Detection of HLA class II-dependent T helper antigen using antigen phage display.

Major histocompatibility complex (MHC) class II-dependent antigens not only activate CD4+ T helper (Th) cells, but also cytolytic T lymphocytes and effector cells of the innate immune system. These antigens therefore are candidate vaccines against cancer and infectious agents. We have developed a novel approach using a model antigen, tetanus toxoid (TT), which provides the basis for the establishment of a novel strategy of cloning Th antigens. In the TT model system, a cDNA library encoding part of the TT light chain which contained a TT-associated Th epitope recognized by TT-specific Th clones was displayed on a phage vector (TT-phage) and presented to TT-specific Th cells by autologous Epstein-Barr virus-transformed B cells (APC). These TT-phages were able to specifically activate two different TT-specific CD4+ Th cell lines as demonstrated both in [3H]thymidine incorporation and cytokine release assays. Th cell stimulation by TT-phages was significant at a ratio of one TT-phage in 50 irrelevant phages. The described approach provides the basis for the development of a novel strategy of cloning MHC class II-dependent Th antigens, using available Th cells. This strategy has several potential advantages over existing antigen cloning methods or biochemical peptide isolation.

Addition of interleukin-12 to GA733 tumor protein vaccine leads to development of tumor protective immunity despite surgical stress.

BACKGROUND: We have previously shown that preoperative vaccination with the GA733 protein does not inhibit tumor growth in mice undergoing open surgery or carbon dioxide insufflation. In this study we assessed the antitumor effect of a combined GA733 and interleukin-12 (IL-12) vaccine. METHODS: For this study, BALB/c mice were immunized with GA733, IL-12, or GA733 and IL-12, or they received no vaccine. Immediately after surgery (laparotomy or insufflation), GA733-transfected CT26 cells (C26-GA733) were injected subcutaneously into all mice. After 5 weeks, the mice were sacrificed, their tumors measured, GA733-specific antibodies determined by enzyme-linked immunoassay, and GA-733-specific cytotoxicity tested by flow cytometry using labeled C26-GA733 cells. RESULTS: Tumors were significantly (p < 0.05) smaller in both the insufflation and open groups that received combined GA733 and IL-12 than in their respective control subjects. Vaccination also induced a significant increase in the antibody and cell-mediated tumor-specific immunity. CONCLUSION: A preoperative vaccine consisting of GA733 and IL-12 inhibited postoperative tumor growth after open and closed surgery and allowed the mice to overcome the immunosuppressive effects of surgery.

Recombinant vaccinia viruses expressing immunoglobulin variable regions efficiently and selectively protect mice against tumoral B-cell growth.

The variable regions of the immunoglobulin (Ig) expressed on the surface of a malignant B cell can be considered tumor-specific antigens and, as such, could be targets for immunotherapeutic approaches. However, because until now the immunization procedures have been complex and have given only partial protection, it was necessary to find new methods of immunotherapy. Here, we present a successful method of vaccination against B-cell tumors in a murine model. We produced recombinant vaccinia viruses (rVV) expressing the heavy and the light chain of surface Ig of a patient's malignant B cells and we tested the ability of these rVV to protect immunized mice against tumor growth of transfectomas producing the same human Ig. The protection of the mice was complete and specific to the variable region of the immunizing heavy chain although specific lymphoproliferative and cytotoxic responses were not detectable in vitro. The protection was strictly dependent on the presence of CD4 T cells and asialo GM1+ cells. Furthermore, tumor protection clearly required gamma-interferon and was partially inhibited by blocking the Fas-Fas ligand interaction. We also show, in a murine syngeneic model, that rVV expressing a poorly mutated Ig protects against the growth of Ig-producing tumor.

Anti-idiotypic antibody and recombinant antigen vaccines in colorectal cancer patients.

