Epitopes Of Carcinoembryonic Antigen Research Articles

Abstract 6317: Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells

Abstract Cancer treatment has improved in the past decades due to advances in targeted therapies and surgical techniques. Immunotherapy, for example, has greatly improved outcomes for people with several types of cancer that typically have a poor prognosis. However, the response rate depends on the type of cancer being treated, with an average response rate between 20 to 50%. Thus, novel approaches to overcome such therapeutic challenges are warranted. Emerging therapies include fusion proteins and immunoconjugates that deliver a cytotoxic payload to the tumor cell with more specificity and potentially less avenues for resistance than exerted by the antibody or toxin alone. Carcinoembryonic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in a high percentage of solid tumors including colon and rectum, lung, prostate, ovary, thyroid, or liver. Thus, oncologic therapies against CEA may promote anti-tumor activity while sparing off-target toxicity to unaffected normal tissue. Our newly developed construct, designated GrB-Fc-huMFE, is a completely human fusion protein that contains the single-chain variable fragment huMFE antibody recognizing a juxta-membrane epitope of CEA remaining after proteolytic cleavage of CEA from the cell surface. The construct rapidly internalized into target cells resulting in granzyme B (GrB)-mediated mitochondrial depolarization and irreversible activation of both apoptotic and necrotic cascade mechanisms of cell death. In vitro cytotoxicity of GrB-Fc-huMFE against an initial subset of CEA+ tumor cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar IC50 cytotoxicity against CEA-negative cell lines. Significantly, GrB-Fc-huMFE cytotoxicity in vitro was unaffected by exogenously-added CEA extracellular domain at doses up to 1000 μg/ml, suggesting that the construct is not competitively absorbed by soluble CEA proteolytically released by tumors and present in the circulation of patients. An in vivo efficacy study against established CEA+ (A-549) tumors in nude mice resulted in significant growth inhibition compared to vehicle controls (1200 mm3 vs <40 mm3). Complete tumor regression was observed in 7/10 tumors in mice treated (IP) with doses of 100 mg/kg of GrB-Fc-huMFE in a Q3Dx5 dose administration schedule. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Pharmacokinetic, stability and in vitro tests against a broader panel of tumor cell lines are ongoing and will be reported. Altogether, GrB-Fc-huMFE appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical and eventual clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Walter N. Hittelman, Michael G. Rosenblum. Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6317.

Selection and characterisation of Affimers specific for CEA recognition

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.

Dendritic Cells Transfected with a DNA Construct Encoding Tumour-associated Antigen Epitopes Induce a Cytotoxic Immune Response Against Autologous Tumour Cells in a Culture of Mononuclear Cells from Colorectal Cancer Patients.

Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour-associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA-transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic-assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA-transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin-positive lymphocytes. In addition, DNA-transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate-loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC-based vaccines.

Correction: Potentiation of Anti-Carcinoembryonic Antigen Immunotoxin Cytotoxicity by Monoclonal Antibodies Reacting with Co-Expressed Carcinoembryonic Antigen Epitopes

The initial step in ricin A-chain (RTA)-immunotoxin-mediated cell cytotoxicity involves binding to the target cell Ag through the antibody moiety. One of the factors influencing this is the affinity of the antibody component for the target cell Ag. Multiple epitopes on carcinoembryonic Ag have been mapped providing a range of mAb of known specificity. These have been used to show that the cytotoxicity of an immunotoxin containing RTA conjugated to an anti-carcinoembryonic Ag mAb (228-RTA) is potentiated by mAb recognizing different epitopes. The potentiating antibodies also increased the level of target cell binding of antibody 228. Cross-linking of cell bound antibody was not involved because monovalent fragments of a potentiating antibody were effective. The potentiating antibodies modified the binding affinity of 228 antibody increasing the t1/2 of antibody at the tumor cell surface. This increased the dwell time of cell bound antibody and using conjugates of 228 linked to albumin-tetramethylrhodamine it was shown to enhance conjugate endocytosis. These investigations indicate that enhanced antibody affinity leads to increased endocytosis of bound immunoconjugate and potentiates cytotoxicity.

