Soluble Carcinoembryonic Antigen Research Articles

Abstract 3637: Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and is the second leading cause of cancer death in the United States. Carcinoembryonic antigen (CEA) is a biomarker commonly used in the management of CRC. It is typically present at low levels in the blood of healthy adults but is elevated in CRC samples and some other cancers. CEA is most commonly used to monitor treatment efficacy and as a critical part of post-treatment surveillance for residual disease, recurrence, and/or progression/metastasis. The CRC recurrence rate averages >25%, and liver metastasis occurs in 25-30% of cases. Improved CEA assays could enable earlier determination of treatment failure, detection of lower levels of minimal residual disease (MRD) after treatment, or earlier detection of recurrence. CEA is a GPI-linked membrane protein secreted from CRC cells mainly by enzymatic cleavage of its GPI anchor. However, we have identified alternative secretion of CEA in association with extracellular vesicles (EVs). We hypothesized that the soluble CEA produced by canonical shedding and the alternatively secreted CEA may provide distinct information about the tumor or its microenvironment and serve as independent but complementary biomarkers of CRC. We developed ultrasensitive, multi-marker electrochemiluminescence (ECL) immunoassays for measuring intact EVs presenting CEA, CD73, or both in human plasma. We observed that plasma levels of CEA+EVs and CD73+EVs were elevated in some late-stage CRC samples compared to controls. CEA and CD73 double-positive EV (CEA+CD73+EVs) levels were also elevated in the same samples, exhibiting a 3-fold improved separation of these samples with CRC from controls, compared to detection of CEA+EVs. Additionally, approximately 25% of stage IV CRC samples had elevated CEA+EV and CEA+CD73+EV levels while exhibiting low soluble CEA. We confirmed this result in an independent sample set that included samples across all CRC stages, and also identified a few earlier-stage samples that exhibited high CEA+EVs and CEA+CD73+EVs without elevated soluble CEA. Since CEA+CD73+EVs and soluble CEA assays identified different subsets of samples, we combined the two assays into a single classifier for late stage CRC and controls, yielding improved receiver operator characteristic (ROC) with area under the curve (AUC) of 0.975 versus 0.927 for the standard soluble CEA assay or 0.788 for CEA+CD73+EVs alone. These results suggest that assaying EVs presenting multiple surface markers can increase specificity for the detection of tumor-derived EVs relative to a single marker and that assaying both soluble and EV-associated forms of CEA in plasma may improve the value of CEA measurement in CRC. Citation Format: Lucie Hebert, Misk Al-Ameen, Talis Spriggs, Collin Nelson, Evan A. Gizzie, Jeffrey L. Franklin, Robert J. Coffey, David A. Routenberg, Jacob N. Wohlstadter. Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3637.

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.

Impact of Different Selectivity between Soluble and Membrane-bound Forms of Carcinoembryonic Antigen (CEA) on the Target-mediated Disposition of Anti-CEA Monoclonal Antibodies.

Carcinoembryonic antigen (CEA) is a tumor-specific antigen overexpressed in multiple cancers. CEA is expressed as a membrane protein, a part of which is cleaved from the cell membrane and secreted into blood. The soluble form of CEA (sCEA) has been shown to accelerate the clearance of anti-CEA antibody, which limits the antibody distribution in the tumor. To overcome this issue, we developed an anti-CEA monoclonal antibody, 15-1-32, which shows a strong affinity for membrane-bound CEA (mCEA) and relatively weak affinity for sCEA. In this study, we compared the effect of sCEA on the pharmacokinetics of 15-1-32 in mice with that of another anti-CEA monoclonal antibody, labetuzumab, showing less selectivity to mCEA than 15-1-32. As expected, the effect of sCEA on the serum concentration of 15-1-32 was much smaller than that of labetuzumab. The decrease in the area under the curve (AUC) of serum concentration was 22.5% for 15-1-32 when it was coadministered with sCEA, while that of labetuzumab was 79.9%. We also compared the pharmacokinetics of these two antibodies in CEA-positive tumor-bearing mice. The AUCs of 15-1-32 and labetuzumab were decreased in tumor-bearing mice compared with non-tumor-bearing mice to a similar extent (approximately 40% decrease). These results suggested that mCEA also contributes to the clearance of anti-CEA antibodies in CEA-positive tumor-bearing mice. Although the increased selectivity to mCEA minimized the effect of sCEA on the pharmacokinetics of 15-1-32, it may be insufficient to improve the pharmacokinetics in CEA-positive cancer patients. SIGNIFICANCE STATEMENT: Because previous studies reported the rapid clearance of anti-CEA antibodies mediated by soluble CEA, we obtained a monoclonal antibody, 15-1-32, selective to membrane-bound CEA and evaluated the effects of CEA on its pharmacokinetics. Although the effect of soluble CEA on the serum concentration of 15-1-32 was very small, the clearance of 15-1-32 in CEA-positive tumor-bearing mice was still rapid, suggesting membrane-bound CEA also contributes to the clearance of anti-CEA antibodies. These results indicated that increasing selectivity to membrane-bound CEA is not enough to improve the pharmacokinetics of anti-CEA antibody.