The colorectal carcinoma (CRC)-associated GA733 antigen (also known as CO17-1A, KS1-4, KSA or EpCAM) has been the target of a phase II/III randomized trial of passive immunotherapy with monoclonal antibody CO17-1A and phase I active immunotherapy trials with polyclonal anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope on the antigen. The CO17-1A antigen was molecularly cloned and the extracellular domain expressed in baculovirus (BV) GA733-2E. Whereas, anti-idiotypic antibody mimics a single epitope on the antigen, BV GA733-2E expresses multiple potentially immunogenic epitopes. In animals, the immunogenicity of BV GA733-2E in aluminum hydroxide was superior to that of anti-idiotype in the same adjuvant. Here, we compared the immunogenicity of anti-idiotypic antibody and GA733-2E antigen in CRC patients. These studies indicate that the antigen is superior to the anti-idiotype antibody in inducing humoral and cellular immunity in CRC patients.

Immunogenicity of recombinant GA733-2E antigen (CO17-1A, EGP, KS1-4, KSA, Ep-CAM) in gastro-intestinal carcinoma patients.

Targeting the GA733 antigen (also known as CO17-1A, EGP, KS1-4, KSA, Ep-CAM) by monoclonal antibody CO17-1A or anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope has induced prolonged survival and specific immune responses to the antigen, respectively, in colorectal cancer (CRC) patients. In pre-clinical studies in mice and rabbits, recombinant baculovirus-derived GA733-2E antigen was superior to anti-idiotypic antibodies at modulating specific immune responses. Our aim was to evaluate the immunogenicity and potential toxicity of alum-precipitated GA733-2E in a phase I trial in patients with resected CRC or pancreatic cancer. Six patients with advanced pancreatic carcinoma and 6 with CRC Dukes' stage A, B or C received between 4 and 7 doses of alum-precipitated GA733-2E at 50, 200 or 800 microg/dose at monthly intervals. Antibody binding to GA733-2E or antigen-positive CRC cells was determined, as were antigen-specific proliferative, cytolytic T-lymphocyte and delayed-type hypersensitivity responses. Six of the 12 patients developed antigen-specific humoral immune responses after immunotherapy, and 8 developed cellular immune responses. The overall immune response rate, including patients with humoral and/or cellular immune responses, was 83%. Median overall survival of the CRC and pancreatic cancer patients was 39.8 and 11.2 months, respectively. Following 18 years of single-epitope targeting of the GA733 antigen, immunization of patients against multiple epitopes of the antigen frequently induces an immune response in the absence of significant toxicity, despite relatively widespread expression of this antigen on normal epithelial cells.

Oligomeric state of the colon carcinoma-associated glycoprotein GA733-2 (Ep-CAM/EGP40) and its role in GA733-mediated homotypic cell-cell adhesion.

The GA733-2 antigen (GA733) is a homotypic calcium-independent cell adhesion molecule (CAM) present in most normal human epithelial cells and gastrointestinal carcinomas. Because oligomerization of some CAMs regulates cell adhesion and signal transduction, the correlation between GA733 oligomeric state and cell-cell adhesion was investigated. Sedimentation equilibrium studies showed that full-length (-FL) GA733 exists as dimers and tetramers in solution, whereas the GA733 extracellular domain (-EC) is a monomer. The Kd of GA733-FL is less than 10 nm for the monomer-dimer association, whereas the dimer-tetramer association is about 1000-fold weaker (Kd approximately 10 microm). Chemical cross-linking of purified GA733-FL in solution resulted in a major product corresponding to GA733 dimers, and minor amounts of trimers and tetramers. However, GA733-EC cross-linked under the same conditions was consistently a monomer. Chemical cross-linking of dissociated colon carcinoma cells produced predominantly GA733 dimers, whereas cross-linking of cells in monolayers yielded some tetramers as well. GA733-FL retained its cell-cell adhesion function as shown by inhibition of cell aggregation, whereas monomeric GA733-EC was inactive. These data show that GA733 exists predominantly as high affinity noncovalent cis-dimers in solution and on dissociated colon carcinoma cells. The lower affinity association of dimers to form tetramers is most likely the head-to-head interaction between GA733 cis-dimers on opposing cells that represents its cell-cell adhesion activity.

Isolation of the melanoma-associated antigen p23 using antibody phage display.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.

Anti-idiotypic antibody (ab2) vaccines: coupling of Ab2 BR3E4 to KLH increases humoral and/or cellular immune responses in animals and colorectal cancer patients.

