Tumor-associated antigens and development of immunotherapeutics strategies

The tumor-associated antigens, up-to-date methods for their identification and immunotherapeutics strategies for cancer treatment have been reviewed. The main attention has been focused on the antigens identified by SEREX (Serological identification of antigens by recombinant expression cloning) metho­ dology and the categories of tumor-associated antigens (i. e. identified by SEREX approach) have been listed. The main immunotherapeutic approaches have been classified, the successes of active specific immunotherapy based on immunodominant peptides of tumor-associated antigens have been described.

Tumor-associated antigens and methods for their identification.The identification and molecular char acterization of tumor antigens in the tumor-bearing host are the major tasks in the tumor immunology.An analysis of humoral and cellular immune responses against such antigens in cancer patients indicates for a long time that tumor-associated antigens (TAA) do exist and are recognized by patient's immune system.These antigens are the potential targets for the antigen specific cancer immunotherapy and cancer diagnostics.
The first useful antigens -alfa fetoprotein, a serum marker for hepatoma and germ cell tumors, and carcinoembryonic antigen (CEA), a serum mar ker for colon and other epithelial cancers were identified by the analysis of heteroimmune sera obtained from rabbits and other animals immunized with human cancer cells [1,2].In the 1970s and 1980s, the monoclonal antibody technology was exp loited for the identification of molecules on tumor cells that could be used as diagnostic markers or as target structures for the immunotherapeutic approa-ches with monoclonal antibodies.Some of these efforts yielded new therapeutic tools, such as the anti-CD20 antibody «rituximab» which showed consider able activity and has been licensed for the treatment of B-cell lymphomas [3 ].However, the approaches of active immunotherapy require the identification of target structures, immunogenic in the autologous tumor-bearing host.There are two different opinions regarding the methods for searching antigens for active immunotherapy.
First, numerous studies on experimental animals showed that the cellular, rather than the humoral, immune response was in charge of the rejection of transplanted tumors or allogenic (genetically dif ferent) tissues.With the exception of antibodies against the growth factor receptors on cancer cells, the administration of antibodies has little effect on the growth of solid tumors.Thus, a significant effort should be directed at identification of the antigens recognized by human T-lymphocytes [4,5].On the other hand, it is now commonly accepted that immune recognition of tumors is a concerted action.Hence, high-titer circulating tumor-associated antibodies of the immunoglobulin G (IgG) class may reflect a significant host-tumor interaction and identify such gene products, against which at least cognate Thelper cells and specific cytotoxic T-cells should exist [6].The present review describes the categories of TAA and methods for their identification are as well as some immunological strategies for cancer immu notherapy, based on molecular identification of TAA.The main attention is focused on the serological approach to search for TAA (SEREX), but T-celldefined tumor antigens and techniques for their identification are also considered.
Since T-cells play an essential role in tumor growth inhibition and cancer cells eradication, first of all some useful techniques have been developed to identify the cancer antigens capable of eliciting cel lular immune response.The genetic approach repre sents the transfection of genomic DNA or cDNA libraries into the cells expressing the appropriate major histocompatibility complex (MHO molecules, followed by the identification of the transfectants using cytokine release or lysis by human T-cells (CTL -cytotoxic T-lymphocytes) with specific anti tumor reactivity [7 ].The biochemical technique offers the elution of peptides from tumor cells or from MHC removed from tumor cells surface and detection of fractions capable of stimulating antitumor T-cells after pulsing purified peptides onto antigen-presenting cells.The triple quadrapole mass spectrometric tech niques have been used to sequence the minute quan tities of peptides obtained [8 ].
Another method is an identification of tumor antigens by in vitro sensitization of T-cells against possible tumor antigens [9].T-cells culture suc cessfully sensitized in vitro against these antigens have been tested for their ability to recognize the intact tumor cells, and that provides strong evidence that these candidate proteins represent the tumor antigens [9].
