Immunological heterogeneity of carcinoembryonic antigen: antigenic determinants on carcinoembryonic antigen distinguished by monoclonal antibodies

Murine monoclonal antibodies against carcinoembryonic antigen (CEA) derived from a colonic tumor were analyzed by radioimmunoassay for reactivity with CEA and the CEA-related antigens, meconium antigen (MA) and nonspecific cross-reacting antigen. Antibody-antigen binding profiles revealed three classes of hybridomas. The Class I antibody, NP-1, recognized an epitope shared among all three antigens, and its affinity for CEA and MA was high but low for nonspecific cross-reacting antigen. The Class II antibodies reacted with sites shared between CEA and MA, while those of the Class III type only bound CEA. The Class III antibody, NP-4, bound less than 50% of the CEA molecules recognized by goat specific anti-CEA antibody and the other classes of monoclonal antibodies. Two Class II antibodies, NP-2 and NP-3, bound similar amounts of CEA and MA with moderate but different affinities for CEA. Studies using labeled monoclonal antibodies for CEA epitope blocking revealed that NP-2 and NP-3 recognize two separate epitopes on CEA within the Class II category. Thus, monoclonal antibodies to CEA can differentiate at least four antigenic sites on colonic cancer CEA. One is shared by CEA, MA, and nonspecific cross-reacting antigen; two others are shared by CEA with MA; and a fourth appears specific for a subpopulation of CEA molecules.     

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.     

Syngeneic anti-idiotype monoclonal antibodies to murine anticarcinoembryonic antigen monoclonal antibodies

BALB/c mice were immunized with either NP-3 or NP-4, two anticarcinoembryonic antigen murine monoclonal antibodies. Each animal produced anti-idiotype antibodies to the corresponding immunogen and no cross-reactivity between anti-NP-3 sera and anti-NP-4 sera was detected. Hybridomas were produced from these animals and two IgG1 anti-idiotype monoclonal antibodies were obtained: CM1 specific for NP-3 and CM11 specific for NP-4. CM1 and CM11 recognized determinants located within the antibody-combining site, since each anti-idiotype antibody inhibited the binding between the corresponding idiotype and carcinoembryonic antigens. Using an immunoblotting technique, neither CM1 nor CM11 reacted with isolated heavy or light chains of NP-3 or NP-4, whereas binding was observed with the intact molecule. This observation indicates that CM1 and CM11 are directed against conformational idiotypes resulting from the association of the variable regions of the heavy and light chains. Taken together, these results suggest that CM1 and CM11 might bear internal images of carcinoembryonic antigen epitopes, and that they are potential candidates as idiotype vaccines against colorectal tumors.     

Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.     

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.     

Protein epitopes in carcinoembryonic antigen. Report of the ISOBM TD8 workshop.

To characterize antigenic sites in carcinoembryonic antigen (CEA) further and to investigate whether there are differences between colon tumor CEA and meconium CEA (NCA-2) that can be detected by anti-CEA monoclonal antibodies (MAb), 19 new anti-CEA MAb were analyzed with respect to specificity, epitope reactivity and affinity. Their reactivities were compared with 10 anti-CEA MAb with known CEA-domain binding specificity that have previously been classified into five nonoverlapping epitope groups, GOLD 1-5. Cross-inhibition assays with antigen-coated microtiter plates and immunoradiometric assays were performed in almost all combinations of MAbs, using conventionally purified CEA (domain structure: N-A1B1-A2B2-A3B3-C) from liver metastasis of colorectal carcinomas, recombinant CEA, meconium CEA (NCA-2), truncated forms of CEA and NCA (CEACAM6) as the antigens. The affinity of the MAbs for CEA was also determined. The new MAbs were generally of high affinity and suitable for immunoassays. Three new MAbs were assigned to GOLD epitope group 5 (N-domain binding), 3 MAbs to group 4 (A1B1 domain), 1 to group 3 (A3B3 domain), 3 to group 2 (A2B2 domain) and 3 to group 1 (also the A3B3 domain). Three MAbs formed a separate group related to group 4, they were classified as GOLD 4' (A1B1 domain binding). The remaining 3 MAbs appear to represent new subspecificities with some relationship to GOLD groups 1, 2 or 4, respectively. Five MAbs, all belonging to epitope group 1 and 3, reacted strongly with tumor CEA but only weakly or not at all with meconium CEA, demonstrating that the two products of the CEA gene differ from each other, probably due to different posttranslational modifications.     