The colorectal carcinoma (CRC)-associated CO17-1A/GA733 antigen (Ag) has been the target of a phase II/III randomized trial of passive immunotherapy with monoclonal antibody CO17-1A (Ab1), and phase I active immunotherapy trials with polyclonal anti-idiotypic antibodies (Ab2) mimicking the CO17-1A or GA733 epitope of the Ag. However, monoclonal rat Ab2 BR3E4 directed against Ab1 CO17-1A was superior to polyclonal Ab2 in inducing antigen-specific humoral and cellular immune responses in mice and rabbits. Various forms of Ab2 BR3E4, i.e., BR3E4-F(ab')2 precipitated with aluminum-hydroxide (alum), BR3E4-F(ab')2 coupled to KLH and precipitated or non-precipitated with alum, and BR3E4-IgG in alum or incomplete Freund's adjuvant were compared for their capacity to induce in rabbits anti-anti-idiotypic antibodies (Ab3) that specifically bind to the CO17-1A Ag. BR3E4-F(ab')2 coupled to KLH and precipitated with alum was shown to induce the highest Ab3 titers, followed by Ab2 BR3E4-IgG in alum. Therefore Ab2 BR3E4 as intact IgG (IgG group) or as F(ab')2 coupled to KLH (KLH group), was administered in a phase I trial to 45 patients with CRC, stage Dukes'D (UICC stage IV), with the goal to modulate patients' immune responses to their tumors. Fifteen of 23 patients in the IgG group developed Ab3 binding specifically to Ab2, and in four of these patients the Ab3 also specifically bound to Ag-positive CRC cells. Lymphoproliferative responses to Ab2 and/or GA733-2E Ag stimulation were observed in three of these patients. Eighteen of the 22 KLH group patients tested developed Ab3 and the Ab3 bound specifically to CRC cells in eight patients. Five of the 15 KLH group patients tested developed lymphoproliferative responses to Ab2 and/or GA733-2E Ag. Thus, there was a trend for the KLH group demonstrating higher immune response rates than the IgG group. Clinical responses were rare in these patients with liver metastases.

Colorectal cancer vaccines: antiidiotypic antibody, recombinant protein, and viral vector.

The colorectal cancer antigen GA733 (also termed CO17-1A, KSI-4, Ep-CAM, KSA) has proved to be a useful target in passive immunotherapy with monoclonal antibody and in active immunotherapy with antiidiotypic antibodies in cancer patients. The GA733 antigen was molecularly cloned and expressed in baculovirus (BV), adenovirus (AV), and vaccinia virus (VV). Recombinant BV-, VV-, and AV-GA733 induced antigen-specific cytotoxic antibodies and proliferative and delayed-type hypersensitive lymphocytes. However, only the AV recombinant induced antigen-specific cytolytic T lymphocytes and regression of established tumors. Cured mice were protected against challenge with antigen-negative tumors, indicating antigen spreading of immune responses. In a model of active immunotherapy against the murine homologue of the human GA733 antigen, murine epithelial glycoprotein (mEGP), BV-derived mEGP protein in various adjuvants did not protect mice against a challenge with mEGP-positive tumors. AV mEGP, only when combined with interleukin-2, significantly inhibited growth of established mEGP-positive tumors. This is in contrast to the same vaccine expressing the human antigen that was effective without interleukin-2. AV GA733, in combination with interleukin-2, is a candidate vaccine for colorectal cancer patients.

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine.

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoaffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector.

Recognition of human colon cancer by T cells transduced with a chimeric receptor gene.

Transduction with chimeric T-cell receptor genes can be used to redirect primary T lymphocytes to recognize specific antigens (Ags), including ovarian and breast cancer Ags. To extend this approach to colon cancer we report here redirection of T cells using a chimeric receptor recognizing the colon cancer-associated Ag EGP40. Chimeric T cell receptors were constructed by ligating single-chain genes of either of two EGP40-specific monoclonal antibodies (CO17.1 A, GA733) to the Fc receptor gamma-signaling chain. Retroviral vectors incorporating these constructs were used to transduce a murine T-cell line and human peripheral blood lymphocytes. These modified T cells were analyzed for specific recognition of colon cancer lines by measuring cytokine release and lytic activity against tumor targets. Murine lymphocytes transduced with the chimeric receptor based on GA733, but not CO17.1A, released cytokine specifically in response to EGP40-expressing colon cancer cell lines. Recognition of colon cancer targets by murine lymphocytes was blocked by the addition of GA733 antibody or soluble EGP40 Ag, confirming that colon cancer recognition is based on specific chimeric receptor-Ag interaction. Human lymphocytes transduced with chimeric GA733 specifically recognized colon carcinoma cells in cytokine release assays and lysed EGP40-expressing tumor cells. Genetic modification of T cells can be used to redirect T cells against EGP40-expressing tumor cells. The expression of chimeric GA733 in the autologous lymphocytes of patients may provide a source of tumor-reactive cells with therapeutic application against colon cancer.