Synergistical toll-like receptors activated dendritic cells induce antitumor effects against carcinoembryonic antigen-expressing colon cancer

Dendritic cell (DC)-based cancer vaccine represents a promising immunotherapy against cancer. There has been recent evidence which have suggested that toll-like receptor (TLR) ligands may be critical for DC preparation; this was usually omitted in the past. Our study is designed to investigate if the vaccination of synergistical toll-like receptors activated DCs can induce more potent cytotoxic T Lymphocytes (CTL) responses and antitumor activity in carcinoembryonic antigen (CEA) transgenic mouse tumor models. We involved combination of TLR3 and TLR7/8 ligands in culture protocol of DCs. The DCs' surface molecules expression, IL-12 secretion and proliferation capacity of lymphocytes were tested. We also investigate the CTL activity against MC38-CEA colon tumor cells and the prophylactic and therapeutic effects of DC vaccination in subcutaneous mouse colon tumor models. Compared with conventionally generated DCs, we showed synergistic TLR-activated DCs exhibited higher surface molecule expression, significantly higher secretion of IL-12 and more potent proliferating capacity of lymphocytes. Synergistic TLR-activated DCs were also able to induce lymphocytes possessing the specific cytotoxicity against MC38-CEA cells in vitro. Vaccination with CEA epitope pulsed TLR-activated DCs elicited antigen-specific preventive effect on MC38-CEA tumors, but failed to cure the tumor-bearing mice, that may be due to the suboptimal epitope selected and host immunosuppression. Our results have proved that combined activation of TLRs can lead to better maturation status of DCs and also induce more effective antitumor immune responses against colon cancer, suggesting this may be a potential strategy to develop more powerful DC cancer vaccines.

Identification of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen

CD4 T cells are important for anti-tumor immune responses. Aside from their role in the activation of CD8 T cells, CD4 T cells also mediate anti-tumor immune responses by recruiting innate immune effectors into the tumor microenvironment. Thus, the search for strategies to boost CD4 T cell immunity is an active area of research. Our goal in this study was to identify HLA-DR epitopes of carcinoembryonic antigen (CEA), a commonly over-expressed tumor antigen. HLA-DR epitopes of CEA were identified using the epitope prediction program, PIC (predicted IC(50)) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-gamma ELIspot assays were used to detect existing immunity against the HLA-DR epitope panel of CEA in breast and ovarian cancer patients. In vitro generated peptide-specific CD4 T cells were used to determine whether the epitopes are naturally processed from CEA protein. Forty-three epitopes of CEA were predicted, 15 of which had high binding affinity for 8 or more common HLA-DR molecules. A degenerate pool of four, HLA-DR restricted 15 amino acid epitopes (CEA.24, CEA.176/354, CEA.488, and CEA.653) consisting of two novel epitopes (CEA.24 and CEA.488) was identified against which 40% of breast and ovarian cancer patients had pre-existent T cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 94% of patients. Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. The degenerate pool of CEA peptides may be useful for augmenting CD4 T cell immunity.

Vaccination with a CEA epitope mimic leads to reduced growth of tumor transplants