An idiotypic replica of carcinoembryonic antigen inducing cellular and humoral responses directed against human colorectal tumours.

A monoclonal anti-idiotypic antibody (anti-Id MAb) 708 (IgG2b), which inhibited the binding of NCRC23 (IgG1) MAb to CEA and prevented radiolabelled CEA from binding to MAb NCRC23, was produced. No recognition of 3 other anti-CEA antibodies, 3 other IgG1 or 2 IgM MAbs was observed with this anti-idiotypic antibody. When an immunoblotting technique was used, 708 anti-Id MAb failed to bind to isolated heavy or light chains of MAb NCRC23, whereas binding was observed with intact antibody. Mouse, rat and human lymphocytes (in vitro) were immunized with 708 anti-Id MAb and the resultant Ab3 antibodies all inhibited binding of labelled 708 anti-Id MAb to MAb NCRC23 and also reacted with CEA, showing that 708 anti-Id MAb induced anti-CEA antibody responses. Similarly, mice immunized with 708 anti-Id MAb could be restimulated in vitro with either CEA or tumour cells expressing CEA which induced specific T-cell proliferative responses. Human tumour-infiltrating lymphocytes isolated from colorectal tumours or peripheral blood T cells from cancer patients were stimulated in vitro with 708 anti-Id MAb or an irrelevant IgG2b antibody. Six days later both sets of lymphocytes were restimulated with CEA, and lymphocytes primed to 708 anti-Id MAb proliferated in response to CEA. These results suggest that 708 anti-Id MAb can act as an idiotypic replica of CEA and stimulate cellular and humoral anti-CEA immune responses. It is therefore of great interest as an idiotypic vaccine against colorectal cancer.     

Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.     

Comparison of therapeutic efficacy and host toxicity of two different 131I-labelled antibodies and their fragments in the GW-39 colonic cancer xenograft model

The in vivo uptake, therapeutic potential, and host toxicity were evaluated for both the intact IgG and F(ab')2 fragment of 2 murine monoclonal antibodies (MAbs) directed against either carcino-embryonic antigen (CEA), called NP-4, or colon-specific antigen-p (CSAp), called Mu-9, in the GW-39 human colorectal carcinoma grown in the hamster cheek pouch. Mu-9 IgG and F(ab')2 were retained longer by the tumor than was the NP-4 IgG or F(ab')2, respectively. Localization of the two antibodies by micro-autoradiography revealed two distinct patterns. Mu-9 was seen densely around whole acini of tumor cells, whereas NP-4 was found around each individual cell, albeit less densely. The anti-tumor effects of 131I-labelled Mu-9 and NP-4 IgG were equal, but the therapeutic effectiveness of Mu-9 F(ab')2 was significantly higher than NP-4 F(ab')2, as measured by change in tumor size. A dose-dependent increase in host toxicity, as measured by change in body weight and change in peripheral white blood cells (p-WBCs), was observed with 0.5 to 3.0 mCi doses of 131I-IgG regardless of the MAb used. A 10-22% loss in body weight lasting 3-7 weeks and a 50-80% loss in pWBCs lasting 6-8 weeks were observed in these animals. In contrast, 3 x 2 mCi doses of 131I-NP-4 or Mu-9 F(ab')2 given at 3-day intervals, a schedule which was equally therapeutic to a single 2-mCi dose of 131I-IgG, resulted in only a 9% loss in body weight and a 50% loss in pWBCs that lasted only 1 week. This was followed by a complete recovery over the next 2-3 weeks. These data suggest that multiple doses of F(ab')2 can be as tumoricidal as a single dose of an intact MAb IgG, but significantly less toxic to the host.     

Anti-idiotype monoclonal antibody carrying the internal image of ganglioside GM3.