The mechanism of eradication of cancer cells by T-lymphocytes involves both CD8 cytotoxic T-cells and CD4 T-helper cells that recognize antigens pre sented as small peptides by the MHC classes I and II.CD8 T-cells recognize peptides derived from intracellular cytoplasmic proteins, digested in proteasomes and presented via the endoplasmatic re ticulum on the cell surface by class I MHC.In contrast, CD4 cells use a different intracellular path way for engulfed extracellular proteins, digested to peptides in intracellular endosomes and presented on cell surface by class II MHC molecules.Since CD8 CTL can lyse tumor cells directly and destroy large tumor masses in vivo, several tumor antigens, pre sented by MHC I, have been defined using the tumorreactive CD8 T-cells.Clinical trials using the tumor antigens have evidenced some therapeutic ef fects inhibiting tumor growth.However, the overall immune response was too weak and transient to eradicate cancer cells in the majority of patients who were immunized.Though a growing body of evidences suggests that CD4 T-cells play a central role in initiating and maintaining immune responses against cancer [10], the optimal vaccination might require the participation of both CD4 and CD8 T-cells to ge nerate a strong and long-lasting antitumor immunity.Although MHC II restricted tumor antigens are clear ly important, some problems exist for their iden tification due to special requirements for MHC II antigen processing and presentation.
The ability of antigen-presenting cells (APC) to endocytose proteins and to present peptides by MHC II has been utilized to identify antigens recognized by CD4 T-cells, although so far few antigens have been identified using this approach [11].A genetic tar geting expression (GTE) approach has been de veloped that allows the screening of cDNA library fused to genes encoding invariant chain (Ii) sequences for identification of MHC II restricted antigens, designed to guide the transfected proteins into the class II presentation pathway [12].
Thus, during the last few years, effective stra tegies of identification of TAA recognized by specific T-cells have resulted in the characterization of various antigens, such as the melanoma differentiation an tigens, CEA, epithelial cell adhesion molecule, HER-2\new, Wilms tumor protein, proteinase 3, NY-ESO-1, antigens of MAGE, BAGE, GAGE, SSX-families, etc. as described by Renkvist et al. [13].The authors have summarized more than 60 MHC class I related TAA, recently identified [13].A completed list of T-cell epitopes can be found in the peptide database of T-cell defined tumor antigens (http://www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm).
SEREX approach and categories of tumor-as sociated antigens.Unfortunately, all the techniques listened above are technically challenging because they require the establishment of autologous CTL lines and tumor cell lines from the same patient, a task not achievable for most epithelial tumor types [14].An alternative technique that circumvents these limitation has been developed by Pfeundschuh and his colleagues [15].They call their approach SEREX for «serological identification of antigens by recom binant expression cloning*.SEREX has been de signed to isolate tumor antigens that elicit high-titer IgG response in human hosts.In their initial ap plication of this method, Pfeundschuh and his col leagues identified MAGE-A1 and tyrosinase, two antigens originally cloned as CTL targets, indicating that SEREX can detect tumor antigens that elicit the CTL-mediated immune response [15].
SEREX allows a systematic and unbiased search for antibody responses and direct molecular definition of immunogenic tumour proteins, based on their reactivity with autologous patient serum.In the SE REX approach, a cDNA expression library is const ructed from a fresh tumour specimen, and cloned into A phage expression vectors.The resulting recombinant phages are then used to infect Escherichia coli.Recombinant proteins expressed during the lytic in fection of the bacteria are transferred onto nitro cellulose membranes, which are incubated with di luted (1:100-1:1000) and depleted from anti E. coli and anti A phage antibodies serum from the auto logous patient.Clones reactive with high-titre anti bodies are identified using an enzyme-conjugated secondary antibody specific for human IgG.Positive clones are subcloned to monoclonality, and the nuc leotide sequence of the cDNA insert is determined.cDNA sequence analysis via database searching is performed to define whether the cDNA inserts are derived from novel genes or cancer-associated known genes.The domains and/or motifs for putative func tion and cellular localisation of the antigens can be also identified.The SEREX approach characterized by several features is reviewed by Sahin et al. [16].The main of them is using fresh tumor specimens that circumvent in vitro the artifacts associated with the short-and long-term tumor cell culture.This elimi nates the need of establishing tumor and CTL cell lines.
