well as the immunologic and clinical effects of this approach. Initial clinical studies of human DC vaccines are generating encouraging preliminary results demonstrating induction of tumor-specific immune responses and tumor regression. Nevertheless, much work is still needed to address several variables that are critical for optimizing this approach and to determine the role of DC-based vaccines in tumor immunotherapy.     

Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: Anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues

Summary Methotrexate (MTX) was coupled to the tumor-targeting monoclonal IgM, anti-SSEA-1 and the nontargeting myeloma IgM, MOPC 104E. At 24-h intervals following injection, drug deposition in MH-15 teratocarcinomas and in several normal tissues was followed by immunoperoxidase microscopy using the M16 monoclonal antibody to MTX. MTX-anti-SSEA-1 was deposited on the surface and in the interior of living tumor cells 24 h after injection; at 48 h and after, only low-level binding to necrotic tissue was found. There was no significant gradation in staining from the outside to the interior of the tumors. In tumors, the control MOPC 104E immunoconjugate was detectable only in necrotic tissue. Binding to SSEA-1-expressing normal tissues was undetectable, except for pericryptal fibroblasts in the small intestine. No significant pathology was found in normal tissues that are SSEA-1 positive. High levels of the immunoconjugate were detected in the liver, where MTX was found predominantly in Kupffer cells and possibly in hepatocytes; again, no significant morphological changes were associated with this retention. Thus tumor-associated antigens can be suitable targets for antibody-drug conjugates even when present in normal tissues and in large quantities, provided that the antigens in normal tissues are inaccessible. Moreover, deposition in viable tumor tissue can be assessed using monoclonal antibodies to methotrexate.Supported by intramural research funds of the VAMC and by the Division of Urological Surgery, University of Pittsburgh     

Glycoengineering of alphaGal xenoantigen on recombinant peptide bearing the J28 pancreatic oncofetal glycotope

In human pancreatic adenocarcinoma, alterations of glycosylation processes leads to the expression of tumor-associated carbohydrate antigens, representing potential targets for cancer immunotherapy. Among these pancreatic tumor-associated carbohydrate antigens, the J28 glycotope located within the O-glycosylated mucin-like C-terminal domain of the fetoacinar pancreatic protein (FAPP) and expressed at the surface of human tumoral tissues, can be a good target for anticancer therapeutic vaccines. However, the oncodevelopmental self character of the J28 glycotope associated with the low immunogenicity of tumor-associated carbohydrate antigens may be a major obstacle to effective anti-tumor vaccine therapy. In this study, we have investigated a method to increase the immunogenicity of the recombinant pancreatic oncofetal J28 glycotope by glycoengineering Galalpha1,3Galss1,4GlcNAc-R (alphaGal epitope) which may be recognized by natural anti-alphaGal antibody present in humans. For this purpose, we have developed a stable Chinese hamster ovary cell clone expressing the alphaGal epitope by transfecting the cDNA encoding the alpha1,3galactosyltransferase. These cells have been previously equipped to produce the recombinant O-glycosylated C-terminal domain of FAPP carrying the J28 glycotope. As a consequence, the C-terminal domain of FAPP produced by these cells carries the alphaGal epitope on oligosaccharide structures associated with the J28 glycotope. Furthermore, we show that this recombinant "alpha1,3galactosyl and J28 glycotope" may not only be targeted by human natural anti-alphaGal antibodies but also by the mAbJ28, suggesting that the J28 glycotope remains accessible to the immune system as vaccinating agent. This approach may be used for many identified tumor-associated carbohydrate antigens which can be glycoengineered to carry a alphaGal epitope to increase their immunogenicity and to develop therapeutic vaccines.     

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

In conclusion  The 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.     

Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24[emsp4 ]h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.     

Animal models of human-derived cancer vaccines

Preclinical cancer vaccine studies must address vaccine safety, immunogenicity, and efficacy, as well as mechanism of vaccine action. Animal models of vaccines employing human tumor-associated antigen or epitopes (TAA, TAE) differ fundamentally from those employing tumor-specific antigens or epitopes (TSA, TSE). TSA and TSE vaccines will most likely demonstrate similar toxicity, immunogenicity, and efficacy in both tumor-bearing animals and patients. In contrast, TAA/TAE immunizations may have to overcome a host’s immunological tolerance to TAA/TAE expressed not only on tumor, but also on normal tissues; immunity to TAA/TAE will potentially target normal tissues and thus may induce autoimmunity. Various experimental models for human-derived TAA/TAE vaccines have been developed. These models include transgenic mice, mice with severe combined immunodeficiency (SCID), and non-human primates. Recently, unique animal models of TAA/TAE cancer vaccines have been developed, taking advantage of the discovery of animal tissue antigens with significant sequence homologies to human TAA/TAE. These models mimic perhaps most closely the situation in cancer patients.     

Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for antitumor vaccination

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.     

Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment

A method for the detection of GM1b-type gangliosides in complex mixtures of gangliosides was developed. The procedure involves separation of gangliosides on high-performance thin-layer chromatography plates, fixation of the silica gel, treatment with neuraminidase from Vibrio cholerae in the absence of detergent, and incubation of the plates with GgOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies are used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolylphosphate. The procedure is capable of detecting as little as 30 ng of gangliosides. Gangliosides from murine T lymphocytes and from human brain served as examples. Besides GM1b, GD1 alpha is also detectable by this method, whereas the human brain gangliosides GM1a, GD1a, GD1b, and GT1b are not, because they are neuraminidase resistant. Since terminally sialylated gangliosides such as GM1b were described as virus receptors, and certain other terminally sialylated gangliosides are discussed as tumor markers, this method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small amounts of material are available.     

Immune based therapies in cancer

Immunotherapy of cancer has become a more promising approach in the past decade. Developments in both basic immunology and tumor biology have increased our knowledge of the interactions between the tumor cells and the immune system. The molecular identification of tumor-associated antigens and understanding of immunological pathways have cleared the way for development of different strategies for anti-tumor vaccines. The success of any cancer vaccine relies on the induction of an effective tumor-specific immune response to break tolerance and to elicit a long lasting anti-tumor immunity. It is also increasingly clear that the interactions of host-tumor are quite complicated leading to tumor escape mechanisms, which add another level of difficulty to this interaction. This review will summarize the recent developments in tumor immunotherapy as well as the clinical trials addressing novel immunotherapeutic approaches to cancer.     

Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas

There have been a number of studies on tumor-specific glycolipid antigens. In particular, neuroectoderm-derived cancers express characteristic ganglioside antigens, some of them have been used as tumor markers and/or target of immunotherapy. Molecules in the signaling pathway activated by gangliosides have been analyzed. Here, we reported results on the functions of molecules involved in the signaling pathway to enhance malignant properties of human melanomas under GD3 expression, and emphasized that those molecules including tumor-associated antigens can be targets of therapeutics for malignant melanomas.     

Identification of a tumor-associated antigen of the guinea pig L2C leukemia by using syngeneic antisera.

Inbred strain 2 guinea pigs immunized with L2C leukemia cells produced antibodies to L2C cells detected by 125I-protein A assay. L2C-associated tumor antigens were reacted with syngeneic antisera and analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. These sera recognized idiotypic determinants on surface IgM molecules of L2C cells but did not recognize any determinants on normal strain 2 spleen cells. Thus, determinants on IgM molecular act as tumor-associated antigens in the L2C system and can be detected by syngeneic sera.     

The potential of DNA vaccination against tumor-associated antigens for antitumor therapy

Conventional treatment approaches for malignant tumors are highly invasive and sometimes have only a palliative effect. Therefore, there is an increasing demand to develop novel, more efficient treatment options. Increased efforts have been made to apply immunomodulatory strategies in antitumor treatment. In recent years, immunizations with naked plasmid DNA encoding tumor-associated antigens have revealed a number of advantages. By DNA vaccination, antigen-specific cellular as well as humoral immune responses can be generated. The induction of specific immune responses directed against antigens expressed in tumor cells and displayed e.g., by MHC class I complexes can inhibit tumor growth and lead to tumor rejection. The improvement of vaccine efficacy has become a critical goal in the development of DNA vaccination as antitumor therapy. The use of different DNA delivery techniques and coadministration of adjuvants including cytokine genes may influence the pattern of specific immune responses induced. This brief review describes recent developments to optimize DNA vaccination against tumor-associated antigens. The prerequisite for a successful antitumor vaccination is breaking tolerance to tumor-associated antigens, which represent "self-antigens." Currently, immunization with xenogeneic DNA to induce immune responses against self-molecules is under intensive investigation. Tumor cells can develop immune escape mechanisms by generation of antigen loss variants, therefore, it may be necessary that DNA vaccines contain more than one tumor antigen. Polyimmunization with a mixture of tumor-associated antigen genes may have a synergistic effect in tumor treatment. The identification of tumor antigens that may serve as targets for DNA immunization has proceeded rapidly. Preclinical studies in animal models are promising that DNA immunization is a potent strategy for mediating antitumor effects in vivo. Thus, DNA vaccines may offer a novel treatment for tumor patients. DNA vaccines may also be useful in the prevention of tumors with genetic predisposition. By DNA vaccination preventing infections, the development of viral-induced tumors may be avoided.     