Use of cholesterol and soluble tumour markers CEA and syndecan-2 in pleural effusions in cases of inconclusive cytology

AimsIn order to improve diagnostics in pleural effusions, additional value of effusion cholesterol, carcinoembryonic antigen (CEA) and syndecan-2 assays to cytology was studied.MethodsBiomarkers were measured in effusion supernatants from 247...

Impact of CEA-targeting Nanoparticles for Drug Delivery in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world, mainly owing to distant metastasis events. Developing targeted strategies to treat and follow individuals in more developed stages is needed. The carcinoembryonic antigen (CEA) is a cell surface-overexpressed glycoprotein in most CRC patients, and the evaluation of its serum levels is recommended in the clinic. These reasons motivated the production of CEA-targeted nanotechnologies for monitorization of CRC progression, but only a few centers have reported their use for drug delivery. The cellular internalization of CEA-linked nanosystems occurs by the natural recycling of the CEA itself, enabling longer retention and sustained release of the cargo. The functionalization of nanoparticles with lower affinity ligands for CEA is possibly the best choice to avoid their binding to the soluble CEA. Here, we also highlight the use of nanoparticles made of poly(lactic-co-glycolic acid) (PLGA) polymer, a well known material, owing to its biocompatibility and low toxicity. This work offers support to the contribution of antibody fragment-functionalized nanoparticles as promising high affinity molecules to decorate nanosystems. The linkers and conjugation chemistries chosen for ligand-nanoparticle coupling will be addressed herein as an elements essential to the modulation of nanosystem features. This review, to our knowledge, is the first that focuses on CEA-targeted nanotechnologies to serve colorectal cancer therapy and monitorization.

Novel anticarcinoembryonic antigen antibody-drug conjugate has antitumor activity in the existence of soluble antigen.

Carcinoembryonic antigen (CEA) is a classic tumor‐specific antigen that is overexpressed in several cancers, including gastric cancer. Although some anti‐CEA antibodies have been tested, to the best of our knowledge, there are currently no clinically approved anti‐CEA antibody therapies. Because of this, we have created the novel anti‐CEA antibody, 15‐1‐32, which exhibits stronger binding to membrane‐bound CEA on cancer cells than existing anti‐CEA antibodies. 15‐1‐32 also shows poor affinity for soluble CEA; thus, the binding activity of 15‐1‐32 to membrane‐bound CEA is not influenced by soluble CEA. In addition, we constructed a 15‐1‐32‐monomethyl auristatin E conjugate (15‐1‐32‐vcMMAE) to improve the therapeutic efficacy of 15‐1‐32. 15‐1‐32‐vcMMAE showed enhanced antitumor activity against gastric cancer cell lines. Unlike with existing anti‐CEA antibody therapies, antitumor activity of 15‐1‐32‐vcMMAE was retained in the presence of high concentrations of soluble CEA.

Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating carcinoembryonic antigen in mice

Immunity against cancer is impeded by local mechanisms promoting development of tumor-specific T cell tolerance, such as regulatory T cells, myeloid-derived suppressor cells, or immunosuppressive factors in the tumor microenvironment. The release of soluble antigens, such as carcinoembryonic antigen (CEA) from colorectal carcinoma (CRC) cells, has been investigated for diagnostic purposes, but not for its immunological consequences. Here, we address the question of whether soluble CEA influences tumor-specific immunity. Mice were injected with soluble CEA protein, and CEA-specific CD8 T cells were analyzed for their phenotype and functionality by means of restimulation ex vivo or antitumor efficacy in vivo. We furthermore characterized the CD8 T cell population in peripheral blood mononuclear cell (PBMCs) from healthy donors and colorectal carcinoma patients. In mice, circulating CEA was preferentially taken up in a mannose receptor-dependent manner and cross-presented by liver sinusoidal endothelial cells, but not dendritic cells, to CD8 T cells. Such systemically circulating CEA promoted tolerization of CEA-specific CD8 T cells in the endogenous T cell repertoire through the coinhibitory molecule B7H1. These CD8 T cells were not deleted but were rendered nonresponsive to antigen-specific stimulation and failed to control growth of CEA-expressing tumor cells. These nonresponsive CD8 T cells were phenotypically similar to central memory T cells being CD44(high) CD62L(high) CD25(neg) . We found T cells with a similar phenotype in PBMCs of healthy donors and at increased frequency also in patients with colorectal carcinoma. Our results provide evidence for the existence of an unrecognized tumor immune escape involving cross-presentation of systemically circulating tumor antigens that may influence immunotherapy of cancer.

T Cells That Target Carcinoembryonic Antigen Eradicate Orthotopic Pancreatic Carcinomas Without Inducing Autoimmune Colitis in Mice

New treatment approaches are needed for patients with pancreatic adenocarcinoma. Carcinoembryonic antigen (CEA) is highly expressed on the surface of pancreatic adenocarcinoma cells; we investigated the effects of cytolytic T cells that recognize CEA in a mouse model of pancreatic carcinoma. Immune-competent mice that expressed the CEA transgene (CEAtg) in the intestinal and pulmonary tracts were given intrapancreatic injections of Panc02 CEA(+) cells (express CEA and click beetle luciferase) and tumors were grown for 10 days. Mice were then given single intravenous injections of T cells engineered to express a chimeric antigen receptor (CAR) with high specificity, but moderate affinity, for CEA and a luminescence marker. Injection of the anti-CEA CAR T cells reduced the size of pancreatic tumors to below the limit of detection in all mice and produced long-term tumor eradication in 67% of mice. T cells also eradicated CEA(+) fibrosarcoma cells injected 45 days later. Bioluminescence imaging revealed the accumulation and persistence of the T cells at the tumor site. The efficacy of the T cells did not require lymphodepletion and was not reduced by soluble CEA. Mice developed some noninflammatory infiltrations of CAR(+) T cells in intestine and lung, but there was no evidence of destruction of CEA(+) healthy tissues. Injection of T cells that target CEA can eradicate tumors grown from CEA(+) pancreatic carcinoma cells in the pancreas of CEAtg mice without autoimmune effects.

In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT

Nanobodies are a novel type of immunoglobulinlike, antigen-binding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and (99m)Tc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEA-transfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with (99m)Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected (99m)Tc-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. This study shows the potency of antigen-binding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.

Abstract 5625: In vitro and in vivo pharmacology of MEDI-565 (MT111), a novel CEA/CD3-bispecific single-chain BiTE antibody in development for the treatment of gastrointestinal adenocarcinomas