CD4(+), HLA class I-restricted, cytolytic T-lymphocyte clone against primary malignant melanoma cells.

The involvement of HLA-class I in target cell lysis by CD4(+) cytolytic T cells (CTL) has been a controversial issue. A CTL clone of CD4 phenotype was derived from the peripheral blood lymphocytes of a patient with primary melanoma. The CTL clone stably lysed the autologous primary melanoma cells for approximately 9 months in culture. Both the Valpha2/Vbeta8 T-cell receptor and CD4 were involved in CTL cytotoxicity. Of a large panel of allogeneic primary and metastatic melanoma or colorectal carcinoma cells, autologous and allogeneic Epstein-Barr virus-transformed B cells and autologous fibroblasts, only allogeneic metastatic melanoma cells matched with the autologous tumor cells for HLA-class I (B57[17]) were lysed and induced IFN-gamma secretion by the CTL clone. Lysis of the autologous tumor cells was significantly blocked by monoclonal antibody to HLA-B17. Importantly, allogeneic, HLA-class I- and class II-unmatched melanoma cells were lysed by the CTL only following transfection of the cells with B57[17] cDNA. Our results provide direct evidence for the involvement of both CD4 and HLA-class I in tumor cell lysis by CD4(+) CTL.

Efficient laboratory-scale production of monoclonal antibodies using membrane-based high-density cell culture technology.

Monoclonal antibodies (MAbs) are important tools used in basic research as well as in the imaging and therapy of cancer. Many countries have limited the use of animals for large-scale production of MAbs, obliging laboratories to find efficient in vitro alternatives to ascites production. In this report we describe a protocol for laboratory-scale production of MAbs by culturing hybridoma cells in the two-chamber cell culture device CELLine 1000. This culture flask supports high cell densities (10(7)-10(8) cells/ml) and generates high concentrations of MAbs (0.7-2.5 mg/ml). Two hybridomas producing MAbs directed against the gastrointestinal antigen GA733-2, GA733 MAb and CO17-1A MAb, were evaluated over culture periods of up to two months using several alternative conditions. Two different sets of conditions are reported; the first using serum-supplemented medium (20% v/v) and the second using serum-free medium (SFM). Average weekly yields of the purified MAbs in serum-supplemented medium were 24 mg and 33 mg, and in SFM were 21 mg and 17 mg for GA733 MAb and CO17-1A MAb, respectively. Experimental variables that can affect antibody production and economy include: nutrient medium and cell compartment medium compositions (cell line dependent), the proportion of the cell compartment medium harvested every 3 days (50% to 80% with 80% optimal) and the frequency of nutrient medium changes (3 to 9 days with 6 days as most cost effective). Protein-A Sepharose purification followed by antigen-specific affinity purification showed that MAbs obtained from serum-supplemented cultures contain less than 0.6% of bovine IgG contamination, while MAbs obtained from serum-free cultures contained no extraneous IgG. In addition, MAbs from both culture media were fully active (essentially 100%) as measured by their ability to bind to an antigen column. In contrast, the same MAbs purified from ascites using Protein-A-Sepharose typically contained a major portion of inactive IgG. This in vitro method for laboratory-scale production of MAbs (10 to 500 mg) proved to be simple, reproducible and cost effective. It represents a useful alternative to the in vivo production of MAbs in mice.

Advances in cancer vaccine development.