3076 Background: The carcinoembryonic antigen (CEA) is a glycoprotein which is overexpressed on nearly 50% of all human tumors. We aimed to establish an active immunotherapy by replacing the poorly immunogenic glycoprotein with highly immunogenic peptides. Methods: Col-1 is a monoclonal antibody directed against CEA on tumor cells. To generate structural mimics of the Col-1 epitope, we used the Col-1 antibody and the biopanning method in order to select immunreactive peptide mimotopes from random phage libraries. Subsequently, immunogenicity of a selected mimotope was examined in BALB/c mice. We assessed antibody-dependent cytotoxicity (ADDC) and complement-dependent cytotoxicity (CDC) mediated by the induced antibodies on CEA expressing HT29 cells. Furthermore, after immunisation the BALB/c mice were transplanted subcutaneously with Meth-A/CEA tumor cells. Results: All peptides selected were exclusively recognized by the anti-CEA antibody. In addition, true mimicry of the peptides with the natural antigen was confirmed by competition ELISA using purified human CEA. The best mimotope was chosen for synthesis in a multiple antigenic peptide (MAP) configuration rendering, therefore, a multiple antigenic mimotope (MAM). BALB/c mice immunized with the MAM developed antibodies against the immunogen and the original antigen CEA. In vitro, the anti-mimotope antibodies were capable of inducing high specific lysis of CEA-expressing HT29 cells via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, after s.c tumor transplantation no tumor growth was seen in MAM immunized mice, compared to the groups receiving a control mimotope or the naïve group. The CEA-mimotope immunized group showed a capsuled necrotic region without tumor cells but granulocytes and limited vascularisation. The non treated group showed tumor cells in different proliferation stages and neovascularisation. Conclusions: We conclude that the Col-1 epitope of the glycoprotein CEA can be translated into an immunogenic peptide mimic. The mimotope-induced antibodies recognize CEA and do effectively inhibit growth of CEA positive tumors. Based on these findings we suggest that generated mimotopes are candidates for active immunotherapy of CEA- expressing tumors. No significant financial relationships to disclose.

Therapy of Established Tumors in a Novel Murine Model Transgenic for Human Carcinoembryonic Antigen and HLA-A2 with a Combination of Anti-idiotype Vaccine and CTL Peptides of Carcinoembryonic Antigen

Induction of potent and sustained antitumor immunity depends on the efficient activation of CD8(+) and CD4(+) T cells. Immunization using dendritic cells loaded with tumor antigens constitute a powerful platform for stimulating cellular immunity. Our previous studies suggested that vaccination with an anti-idiotype antibody 3H1, which mimics a specific epitope of carcinoembryonic antigen (CEA), has the potential to break immune tolerance to CEA and induce anti-CEA antibody as well as CEA-specific CD4(+) T-helper responses in colon cancer patients as well as in mice transgenic for human CEA. Here, we have combined the anti-idiotype 3H1 with the CTL peptides of CEA to augment both T-helper and CTL responses in a clinically relevant mouse model, which is transgenic for both CEA and HLA-A2. We have evaluated the potential of two different HLA-A2-restricted epitopes of CEA pulsed into dendritic cells in a therapeutic setting. The overall immune responses and survival were enhanced in groups of mice immunized with agonist peptide for CEA(691) (YMIGMLVGV)-pulsed dendritic cells or CAP1-6D (YLSGADLNL, agonist peptide for CAP-1)-pulsed dendritic cells. Mice immunized with peptide-pulsed dendritic cells along with 3H1-pulsed dendritic cells resulted in significant increase in survival compared with mice immunized with peptide-pulsed dendritic cells alone (P < 0.02). IFN-gamma ELISPOT and (51)Cr-release assays showed that HLA-A2-restricted, CEA-specific CTL responses were augmented by combined dendritic cell vaccinations. The combined vaccination strategy resulted in increased antigen-specific proliferation of splenocytes and secretion of Th1 cytokines by CD4(+) T cells that correlated with increased survival. These results suggest the potential use of this vaccination strategy for future clinical applications.

Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N–A1 domains of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N–A1 to test this hypothesis. The N–A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His 6-tag) in the yeast Pichia pastoris. His 6-tagged N–A1 was captured from the supernatant by batch purification with copper-loaded Streamline™ Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N–A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N–A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His 6-tag. These were separated by concanavalin A chromatography followed by HiTrap™ IMAC. The procedure resulted in single-band-purity, mannose-free N–A1. The binding interaction of MFE-23 to N–A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 × 10 −9 M for the P. pastoris expressed, native N–A1, and 5.33 × 10 −9 M for the Endo H-treated N–A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N–A1.

CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice.

