Selecting and screening recombinant antibody libraries.

During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Direct detection and quantitation of a distinct T-cell epitope derived from tumor-specific epithelial cell-associated mucin using human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

The recent characterization of MHC-displayed tumor-associated antigens that recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy, as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, specific T-cell epitopes derived from the Mucin-1 tumor-associated antigen (MUC1) that are widely expressed in many cancers were identified and shown to be recognized by CTLs. We selected a large nonimmune repertoire of phage Fab antibodies on recombinant human class I HLA-A2 complexes displaying an antigenic T-cell epitope derived from MUC1. High frequency of anti-MHC-peptide binders was observed (84%), and surprisingly, a high percentage (80%) of antibodies was fully specific for the MUC1 epitope. We isolated a surprisingly large panel of 16 different high-affinity human recombinant Fab antibodies that exhibited peptide-specific, MHC-restricted binding characteristics of T cells. The analyzed Fabs not only recognize the cognate MHC-peptide complex in a recombinant soluble form but also the native complex as displayed on the surface of antigen-presenting cells and breast tumor cells. Therefore, these findings demonstrate the ability to transform the unique fine specificity but low intrinsic affinity of T-cell receptors on T cells into high-affinity soluble antibody molecules endowed with a T-cell antigen receptor-like specificity. These molecules may prove to be very important and widely applicable for monitoring the expression of specific MHC-peptide complexes on the surface of tumor and immune cells for structure-function studies of T-cell receptor-peptide-MHC interactions, as well as for developing new targeting agents for immunotherapy.

Isolation and characterization of human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells directed toward the widely expressed tumor T-cell epitopes of the telomerase catalytic subunit.

The recent characterization of MHC-displayed tumor-associated antigensthat recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, recently specific T-cell epitopes derived from the telomerase catalytic subunit (hTERT) that are widely expressed in many cancers were identified and shown to be recognized by CTLs derived from cancer patients. We selected a large nonimmune repertoire of phage Fab antibodies on recombinant human class I HLA-A2 complexes displaying two distinct antigenic T-cell epitopes derived from hTERT. We isolated a surprisingly large panel of high-affinity human recombinant Fab antibodies that exhibited peptide-specific, MHC-restricted binding characteristics of T cells. The analyzed Fabs not only recognize the cognate MHC-peptide complex in a recombinant soluble form but also the native complex as displayed on the surface of antigen-presenting cells and hTERT-expressing tumor cells. These findings demonstrate for the first time the ability to transform the unique fine specificity but low intrinsic affinity of TCRs on T cells into high-affinity soluble antibody molecules endowed with a T-cell antigen receptor-like specificity. These molecules may prove to be very important and widely applicable for monitoring the expression of specific MHC-peptide complexes on the surface of tumor and immune cells, for structure-function studies of TCR-peptide-MHC interactions, as well as for developing new targeting agents for immunotherapy.

Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.

Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.

Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries.

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.

Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries.

Human antibodies selectively targeting angiogenic vessels hold great promise for the immunotherapy of human malignancies and can help to elucidate the molecular mechanisms regulating angiogenesis. By selecting a large antibody phage display library on proliferating stimulated HUVEC, we have isolated 15 human antibodies that bind to HUVEC in flow cytometric analysis, 11 of which target the vasculature of colorectal carcinomas as demonstrated by immunohistochemical analysis. The four most specific antibodies, TEM-A, TEM-C, TEM-M and TEM-Q, also stain the vasculature of a series of carcinomas derived from liver, ovary, kidney, bladder, lung and breast, and either react weakly or not all with the vasculature of corresponding normal tissues. All four antibodies react with the vasculature of endometrium, but only TEM-M and TEM-Q react with the vasculature of placenta. As shown by non-reducing western blot analysis, 9/15 of the antibodies recognize either one or two distinct bands on HUVEC cell lysates, with molecular weights of 175 and/or 110-125 kDa. Antibodies identified by this approach may be used for the identification of new markers of angiogenesis and/or tumor vasculature. The selected antibodies may prove useful as new prognostic markers, for in vivo tumor imaging purposes and for further development of targeted therapies.

MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing.

MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 1100-2600 [corrected] ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10- to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 16000-52000 [corrected] GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.

Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy.

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.

Optimizing the exogenous antigen loading of monocyte-derived dendritic cells.

Dendritic cell (DC) vaccination, i.e. the adoptive transfer of antigen-loaded DC, is still at an early stage and requires standardization. In this study, we investigated the exogenous loading of monocyte-derived DCs with HLA class I- and II-restricted peptides, as despite widespread use, little effort has been put into its pre-clinical validation. We found that only mature DCs (m-DC) but not immature DCs (im-DC) could be sufficiently loaded with exogenous class I-restricted peptides and were by far superior in expanding CD8(+) primary (Melan-A.A2 peptide-specific) and recall [Influenza matrix peptide (IMP) A2-specific] T cell responses. Primary stimulation with peptide-loaded im-DCs even down-regulated antigen-specific T cell responses. Our results indicate that stimulation with m-DCs is superior in terms of quantity and quality compared with im-DCs, supporting their preferred use in clinical DC trials. Loading of m-DCs with high (10 microM) concentrations generated clearly more Melan-A effectors than loading with 1 or 0.1 microM without any negative effect on the quality (affinity) of the resulting T cells. In contrast to the findings with the Melan-A peptide loading with 10 microM IMP was counter-productive, induced apoptosis and yielded fewer specific T cells of inferior affinity as compared with loading with 1 or 0.1 microM. In sharp contrast to the situation for HLA class I, much higher levels and longer half-lives of peptide-HLA class II complexes were obtainable upon loading of im-DCs with exogenous peptide, but m-DCs were functionally preferable to induce T(h)1 responses in vitro. Another surprising finding was that, while presentation to T cells upon simultaneous loading of several peptides with highly varying affinities and competing for the same class I or II molecule was possible, in priming experiments peptide competition clearly inhibited T cell induction. Although peptides will obviously vary in their individual properties, our study clearly points to some important principles that should be taken into account.