Traditionally, cancer vaccines have used whole tumour cells administered in adjuvant or infected with viruses to increase the immunogenicity of the cells. With the identification of tumour-associated and tumour-specific antigens (TAA, TSA), antigen and epitope-specific vaccines have been designed. Compared to tumour cell vaccines, antigen and epitope vaccines are more specific and easier to produce in large quantities but may display lower immunogenicity and lead to the in vivo selection of antigen or epitope-negative escape tumour variant cells. The optimal vaccine will elicit both humoral and cellular immunity in the patients as both parameters have been positively correlated with the induction of beneficial clinical responses. The choice of adjuvant, costimulation and delivery mode greatly determines the outcome of vaccinations and may favour the induction of T-cell responses of T helper (Th)1, Th2, or both Th1 and Th2 types. Animal models of TAA vaccines must take into account the normal tissue expression of TAA, which may induce immunological tolerance to TAA. With the identification of homologues of human TAA in animals, novel experimental models of cancer vaccines which mimic the condition in patients are now available. Several vaccines comprising tumour cells, TAA or anti-idiotypic antibodies mimicking TAA have recently entered phase III of clinical evaluation.

Colorectal carcinoma invasion inhibition by CO17-1A/GA733 antigen and its murine homologue.

BACKGROUND: The gastrointestinal carcinoma antigen GA733 is a potential target for passive and active immunotherapy for patients with colorectal carcinoma. This antigen has been characterized previously as a homophilic adhesion (i.e., adhesion to self) protein, but the functional consequences of homophilic adhesion for tumor growth and invasion are unknown. The availability of a murine homologue of GA733, i.e., murine epithelial glycoprotein (mEGP), allows for functional analysis of cell adhesion as it relates to tumor growth and invasion, both in vitro and in vivo. METHODS: CT-26 murine colorectal carcinoma cells were transfected with complementary DNAs encoding either the human or the murine antigen. GA733- or mEGP-producing cells were evaluated for homophilic adhesion, growth on plastic surfaces, colony formation in soft agar, and invasion through a reconstructed basement membrane (Matrigel). mEGP-producing cells were also examined for their capacity to metastasize in mice. Reported P values are two-sided. RESULTS: Compared with control cells, mEGP-producing cells showed significantly lower growth rates, colony formation, and invasion through Matrigel in vitro (all P values <.05). Compared with vector-only transfected cells and parental cells, mEGP-producing cells showed a reduction in metastatic potential in syngeneic immunodeficient and immunocompetent mice (all P values <.05). In contrast to mEGP-transfected cells, GA733-transfected cells did not exhibit significantly reduced growth or colony formation in vitro (all P values >.05). However, GA733-transfected cells did show reduced invasion through Matrigel compared with vector-only transfected cells or parental cells (all P values <.05). CONCLUSION: The adhesion proteins GA733 and mEGP inhibit invasion of tumor cells.

[1st successful immunization of pancreas and colorectal carcinoma patients with CO17-1A-vaccine]. / Erste erfolgreiche Immunisierung von Pankreas- und kolorektalen Karzinompatienten mit CO17-1A-Vakzin.

The baculovirus-derived extracellular domain of the CO17-1A antigen induced cellular and/or humoral immune responses in curatively resected colorectal and pancreatic cancer patients after at least three applications of the vaccine. The vaccine application was safe and non-toxic. The clinical efficacy of the vaccine needs to be evaluated in larger, randomized clinical trials.

Human colorectal cancer (CRC) antigen CO17-1A/GA733 encoded by adenovirus inhibits growth of established CRC cells in mice.

The human colorectal carcinoma (CRC)-associated Ag CO17-1A/GA733, originally defined by mAbs CO17-1A and GA733, has been a useful target in passive immunotherapy of CRC patients with mAb and in active immunotherapy with anti-idiotypic Abs mimicking the CO17-1A or GA733 epitope. Both approaches have targeted single epitopes. We investigated the capacity of full-length CO17-1A/GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant adenovirus 5 (Ad5 GA733-2) to induce humoral, cellular, and/or protective immunity in mice. Ad5 GA733-2 induced Ag-specific Abs that reacted predominantly to CO17-1A- and GA733-unrelated epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with effector cells. Ad5 GA733-2-immune mice developed Ag-specific, proliferative lymphocytes of Th1 type and cytolytic lymphocytes. The use of Ad5 GA733-2 to immunize mice bearing established syngeneic CRC cells transfected with the human Ag induced significant and specific tumor regression. Cured mice resisted rechallenge with human CO17-1A/GA733 Ag-negative parental CRC cells, suggesting that targeting the human Ag on the murine transfectants induced protective immunity to other Ag expressed by the parental tumor. These results may explain the high potency of the recombinant vaccine. Thus, rAd5 GA733-2 may have potential as a vaccine for CRC patients.