A murine monoclonal anti-idiotype (Id) antibody, 3H1 has been developed and characterized previously. Anti-Id 3H1 mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate antigen for CEA. 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colon cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. In this study, we have evaluated the efficacy of 3H1 as a tumor vaccine when admixed with a select CpG ODN 1826 in transgenic mice that express human CEA. The vaccine potential of 3H1 was also assessed in the presence of another widely used adjuvant, QS-21. 3H1 coupled to keyhole limpet hemocyanin (KLH) and mixed with Freund's adjuvant (FA) was used as a gold standard in this system. 3H1 vaccination with different adjuvants induced both humoral and cellular anti-3H1, as well as anti-CEA immunity in CEA transgenic mice. The immune sera could lyse CEA-transfected murine colon carcinoma cells, C15 effectively in an antibody-dependent cellular cytotoxicity assay. The anti-CEA antibody responses were somewhat comparable in each adjuvant-treated group of mice, whereas cellular immune responses were significantly greater when CpG was used as an adjuvant. Splenocytes obtained from 3H1-CpG-immunized mice showed an increased proliferative CD4(+) Th1-type T-cell response when stimulated in vitro with 3H1 or CEA and secreted elevated levels of Th1 cytokines (IL-2, IFN-gamma). This vaccine also induced MHC class I antigen-restricted CD8(+) T-cell responses. In a solid tumor model, C15 tumor growth was significantly inhibited by 3H1 vaccinations. In 3H1-CpG-vaccinated mice, the duration of survival was, however, longer compared to the 3H1-QS21-vaccinated mice. These findings suggest that 3H1-CpG vaccinations can break peripheral tolerance to CEA and induce protective antitumor immunity in this murine model transgenic for human CEA.

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers

BackgroundHuman monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.MethodsXenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.ResultsFlow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.ConclusionOur human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.

Immunostimulatory CpG oligonucleotides enhance the immune response of anti-idiotype vaccine that mimics carcinoembryonic antigen.

We have developed and characterized a monoclonal anti-idiotype (Id) antibody, designated 3H1, which mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate for CEA. Anti-Id 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colorectal cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Immunostimulatory oliogonucleotides containing the unmethylated CpG motif (CpG ODN) are potent inducers of both innate and adaptive immunity and can serve as suitable vaccine adjuvants. In this study, using the CEA-transduced MC-38 murine colon carcinoma model in syngeneic C57BL/6 mice, we assessed whether a select CpG ODN (1826) can function as immune adjuvant in immunization of mice with anti-Id 3H1. Complete Freund's adjuvant (FA) was used as a gold standard in this system. A single immunization of 3H1 mixed with CpG ODN 1826 was sufficient to induce measurable anti-CEA immunity in naïve mice. However, 3 immunizations every other week were necessary to obtain and sustain peak immune reactivity over a long period of time. With FA and 3H1, single immunization was ineffective and multiple immunizations (5 to 6) were needed to achieve and sustain peak immunity. Anti-CEA antibody reactivity was comparable in both groups, but cellular immune reactivity as measured by immune splenic lymphocyte T cell proliferation and cytoxicity assay was slightly higher in the CpG ODN group. Mice immunized with 3H1 and either CpG ODN or FA were protected from challenge by lethal doses of MC-38-CEA cells. However, the degree of protection was slightly higher and the duration of survival was somewhat longer in the group of mice treated with 3H1 plus CpG ODN. Thus, CpG ODN 1826 was faster than FA in increasing anti-tumor immunity induced by anti-Id 3H1 immunization in this prophylactic model.

Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review.