However, the data accumulated over recent years evidence that antigen repertoire detectable by the conventional SEREX approach to be limited [6 ].This might be in part attributed to the fact that the potential antigens modificated posttranslationally re main undetected in bacterial expression systems.The posttranslational modifications play an important role in the proper function of many proteins and also affect their immunogenicity.To overcome some of the inherent problems with the conventional SEREX sys tem, recombinant antigen expression on yeast sur face* (RAYS) has been proposed recently to establish a eukaryotic expression system in yeast.Yeasts have the advantage that recombinant proteins can be expressed on the cell surface in a more naturally folded, partially glycosylated manner [17].A number of other modifications of the original method have been proposed recently.Subtractive approaches allow enrichment of the cDNA library for tumor specific transcripts [18 ]. cDNA Libraries may also be prepared from sources of specific interest, such as amplified chromosomal regions obtained by microdissection [19].Another alternative cloning strategy for TAA with a possibility of high throughput analysis of patient sera and tumor libraries has been projected newly.The pJuFo phage surface display, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, has been explored for cloning TAA in prostate cancer [20 ].
In 1995 the SEREX program was initiated under supervision of Dr L. Old, the head of Ludwig Institute for Cancer Research (LICR).The SEREX colla borative group involve the investigators from the University of Saarland (Hamburg, Germany), Ludwig Institute Branches in New York, San Diego (USA), Melbourne (Australia), and London (UK, University College); Aichi Cancer Center (Naoya, Japan), Krankenhaus Nordwest (Frankfurt, Germany), Mie Uni versity School of Medicine (Mie, Japan), Moscow State University (Moscow, Russia), and the Institute of Molecular Biology and Genetics (Kyiv, Ukraine).
During the last few years, SEREX has been applied to a wide range of tumor types, including melanoma, renal cancer, astrocytoma, Hodgkin's di sease [15], oesophageal cancer [21], gastric cancer [22], colon cancer [23], lung cancer [24], breast cancer [25], prostate cancer [26], leukaemia [27] and thyroid cancer [28].For the systematic docu mentation and archiving of sequence data and im munological characteristics of identified antigens, the electronic SEREX database, established by the LICR, is accessible to the public http://www.licr.org/SE-REX.html.To date more than 2,000 antigens have been listed in the SEREX database [6 ].According to their expression patterns in normal and malignant tissues, several categories of tumor antigens can be distinguished.The table 1 lists the categories of the tumor antigens identified.tricted to male germ cells in the testis, in some cases in ovary and in throphoblast, but not in adult somatic tissues.In malignancy, the gene regulation is dis rupted, resulting in CT antigen expression in tumors of various type.Many of the genes coding for these antigens are mapped to chromosome X and exist as multimember families.The representatives of this category, MAGE [29], BAGE [30], and GAGE [31], were initially identified as targets for the cytotoxic T-cells.The HOM-Mel-40 antigen (gene SSX-2), detected in the melanoma library, is the first CT antigen identified by SEREX [32].The SEREX analysis of esophageal cancer resulted in the iden tification of the NY-ESO-1 gene with restricted exp ression in testis and various cancers [21 ].Differentiationantigens are expressed in tumor in a lineage-associated manner, but found also in normal cells of the same origin.Melan A/MART-1 [34,35], tyrosinase [36], gplOO [37,38], gp75/TRP-l [39] antigens have been found in malignant melanomas, a particularly rich source of differentiated antigens, the expression of which is limited to melanomas as well as melanocytes, the cells of this tumor origin.The next examples are glial fibrillary acidic protein (GFAP), which is antigenic in malignant glioma, but also expressed in brain cells [40], and recently found NY-BR-1 expressed in 70-80 % of breast cancer, 25 % of prostate cancer as well as in normal breast, prostate and testis tissues [41 ].