Cytotoxic T-cell clone against rectal carcinoma induced by stimulation of a patient's peripheral blood mononuclear cells with autologous cultured tumor cells.

In an effort to establish cytolytic T lymphocytes (CTLs) against colorectal carcinoma (CRC) by stimulating patients' lymphocytes with autologous tumor cells, we used peripheral blood mononuclear cells (PBMC) from a patient with minimal residual rectal carcinoma following removal of the primary lesion and involved regional lymph nodes as a source to generate CTLs in culture. A CTL line and clone were established from the patient's PBMC following stimulation of PBMC with autologous, cultured tumor cells and interleukin-2. The CTL line and the clone consisted predominantly of CD4+ lymphocytes. The CTL clone expressed two T-cell receptor variable alpha chains (V alpha11 and V alpha22) and one beta chain (Vbeta14). The cytokine secretion pattern of the CTL line was of the Th1-type. Both the CTL line and the clone lysed the autologous rectal carcinoma cells, but not the allogeneic, partially human lymphocyte antigen (HLA)-matched or nonmatched CRC cells, autologous Epstein-Barr virus-transformed B cells, K562 (natural killer target) cells or Daudi (lymphokine-activated killer target) cells. Lysis of autologous tumor cells most likely was HLA class I-restricted. Our unique success in generating CTLs against this tumor type may rest in the inclusion of a patient with minimal residual, rather than advanced, disease.

A model system for detection and isolation of a tumor cell surface antigen using antibody phage display.

To establish a screening procedure for tumor cell-surface reactive Fabs, we used a model antigen/antibody system including the epidermal growth factor receptor (EGF-R) and the anti-EGF-R monoclonal antibody 425. The 425 Fab was displayed on the surface of M13 filamentous phage. In a screening assay for 425 phage binding to tumor cell surfaces, biotinylated 425-phage bound specifically to EGF-R-positive A431 epidermoid carcinoma cells and not to K562 non-expressor erythroleukemia cells. With a model library, the sensitivity of phage enrichment by phage binding to cell surfaces was one 425-phage in 20,000 unrelated phages after 4 rounds of panning on A431 cells. In a phage tissue screening assay, 425-phage, but not unrelated phage, bound specifically to melanoma cells expressing EGF-R. Epitope and idiotope specificity of 425-phage was demonstrated in phage competition assays, using as targets A431 cells and anti-idiotypic antibodies to monoclonal antibody 425, respectively. Finally, the EGF-R protein was directly isolated from A431 cell extracts, using biotinylated 425-phage. The data obtained with the 425 model library system demonstrate the usefulness of antibody phage display for the rapid identification and isolation of tumor or other disease-related cell surface antigens.

Combinatorial antibodies against human malignant melanoma.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma antigens in patients. We have attempted to identify melanoma-associated antigens recognized by patients' B cells using an antibody phage display approach. Antibody display on filamentous phages allows direct screening of cDNA libraries for expression of cell-surface-reactive antibodies, without the need for antibody production and purification using bacteria or eukaryotic cell systems. This approach was used to identify melanoma-associated cell-surface antigens recognized by patients' B cells. Antibodies produced by the B cells of a melanoma patient (in remission for > 7 years following periodic vaccination with allogeneic melanoma cell vaccine) were displayed as Fabs on the surfaces of filamentous phages. A library of 10(8) phages was absorbed to normal melanocytes, followed by phage binding to and elution from melanoma cells (human lymphocyte antigen nonmatched and vaccine melanoma cells). Phages were further selected for reactivities with tunicamycin-treated melanoma cells. These procedures resulted in a > 10(6)-fold enrichment of tumor-specific phages from the original phage library. One phage-Fab bound to melanoma cells, other tumor cells, and a few normal cells in cultured cell lines and in tissue sections.