To describe the features of carcinoembryonic antigen (CEA) that are important for its use in vaccination approaches and review the clinical experience with therapeutic vaccines targeting CEA. A PubMed search was performed on CEA, along with various qualifiers such as cancer vaccines, epitopes, and function. Relevant articles were reviewed. CEA is a member of the immunoglobulin supergene family and may play a role in tumorigenesis. CEA protein is processed and presented on major histocompatibility complex (MHC) proteins for multiple alleles, including HLA A2, A3, and A24. T lymphocytes from healthy volunteers and cancer patients can recognize the processed epitopes of CEA and can become activated to lyse CEA-expressing tumors. Therapeutic vaccination approaches that have targeted CEA include vaccination with recombinant CEA protein, CEA anti-idiotype antibodies, and dendritic cells pulsed with agonist epitopes of CEA. Humoral responses have predominantly been induced with the first two approaches, whereas CD4 and CD8 responses, disease stabilization, and even objective clinical responses have been seen with the dendritic cell approach. Recently, CEA-poxvirus vectors encoding CEA and costimulatory molecules such as B7.1 have been shown to be safe and to induce increases in the frequency of T-cell precursors that recognize processed epitopes of CEA presented on MHC class 1 molecules. Disease stabilization has been seen in up to 37% of patients treated with these vaccines. Tolerance to CEA in patients with cancer can be overcome with several different vaccination approaches, and such vaccinations are safe and immunologically active. Poxvirus-based vaccines can reproducibly generate T-cell responses to CEA and to tumors expressing CEA. Clinical activity has been seen with poxvirus or dendritic cell approaches. Other approaches are also being explored.

The carcinoembryonic antigen (CEA) family (CD66) expressed in melanocytic naevi is not expressed in blue naevuscell naevi in dendritic type.

Although sporadic reports have regarded the expression of the carcinoembryonic antigen (CEA) family in melanoma, there has been no information about the expression in precursor lesions of melanoma such as melanocytic naevi and blue naevi. The expression was immunohistochemically studied in frozen biopsy specimens of 45 acquired and 16 congenital melanocytic naevi and 20 blue naevi, using a panel of monoclonal and polyclonal antibodies that recognize different epitopes of CEA and related molecules. Members of the CEA glycoprotein family were strongly expressed in all of the subtypes of melanocytic naevus. A reduced expression of the CEA glycoproteins with increased dermal depth or acquisition of a spindled morphology of naevus cells was apparent. The expression was not seen in the present blue naevi and normal epidermal melanocytes. Although the significance of the expression was not clarified, this report has clearly demonstrated that the CEA family is strongly expressed in melanocytic naevi and immunoreactivity is divergent between melanocytic naevi and blue naevi and between dermal naevus cell types, suggesting that the expression may be altered with architectural changes in the melanocyte-lineage cells.

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA).

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.

Identification of HLA‐A24 epitope peptides of carcinoembryonic antigen which induce tumor‐reactive cytotoxic T lymphocyte

Carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy. HLA-A24 is the most frequent allele among Japanese and is also frequently present in Asians and Caucasians. We tested CEA-encoded HLA-A24 binding peptides for their capacity to elicit anti-tumor cytotoxic T lymphocytes (CTL) in vitro. For this purpose, we used CD8+ T lymphocytes from peripheral blood mononuclear cells (PBMC) of a healthy donor and autologous peptide-pulsed dendritic cells as antigen-presenting cells. This approach enabled us to identify 2 peptides, QYSWFVNGTF and TYACFVSNL, which were capable of eliciting CTL lines that lysed tumor cells expressing HLA-A24 and CEA. The cytotoxicity to tumor cells by the CTL lines was antigen-specific since it was inhibited by peptide-pulsed cold target cells as well as by anti-class I major histocompatibility complex (MHC) and anti-CD3 monoclonal antibodies (MAbs). The antigen specificity of the 2 CTL lines was examined using several tumor cell lines of various origins and for their peptide-dose responses. The identification of these novel CEA epitopes for CTL offers the opportunity to design and develop epitope-based immunotherapeutic approaches for treating HLA-A24+ patients with tumors that express CEA. Int. J. Cancer 80:92–97, 1999. © 1999 Wiley-Liss, Inc.

Identification of HLA-A24 epitope peptides of carcinoembryonic antigen which induce tumor-reactive cytotoxic T lymphocyte.

Carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy. HLA-A24 is the most frequent allele among Japanese and is also frequently present in Asians and Caucasians. We tested CEA-encoded HLA-A24 binding peptides for their capacity to elicit anti-tumor cytotoxic T lymphocytes (CTL) in vitro. For this purpose, we used CD8+ T lymphocytes from peripheral blood mononuclear cells (PBMC) of a healthy donor and autologous peptide-pulsed dendritic cells as antigen-presenting cells. This approach enabled us to identify 2 peptides, QYSWFVNGTF and TYACFVSNL, which were capable of eliciting CTL lines that lysed tumor cells expressing HLA-A24 and CEA. The cytotoxicity to tumor cells by the CTL lines was antigen-specific since it was inhibited by peptide-pulsed cold target cells as well as by anti-class I major histocompatibility complex (MHC) and anti-CD3 monoclonal antibodies (MAbs). The antigen specificity of the 2 CTL lines was examined using several tumor cell lines of various origins and for their peptide-dose responses. The identification of these novel CEA epitopes for CTL offers the opportunity to design and develop epitope-based immunotherapeutic approaches for treating HLA-A24+ patients with tumors that express CEA.

Immunohistochemical demonstration of carcinoembryonic antigen and related antigens in various cutaneous keratinous neoplasms and verruca vulgaris.

Carcinoembryonic antigen (CEA), which is a well-known marker for the normal sweat gland apparatus and its neoplasms in the skin, was recently demonstrated in sebaceous neoplasms. The aim of this study was to examine the expression of CEA and related antigens in the other cutaneous keratinous neoplasms and verruca vulgaris. Normal adult skin, squamous cell carcinoma (SCC), senile keratosis, Bowen's disease, basal cell carcinoma (BCC), seborrhoeic keratosis and verruca vulgaris were stained immunohistochemically with a panel of monoclonal and polyclonal antibodies that recognize different epitopes of CEA and related molecules. Localization of the antigens was compared with that of involucrin and proliferating cell nuclear antigen. The strongest expression of CEA-related antigens, other than non-specific cross-reacting antigen (NCA) -50/90, was seen in SCC and verruca vulgaris, while no detectable expression was seen in BCC. Senile keratosis, Bowen's disease and seborrhoeic keratosis showed the predominance of the CEA-related antigens over CEA weakly expressed. Strong expression of both CEA and NCA-50/90 was seen only in SCC. All the expressions were limited to the cells situated in the upper epidermal cell layers of the tumours, at the centre of tumour islands in SCC and along the pseudohorn cysts in seborrhoeic keratosis, where involucrin was coexpressed. We suggest that CEA and related antigens are not only markers for sweat gland differentiation in the skin, as currently accepted, but are also expressed in various cutaneous keratinous neoplasms and verruca vulgaris. The expression may correlate with the terminal differentiation of the tumour cells, the strong coexpression of CEA and NCA-50/90 may correlate with the malignant potential of the tumour types, and the mechanisms that control the expression of CEA and related antigens in the neoplasms may be similar to those operative in verruca vulgaris.

Restricted epitope of carcinoembryonic antigen (rCEA) may be a good marker for detecting precancerous lesions and early stage of colonic and gastric carcinomas

CEA is a member of a rather large family of glycoproteins found in a variety of normal tissues, including colon and gastric mucosa. Therefore, the clinical application of CEA detection in GI tissue sections for cancer diagnosis are limited. In this study, we report the normal and pathological GI tissues reactivity to a monoclonal antibody COL-l, which only reacts the restricted CEA antigen epitope (rCEA) but not cross reacts with nonspecific crossreacting antigens (NCA), including normal colorectal and gastric mucosa. MATERIALS and METHOD: All specimens were formalin fixed, parhffln embedded tissues obtained from surgical resection or biopsy. There are 180 cases of colonic tissues, including normal colonic mucosa, remote colonic mucosa at least 6 cm away from the cancer site. colorectal cancer and colonic polyps. 140 cases of gastric tissues, including normal gastric mucosa, chronic superficial gastritis, Chronic Atrophic Gastritis (CAG), with or without Intestinal Metoplasia (IM) or Dysplasia (DYS), and gastric carcinoma. LAB-SA (SP) method was applied for immunostaining. RESULTS:

Gene therapy of cancer

Gene therapy of cancer