Splice variants antigens have been detected by serological approach, for example, the Hodgkin's disease-associated variant of restin [15] and gastric cancer associated splice variant of TACC1 antigen [45].
Virus encoded antigens are expressed in different malignancies in humans.Many of viruses associated with oncogenesis can present proteins on the induced cancer antigens that can serve as the targets for immune attack.These viral antigens can be also used as targets for preventive or therapeutic vaccination.However, the induction of neutralizing antibodies against envelope viral proteins can prevent the an tigen-specific immune response in cancer patients immunized with the recombinant viruses vaccines [46].
Cancer-related autoantigens art expressed ubiq uitously and at similar levels in both healthy and malignant tissues.Cancer-related autoantigens, in contrast to non-cancer-related antigens, that elicit antibody response only in cancer patients, and not in healthy individuals that elicit antibody response with similar frequencies in both healthy individuals and cancer patients.This might result from tumor-as sociated post-translational modifications or changes in antigen processing and/or presentation in tumor cells.An example is HOM-MEL-2.4that represents the CCAAT enhancer binding protein [7 ].
Thus, systematic application of antibody-based (SEREX) and CTL-based methods has led to the definition of growing number of TAA.The multitude of tumor-specific antigens identified by the SEREX approach demonstrates that there is ample immune recognition of human tumors by the autologous host's immune system.Together with the identification of T-lymphocyte epitopes, a picture of immunological profile of cancer is emerging.Knowledge of cancer immunome (i.e. the sum of all the proteins expressed by tumor-bearing autologous host) provides a new basis for understanding tumor biology and for deve lopment of new diagnostic and therapeutic strategies for cancer.Besides their roie as targets for cancer vaccination, the identified antigens may be useful as new molecular markers of malignant diseases.The value of each of these markers or a combination of them for diagnostic or prognostic evaluation of cancer patients has to be determined by correlation between markers expression and clinical data.Furthermore, the immune system is a sensitive biodetector, which may determine structural and regulative alterations and may therefore point to gene products of sig nificance for neoplastic transformation or tumour progression [47].
Development of immunotherapeutic strategies.The first clear indication that immunological mani pulation could cause the regression of established invasive human cancers came from the studies on administration of interleukin-2 (IL-2) to patients with metastasis kidney cancer or melanoma [48].The discovery of tumor antigens and the identification of their immunodominant epitopes have shifted emphasis to the utilization of these antigens to mediate the destruction of growing cancers in humans.Attempts are in progress to develop immunotherapies based on the specific stimulation of immune reactions against defined tumor antigens rather than on the non specific stimulation of the immune system that is characteristic of the prior approaches.

Specific as well as non-specific immunotherapies
fall into either adoptive or active immunotherapy (tabl.2).Early studies on non-specific adoptive immunotherapy of tumor-bearing patients involved the transfer of limphokine-activated killer (LAK) cells that recognize and lyse cancer cells in vitro [49 ].For these purposes TIL (tumor infiltrated lymphocytes from respective tumors) could be expanded to 10 10 -10" cells and then adoptively transferred along with IL-2 into melanoma patients [50 ].Non-specific active immunotherapy used either autologous or allogenic whole cancer cells or cancer cells extracts for immu nization of cancer patients.As reviewed by Rosenberg [46 ], the vaccines based on cancer cells were derived from: whole cancer cells (both autologous and allo genic preparation), gene-modified cancer cells (genes encoding cytokines or co-stimulatory molecules) or cancer cell extracts (lysates, membranes and heatshock proteins) and cancer cells fused to APC.Measurable specific immune responses were rarely detected following this approach, and the efficacy of immunization was reflected only by clinical deve lopment [51 ].