Abstract MEDI-565 (MT111) is a novel bispecific single-chain antibody of the BiTE (Bispecific T cell engager) class that transiently links carcinoembryonic antigen (CEA) on cancer cells with human CD3 on T cells. MEDI-565 specifically binds to human CEA with an affinity of 8.5 nM but not to any other member of the CEACAM family. In targeted expression studies on cryopreserved and formalin-fixed paraffin-embedded sections, MEDI-565 demonstrated membrane-localized binding specifically to epithelial cells in human cancerous tissues. The highest prevalence of MEDI-565 binding (∼90%) was in adenocarcinomas of gastrointestinal origin. In in vitro killing assays, MEDI-565 recruited T cells via the CD3 antigen (affinity of 310 nM) in a process that required concomitant binding to CEA positive tumor cells for T cell activation. As a consequence, T cells expanded, increased cell surface expression of the activation markers CD69 and CD25, and released perforin and granzymes. The release of cytotoxic granule content led to a Ca2+-dependent activation of pro-caspases and subsequent apoptosis of CEA-expressing tumor cells. Efficient target cell lysis by MEDI-565 was predominantly mediated by CD8+ T cells and occurred within a wide range of effector-to-target ratios (80:1 to 5:1) for T cells derived from various human donors. BiTE mediated killing was not affected by the presence of soluble CEA (≤5 µg/mL). The in vivo activity of MEDI-565 was investigated in subcutaneous xenograft models using immunocompromised SCID mice inoculated with mixtures of human T cells and human cancer cell lines. The antibody was eliminated from the serum with a half-life of a few hours following a single intravenous (IV) or subcutaneous (SC) injection. Despite a relatively short serum half-life, daily IV or SC bolus treatments over 5 days provided sufficient levels of exposure to inhibit the growth of CEA-positive tumors in a dose-dependent manner. Inhibition of growth was observed for tumors of different tissue origins and was dependent on the presence of human T cells and CEA expression by tumor cells. There were no MEDI-565-related in-life observations following treatment. These studies demonstrated that MEDI-565 has potent and selective anti-cancer activity in vitro and in vivo and provides evidence that MEDI-565 may be an effective monotherapy to treat CEA-expressing malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5625.

AN ELISA FOR QUANTIFICATION OF T84.66, A MONOCLONAL ANTI-CEA ANTIBODY, IN MOUSE PLASMA

T84.66 is a monoclonal antibody with high affinity and specificity for tumor-associated carcinoembryonic antigen (CEA). In this work, we have developed an enzyme linked immunosorbent assay to determine T84.66 concentrations in mouse plasma. The assay was validated with respect to precision and accuracy by evaluating the recovery of T84.66 from mouse plasma. The working range of the assay is 25–200 ng/mL, and the limit of quantification is 2.5 μg/mL. Intra-assay recoveries ranged from 90.6 to 97.4%, and intra-assay precision reported as the percent coefficient of variation (CV%), ranged from 4.58 to 12.6%. Inter-assay recoveries were between 92.6 to 98.1% and the CV% ranged from 4.9–6.5%. The assay was tested for possible interference from soluble CEA. Soluble CEA, at concentrations up to 5 ng/mL, did not influence the recovery of T84.66. The assay was applied to study the pharmacokinetics of T84.66 in athymic Foxnu mice.

Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T-cell killing mediated by CEA/CD3-bispecific T-cell-engaging BiTE antibody

Background:Novel technologies to redirect T-cell killing against cancer cells are emerging. We hypothesised that metastatic human colorectal cancer (CRC) previously treated with conventional chemotherapy would be sensitive to T-cell killing mediated by carcinoembryonic antigen (CEA)/CD3-bispecific T-cell-engaging BiTE antibody (MEDI-565).Methods:We analysed proliferation and lysis of CEA-positive (CEA+) CRC specimens that had survived previous systemic chemotherapy and biologic therapy to determine whether they could be killed by patient T cells engaged by MEDI-565 in vitro.Results:At low concentrations (0.1–1 ng ml−1), MEDI-565+ T cells caused reduced proliferation and enhanced apoptosis of CEA+ human CRC specimens. High levels of soluble CEA did not impair killing by redirected T cells and there was no increase in resistance to T-cell killing despite multiple rounds of exposure.Conclusions:This study shows for the first time that metastatic CRC specimens derived from patients previously treated with conventional chemotherapy can be lysed by patient T cells. Clinical testing of cancer immunotherapies, such as MEDI-565 that result in exposure of tumours to large numbers of T cells, is warranted.

Carcinoembryonic Antigen (CEA) Regulates Tumor-Angiogenesis in Colon Carcinomas.

Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation and secretion of soluble CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can affect tumor cell behavior including the inhibition of cell differentiation and apoptosis. However, any functional effects on angiogenic endothelial cell behavior are so far unknown. In the present work we found that in endothelial cells exogenous CEA led to a time and dose dependent increase in ERK phosphorylation, which was inhibited by the specific MEK inhibitor U0126. Thereby, the observed CEA effect was comparable in time and intense with the canonical angiogenic growth factor VEGF. The CEA-induced ERK phosphorylation was not affected by the blockage of VEGFR-2 / flk-1 using a specific inhibiting peptide (CBO-P11), which indicates a VEGF-independent mechanism. Furthermore, co-stimulation of endothelial cells with VEGF and CEA shows synergistic effects on ERK phosphorylation. While in endothelial cells no endogenous expression of CEA is detected, its putative receptor, the CEA receptor (CEAR), is highly expressed as shown by immunohistochemical staining of paraffin-embedded colon carcinoma sections as well as in biochemical analyses. When an activating antibody against CEAR was used, CEA-induced ERK phosphorylation was mimicked, while downregulation of CEAR by siRNA diminished CEA-induced signal transduction, significantly. To test a biological relevance of our findings, we first measured endothelial cell proliferation: CEA led to a dose dependent increase in endothelial cell proliferation in vitro, which again revealed a synergistic effect with VEGF. Thereby, CEA-induced endothelial cell proliferation was again independent of VEGFR-2 / flk-1. A biological role of CEA in tumor-angiogenesis was reflected by an in vivo model using CEA Mimotope immunized BALB/c mice, which were transplanted with MethA/CEA overexpressing tumor cells. Immunohistological analyses of these tumors revealed a significantly reduced vascular density, which was accompanied with diminished tumor growth. Our data provide first evidence of CEA as a novel pro-angiogenic activator of endothelial cells, which results in an increase in endothelial cell proliferation, independent of VEGFR-2. Furthermore, by targeting CEA in an in vivo mouse model, tumor-angiogenesis was markley reduced, indicating a potential therapeutic target in cancer.

Antitumor activity of chimeric immunoreceptor gene‐modified Tc1 and Th1 cells against autologous carcinoembryonic antigen‐expressing colon cancer cells

To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

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.

Serum tetranectin is an independent prognostic marker in colorectal cancer and weakly correlated with plasma suPAR, plasma PAI‐1 and serum CEA

Soluble tetranectin (TN) was measured preoperatively in serum from 567 patients with primary colorectal cancer and levels were tested for association with prognosis. The prognostic significance of TN was also compared to that of plasminogen-activator inhibitor-1 (PAI-1), urokinase plasminogen activator (uPAR) and carcinoembryonic antigen (CEA). Significantly shorter survival was found for patients with TN levels below a cut-off point of 7.5 mg/l compared to patients with levels above, as illustrated by Kaplan-Meier curves. By Cox analyses, log TN, log soluble uPAR as well as log CEA were found to have an independent prognostic value for survival (log TN: HR = 0.47, 95% CI: 0.29-0.76); log soluble uPAR: HR = 1.65, 95% CI: 1.18-2.31; log CEA: HR = 1.I1, 95% CI: 1.03-1.20). Based on the multivariate model, a patient with a combination of low levels of TN and PAI-1 and elevated levels of soluble uPAR and CEA had a 2.43 increased risk as compared to a patient with median levels of these biochemical markers. Significant correlations were found with Dukes' stages for all the biochemical markers and between the respective biochemical markers. The findings confirm that TN is a strong prognostic factor in patients with colorectal cancer. TN may be valuable as a prognostic variable in future studies evaluating new treatment strategies for colorectal cancer.

Detection of epithelial tumour RNA in the plasma of colon cancer patients is associated with advanced stages and circulating tumour cells.

Although circulating tumour DNA has been detected in patients with different types of cancer, little is known of free RNA in cancer patients. We investigated the presence of RNA from epithelial tumours in plasma from patients with colorectal carcinomas, and its correlation with tumour characteristics and circulating tumour cells. beta-actin mRNA was analysed to assess the viability of plasma RNA in samples from 53 patients with colonic cancer and 25 controls. Subsequently, nested primers were used to detect the presence of cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) RNA in the same samples. Nine clinicopathological parameters were studied to correlate the molecular and clinical parameters. Additionally, we investigated for micrometastases in blood in 18 of these patients and in 10 of the controls samples. All samples had detectable quantities of beta-actin RNA. In the controls, one case (4%) was positive for CEA and five (20%) for CK19 RNA; of the 53 patients, 17 cases (32%) were positive for CEA and 39 (73.6%) for CK19 RNA. This was statistically significant (p=0.000001). Advanced stages (p=0.03) and soluble CEA status (p=0.03) were associated with the presence of CEA, CK19, or both RNAs in plasma. Lymph node metastases (p=0.06) and vascular invasion (p=0.07) were almost significant. On the basis of these results, we examined the possible presence of micrometastases in blood in several of these patients. The presence of plasma tumour RNA was found to be associated with circulating tumour cells in blood (p=0.04). Epithelial tumour RNA is detectable in plasma from colon cancer patients. This molecular event is associated with advanced stages and circulating tumour cells. Our results could offer new approaches in the diagnosis and monitoring of colon cancer.