Malignant Glial Neoplasms: Definition of a Humoral Host Response to Tumor-Associated Antigen(s)

There is increasing evidence that human tumors possess tumor-associated neo-antigens. The host mounts an immunological response to these antigens, as evidenced by the detection of circulating humoral antibodies in a variety of human neoplasia.An indirect immunofluorescent antibody technique was employed to detect antibodies to tumor-associated antigens in the sera of patients with malignant gliomas. Viable single cell suspensions were used to demonstrate antibodies to surface contents of tumor cells and cell preparations were snap-frozen at −160° C to demonstrate antibodies to cytoplasmic components of tumor cells. After incubation with serum, the preparations were treated with polyvalent sheep antihuman globulin conjugated to isomer-1-fluorescein isothiocyanate, washed, and examined with a Leitz incident fluorescent microscope.Of the 17 sera from histologically proven malignant glial neoplasm patients, 2 (11%) were positive for an autologous surface antibody reaction. Five (23%) of 21 were positive for an autologus cytoplasmic antibody, however, 10 (47%) of 21 of the sera gave a positive reaction for cross-reacting cytoplasmic antibodies when tested with a battery of tumor cells obtained from different patients with malignant glial tumors.No reaction was observed with normal brain tissue. Absorption studies indicated the presence of a tumor-associated antigen.This study demonstrated that certain patients with malignant gliomas possess circulating antibodies to cytoplasmic components of their own tumor cells. The fact that a number of sera cross-reacted with tumor cells obtained from different patients suggests that antigenic cross-reactivity exists between malignant glioma cells from different patients. It is suggested that with further refinement, immunofluorescent detection of antibodies could evolve as a useful diagnostic adjunct in malignant glioma.     

Generation of antigen-specific CTL responses using <Emphasis Type="Italic">RGS1</Emphasis> mRNA transfected dendritic cells

Advances in tumor immunology and Identification of tumor-associated antigens (TAAs) provide a basis for the development of novel immunotherapies to treat malignant diseases. In order to identify novel TAAs, we performed comparative microarray analysis of (heterogeneous) tissues and found regulator of G protein-signaling 1 (RGS1) extensively up-regulated in renal cell carcinoma (RCC) tissues. To examine the possible function of this molecule as a novel, broadly applicable TAA, synthetic full-length RGS1-mRNA was synthesized for the transfection of monocyte-derived dendritic cells (DCs). These modified antigen-presenting cells (APCs) were then used to induce RGS1-specific cytotoxic T cells (CTLs) in vitro. The CTLs generated from several healthy donors and a patient with chronic lymphocytic leukemia (CLL) elicited an antigen-specific and HLA-A2- and -A3-restricted cytolytic activity against tumor cells endogenously expressing the RGS1 protein including renal cell carcinomas (RCCs), melanoma, ovarian carcinoma and the primary autologous CLL-blasts. In conclusion, our study demonstrates that the in vitro induction of RGS1-specific CTLs by RNA-transfected DCs is feasible and highly effective. Since this molecule is (over-) expressed in a broad variety of malignancies it might represent an interesting novel TAA in the context of cancer vaccines designed to target RGS1 expressing tumor cells.     

Antibodies to glycolipids in demyelinating diseases of the human peripheral nervous system

Antibodies to complex glycolipids occur in patients with a variety of diseases of the peripheral nervous system. Many patients with demyelinating neuropathy occurring in association with IgM paraproteinemia have a monoclonal antibody that reacts with a carbohydrate determinant shared between sulfate-3-glucuronyl paragloboside (SGPG), the myelin-associated glycoprotein and other glycoproteins of peripheral nerve. Other patients with neuropathy in association with IgM paraproteinemia have monoclonal antibodies reacting with carbohydrate determinants on various gangliosides. More than 80% of the IgM monoclonal antibodies from patients of this type that have been screened in our laboratory react with SGPG or ganglioside antigens. High levels of antibodies reacting with ganglioside antigens are also found in some patients with inflammatory neuropathies such as Guillain-Barré Syndrome and chronic relapsing inflammatory polyneuropathy. The pathogenetic significance of these antibodies reacting with acidic sphingoglycolipids remains to be established.