The identification of human cancer antigens has opened a new era of specific cancer immunotherapy.To date specific cancer immunotherapy (tabl.2) based either on the adoptive transfer of the in vitro generated lymphocytes with high level of specific reactivity against tumor antigens (passive or adoptive immunotherapy) or on immunization against specific cancer antigens (active immunotherapy), represents attractive approaches.Cancer antigens has facilitated the in vitro generation of anti-tumor T-cells which can be expanded and used for specific adoptive immu notherapy [52].The genetic modification of these lymphocytes to improve their anti-tumor efficacy (for instance, by inserting genes, encoding anti-tumor or chemokine receptors, or genes, encoding anti-tumor cytokines) is under active investigation [53 ].In vitro sensitization techniques, using repeated in vitro sti mulation against immunodominant peptides or uti lizing tumor antigens pulsed onto professional APC, have resulted in the in vitro generation of T-cells with extremely high antitumor activity.Clinical protocols utilizing the specific adoptive transfer of these in vitro sensitized cells, are in progress as well [24 ]. Specific active immunotherapy suggests using purified cancer antigens (natural or recombinant), synthetic peptides alone or pulsed onto APC, «naked» DNA (for exam ple, plasmids), recombinant viruses (adenovirus, vac cinia or avipox) and recombinant bacteria (Bacille Calmete-Guerin and Listeria) as cancer vaccine, and represents the most perspective and promising ap proach.The advantage of antigen-specific vaccination is a possibility of monitoring specific vaccine-induced immune responses that is for the identification of mechanisms mediating tumor regression in vivo [46 ].The efficacy of cancer vaccines is affected by the immunogenicity of antigens, the route of immu nizations and tumor features making them susceptible to immunologic recognition [51 ].The main goal of cancer vaccination is the induction of an effective specific response against tumor cells which spares the cells of normal tissues.With respect to specificity, several classes of antigens may be suitable targets; in addition to the CT antigens, these include diffe rentiation antigens, tumor associated overexpressed gene products, mutated gene product and tumorspecific splice variants [6].
A number of vaccines based on tumor associated antigens, i. e. SEREX-defined antigens, have been developed for the needs of specific active immu notherapy.Several steps of the analysis are required before SEREX-defined antigens become potential tar gets for the active immunotherapy trials in cancer patients as reviewed by Jager et al. [54 ].
As the first step, a careful expression analysis of each antigen has to be performed by comparing the cDNA sequence with EST databases.The antigens with restricted expression according to the database analysis are being tested by RT-PCR using a panel of normal and tumor tissues cDNAs to confirm the restricted mRNA expression pattern.
The antigens that show a tumor/tissue-restricted expression pattern, undergo a serological analysis to define the frequency of humoral immune responses in sera derived from cancer patients and normal indi viduals.A limited number of sera can be tested using the phage-plaque assay; for large-scale serology, the recombinant protein has to be produced and tested in Western blot assays or ELISA.
The antigens with tumor/tissue-specific expres sion and detectable humoral immune responses in tumor patients have to be analyzed for T-cell recog nition.An approach called the reverse immunology approach allows testing the entire protein sequence of the respective antigen for potential MHC I binding sequences (see CancerlmmunomeDB, SYFPEITHI database of MHC ligands and peptide motifs at http: / /www.bmi-heidelberg.com/syfpeithi/).Peptidespecific CTL can be generated upon in vitro sti mulation, and recognition of target cells expressing the antigenic protein will be documented.The tumor antigens that represent the target antigens for CD8 T-cells can be used to immunize the patients with antigen-positive tumors.
The following list summarizes the steps of ana lyzing SEREX-defined antigens as potential targets for CD8 T-cells: SEREX analysis -Database ana lysis -Expression analysis -Serology-MHC-motif search for epitopes -ELISPOT assays (T-cell reac tivity) -Immunotherapy trial [54 ].