A major histocompatibility complex-peptide-restricted antibody and t cell receptor molecules recognize their target by distinct binding modes: crystal structure of human leukocyte antigen (HLA)-A1-MAGE-A1 in complex with FAB-HYB3.

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 A resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1.MAGE-A1. Fab-Hyb3 employs a diagonal docking mode resembling that of T cell receptors. However, other than these natural ligands, the antibody uses only four of its six complementarity-determining regions for direct interactions with the target. It recognizes the C-terminal half of the MAGE-A1 peptide, the HLA-A1 alpha1-helix, and N-terminal residues of the alpha2-helix, accompanied by a large tilting angle between the two types of molecules within the complex. Interestingly, only a single hydrogen bond between a peptide side chain and Fab-Hyb3 contributes to the interaction, but large buried surface areas with pronounced shape complementarity assure high affinity and specificity for MAGE-A1. The HLA-A1.MAGE-A1.antibody structure is discussed in comparison with those of natural ligands recognizing HLA.peptide complexes.

Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity.

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC-peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC-peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy. To generate molecules with such a unique fine specificity, we selected a large nonimmune repertoire of phage Fab antibodies on recombinant HLA-A2 complexed with three common antigenic T cell, HLA-A2-restricted epitopes derived from the melanoma differentiation antigen gp100. We were able to isolate a surprisingly large panel of human recombinant Fab antibodies that exhibit a characteristic TCR-like binding specificity to each of the three gp100-derived epitopes, yet unlike TCRs, they did so with an affinity in the nanomolar range. These TCR-like antibodies recognize the native MHC-peptide complex expressed on the surface of antigen-presenting cells. Moreover, they can detect the specific MHC-peptide complexes on the surface of melanoma tumor cells. These results demonstrate the ability to isolate high-affinity human recombinant antibodies with the antigen-specific, MHC-restricted specificity of T cells, and this ability was demonstrated for three different epitopes of the same melanoma-derived antigen.

Target validation for genomics using peptide-specific phage antibodies: a study of five gene products overexpressed in colorectal cancer.

Genomic approaches are providing a wealth of information on differential gene expression in cancer. To identify the most interesting genes amongst the many identified, high-throughput methods for analysis of genes at the translational level are required. We have used a rapid method for the in vitro selection of antibodies to peptide antigens for the generation of probes to 5 gene products that we have found to be overexpressed in colorectal cancer. The rationale of our study was to select a non-immune phage displayed human antibody library on peptides designed from the coding regions of the gene sequences and to verify whether such antibodies would be suitable probes for the parental protein in immunohistochemical and Western blot analysis. After the generation of a profile of genes overexpressed in primary colorectal cancer (CRC) we selected 5 genes, Ese-3b, Fls353, PBEF, SPARC and Smad5 for a more detailed analysis using phage display-derived antibodies. For these 5 antigens we designed 14-20 amino acid peptides predicted to be exposed on the surface of the parental protein. Selection of a large phage displayed antibody library resulted in specific antibodies for 6 of 8 different peptides with between 2 and 15 different antibodies isolated per peptide. Of 20 antibodies tested, 2 antibodies recognized the putative parental protein from primary CRC tissue. An antibody specific for a PBEF-derived peptide (Fab/PBEF-D4) was shown to recognize a protein product of the expected molecular weight in Western blotting and showed overexpression in n = 6/8 matched tumor/normal protein lysates. Furthermore, in immunohistochemistry this antibody showed restricted staining of the tumor stromal compartment with no detectable staining of epithelial cells. The discovery that PBEF is overexpressed in cancer is unexpected given that the normal function of PBEF is as a cytokine required for the maturation of B cell precursors. We also report on the isolation of an antibody (Fab/SMAD-50) specific for a Smad5-derived peptide that showed cytoplasmic staining of epithelial cells in both CRC tumor and matched normal mucosa. Fab/SMAD-50 also bound to a group of proteins in Western blotting with molecular weights consistent with belonging to the Smad family. These antibodies may be suitable probes for further investigation of the roles of PBEF and Smad5 in cancer. The amenability of phage display to automation suggests that this approach may be developed for implementation on a genomics scale. Indeed, the large-scale generation of antibody probes that can be used to study protein expression in situ would be of great value in target validation for functional genomics.

A human immunoglobulin G1 antibody originating from an in vitro-selected Fab phage antibody binds avidly to tumor-associated MUC1 and is efficiently internalized.

We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.

TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity.

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library.

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.