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo.

The gene transfer of tumor-specific chimeric immunoglobulin T-cell receptors (cIgTCRs) combining antibody-like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human natural killer (NK) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the human carcinoembryonic antigen (CEA). The cIgTCR was constructed of a CEA-specific humanized single-chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 zeta chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR-expressing cells were enriched by immunoaffinity purification. cIgTCR-expressing YT cells specifically lysed CEA(+) colon carcinoma cell lines, which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. Receptor gene-modified YT cells retained their CEA-specific cytolytic activity after gamma-irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into NOD/SCID mice. This gene-modified NK cell line available in unlimited source might be useful in clinical immunotherapy of CEA(+) cancer.

Primary polyclonal human T lymphocytes targeted to carcino-embryonic antigens and neural cell adhesion molecule tumor antigens by CD3zeta-based chimeric immune receptors.

Antigen-specific T lymphocytes are attractive as potential anticancer agents. The generation of large numbers of antigen-specific T cells is possible through the use of gene therapy to express targeting receptors on the T lymphocyte. Activated T lymphocytes were transduced to express carcino-embryonic antigen or neural cell adhesion molecule targeted CD3zeta chimeric immune receptors. The chimeric receptors were expressed as homodimers and also as heterodimers with the native CD3zeta. T lymphocyte populations were expanded in the absence of selection for the modified cells and were shown to produce cytokines when cultured in the presence of immobilized purified protein antigen. These lymphocytes also responded by cytokine production and cytolytic activity when challenged with tumor-cell lines expressing the antigen recognized by the chimeric immune receptor. The cytolytic activity appears to be largely perforin mediated. Furthermore, soluble carcino-embryonic antigen did not interfere with the functional activity of the carcino-embryonic antigen-targeted lymphocytes. Long-term (5-day) stimulation of the modified lymphocytes by protein antigen resulted in reduced viability similar to that induced by anti-CD3 antibodies alone. Viability was improved by a costimulatory signal indicating that such signals may be vital in the maintenance of long-term functional activity of receptor modified T lymphocytes.

Morphometric analysis of liver macrophages in patients with colorectal liver metastasis.

Kupffer cells, residential liver macrophages, have binding sites for carcinoembryonic antigen (CEA), but their role in metastatic liver tumor progression has not been well addressed clinically. Liver macrophages were analyzed morphometrically and their relationship with CEA and tumor microvessel density (MVD) was examined in 71 patients who underwent macroscopic curative hepatectomy for metastatic liver tumors from colorectal cancer. In paraffin-embedded sections, MVD was evaluated by CD34-positive cell counts, liver macrophages visualized using anti PG-M1 (CD68) antibody were analyzed, and membrane-bound CEA was assessed by immunoreactivity of tumor cells for CEA. The area of liver macrophages in peritumoral regions (43.0 +/- 11.6 microm2) was significantly larger than that in non-tumor regions (25.2 +/- 24.2 microm2) (P < 0.0001). The liver macrophage density in peritumoral regions was 154 +/- 49 per field, and was significantly higher than in non-tumor regions (74 +/- 24 per field) (P < 0.001). The density and the area of liver macrophages had no correlation with serum CEA levels and the degree of tumor CEA expression. A weak positive correlation was observed between MVD and liver macrophage density (P = 0.0104, Spearman rank correlation coefficient = 0.31). Cox multivariate regression analysis showed that MVD greater than 50 (P = 0.0233, hazard ratio = 2.463), and the area of liver macrophages larger than 40.9 microm2 (P = 0.0485, hazard ratio = 2.127), were significant independent prognostic factors. The present morphometric analysis suggests that the liver macrophages are accumulated and activated in peritumoral regions in patients with colorectal liver metastasis and this accumulation and activation of liver macrophages, with which soluble or membrane-bound CEA might not be associated, are related with patients' poor prognosis.