Among a number of cancer vaccine trials per formed with tumor antigens, «naked» DNA, APC, tumor cells, cytokines, co-stimulatory molecules and so on, the immunization strategy with immuno dominant peptides derived from tumor antigens is the most attractive.Promising immunologic and clinical results were observed after vaccination with the peptides derived from the CT antigens MAGE-3 [55 ] and NY-ESO-1 [56].The detectable peptide-specific CD8 T-cell responses were associated with the reg ression of metastatic lesions in single patients [55].The objective tumor regression was observed in single patients under continued immunization with the pep tide derived from melanocyte differentiation antigens MelanA/MART-1 [57] and tyrosinase [58].The overexpressed antigens, such as HER-2\new, wildtype 53 and viral antigens, for example, the proteins of Human pappiloma viruses may also represent useful targets for therapeutic and/or preventive im munologic intervention [51 ].The immunization stra tegies with MHC II restricted peptides are incre asingly studied in vaccine trials [59,60].The anti genic peptides derived from different cancer antigens (used either alone or combined with cytokines i. e., IL-2, IL-12, GM-CSF or adjuvants i. e., incomplete Freund's adjuvant, QS21) lead to the induction of strong specific CD T-cell responses [51 ].The immunogenicity of peptide epitopes may be signficanty increased in vitro by modifying specific amino acid anchoring residues (e. g., exchange of cysteine with valine or alanine) [61 ].Enhanced immunologic ef fects were observed in clinical studies using dendrite cells loaded with the peptide derived from cancer antigens, i. e. MAGE-3, MelanA\MART-l, that were related with disease stabilization and regression of metastasis melanoma lesions in single patients [62].The local immune reactions, i. e., DTH reactions, inflammatory enlargement of lymph nodes, were ob served after the antigen administration depending on the route of immunization: intradermal, intralymphatic or subcutaneous.The overall goal of clinical vaccine trials is the induction of CD8 and/or CD4 T-cell responses against the defined cancer antigens.Currently, the ELISPOT assay and structure-based peptide-MHC-tetramer assay measuring the mag nitude of CD8 T-cell responses in the peripheral blood, in tumor-infiltrating lymphocytes, and in drai ning lymph nodes were established to be the most sensitive methods to determine the function and the quantity of the vaccine induced effector cells.In the function-based ELISPOT assay, antigen-specific cells are detected on the basis of proximal cytokine pro duction [63].The structure based peptide-MHCtetramer assay is the method of identifying antigenspecific T-cells on the basis of T-cell receptor recog nition by its ligand, the peptide-MHC complex [64].Novel immunological tools that can be used not only to quantify the antigen-specific response, but also to analyze the phenotype and function of individual effector cells are described in the review of Yee and Greenberg [65].
In response to effective immunotherapy, the tu mor cells may evolve the mechanisms that allow them to escape immune recognition.Such immunoselection can cause an outgrowth of the tumor cell population that has lost the expression of a given target antigen [66].A significant form of the immune escape is an ability of tumor cells to evolve the mechanisms that impede the antigen processing and presentation, in cluding the alteration or loss of the expression of MHC alleles, a 2 -microglobulin and transporter mo lecules associated with the antigen processing (TAP).Tumor cells can also secrete the immunosupressive cytokines such as transforming growth factor (TGF)-(3 and IL-10, which can inhibit the T-lymphocyte effector function [67].Usage of polyvalent vaccines, specific for several tumor-associated antigens, or vaccination with antigens required by the tumor for maintenance of it's malignant phenotype (e. g. telomerase), can circumvent this form of the immune escape [67,68].The information on the composite expression of different antigens in a given tumor is crucial for the design of polyvalent vaccine strategies.It is important to assess the number of patients that are suitable for a combined or sequential vaccination with two or more tumor antigens, because such a strategy could have the potential of reducing or even preventing the in vivo selection of antigen loss tumor cell variants [67].
Hence, during last two decades the successes of immunotherapy resulted in the development of diverse modern immunotherapeutic strategies for cancer tre atment.The identification of tumor-associated anti gens has opened a new era of tumor immunology and unlimited possibilities for development of active spe cific immunotherapy.Novel immunotherapeutic ap proaches to cancer treatment and vaccines of new generation have been developed due to the successes in the field of molecular biology, cell immunology and genetic engineering.Modern methods of monitoring immune response make possible to investigate the molecular mechanisms of immune response during immunotherapeutic manipulations that allow to under stand the origin of cancer and to improve the treat ment of cancer diseases.
Cancer-testis(CT) antigens are a fascinating category of tumor antigens with the expression res-