Murine anti-idiotype monoclonal antibodies were generated against a human IgM monoclonal antibody (L612) that recognizes ganglioside GM3 on human melanoma. Hybridomas secreting antibodies that bound specifically to L612 were selected by enzyme-linked immunosorbent assay using L612 and three negative control human IgMs, including monoclonal anti-GM2 and anti-GD2 antibodies, as well as purified serum IgM, as antigen sources. GM3-binding inhibition and cell-binding inhibition assays were used to identify seven anti-idiotype monoclonal antibodies that recognized determinants located within the antigen-combining sites of L612. To determine whether these anti-idiotype monoclonal antibodies possessed the internal image of the original antigen, we immunized syngeneic BALB/c mice with one of the anti-idiotype monoclonal antibodies, 4C10, coupled with keyhole limpet hemocyanin. Sera from the immunized mice reacted strongly with an antigen-positive M12 melanoma cell line and with purified GM3. Because L612 detects and kills melanoma tumor cells in vitro and in vivo in the presence of complement without affecting normal tissues, anti-idiotype monoclonal antibodies carrying the internal image of GM3 may be an effective tool for active specific immunotherapy in patients with melanoma.     

Determination of the specificities of monoclonal antibodies recognizing members of the CEA family using a panel of transfectants

Carcinoembryonic antigen (CEA), one of the most clinically important tumor markers, is mainly used in the post-surgical surveillance of patients with colorectal carcinomas. CEA belongs to a large protein family, which includes cross-reacting antigens, e.g., non-specific cross-reacting antigens (NCAs) and biliary glycoprotein (BGP) as well as pregnancy-specific glycoproteins (PSGs). The genes encoding these proteins can be subdivided into the CEA and PSG subgroups. The members of the subgroups share antigenic determinants and show high similarity in amino-acid sequences. Their derived secondary structures show them to belong to the Immunoglobulin superfamily. Due to the close relationship of the members of the CEA subgroup, it is very difficult to distinguish between the individual members with MAbs. Here we have used flow cytometric analysis of transfectants expressing individual members of the CEA subgroup as an alternative approach to determine the specificities of 13 MAbs. This allows us to examine the specificities of these antibodies for members of the CEA family, even of those which have not yet been characterized at the protein level. In addition, binding of the MAbs to NCAs expressed by polymorphonuclear cells (PMN) was tested by Western-blot analysis, immunoprecipitation and flow cytometry. Four antibodies bound exclusively to NCA-50/90 and one MAb (80H3) only to NCA-95. MAb 4/3/17 recognizes CEA and BGP on the surface of transfectants and NCA-160 from granulocytes. We assume that NCA-160 is a product of the BGP gene. On granulocytes, which do not express CEA, MAb 4/3/17 is specific for NCA-160 (BGP). Mutual inhibition of the MAbs binding to NCA-50/90 revealed 3 different epitope groups.     

Monoclonal antibody recognizing a carcinoembryonic antigen epitope differentially expressed in human colonic carcinoma versus normal adult colon tissues

Murine hybridomas were generated against purified carcinoembryonic antigen (CEA), and their monoclonal antibodies (MAb) were assayed for their ability to recognize specific CEA epitopes. One of the MAb, designated 7F, was found to recognize an epitope of CEA that was expressed in human colonic carcinoma tissues but not in normal colonic tissues. When extracts of 13 colonic carcinoma tissues and 9 normal colonic tissues were examined with the Western blot technique using MAb 7F, 11 colonic carcinoma tissues (85%) reacted with 7F, but none of the 9 normal colonic tissues (including many obtained from the areas adjacent to the colonic carcinomas) did. Western blot analysis indicated a Mr 180,000 band in 11 of 13 (85%) colonic carcinoma extracts. The limit of detectability of CEA was 0.5 micrograms/lane. According to immunohistochemical techniques, 16 of 18 (89%) formalin-fixed paraffin-embedded sections of colonic carcinomas reacted with MAb 7F, whereas 9 of 9 (100%) frozen sections of colonic carcinomas reacted with the antibody. Of the 32 paraffin-embedded and frozen sections of normal colonic tissues examined, none showed any reactivity with MAb 7F. MAb 7F did not react with nonspecific cross-reacting antigen either. This antibody has been examined for its usefulness in detecting CEA in suspension and has been found to work both in sandwich enzyme-linked immunosorbent assays and in sandwich radioimmune assays. The detection of CEA in colon tumors but not in normal colonic tissues may be attributed to two possible explanations. It is possible that the level of CEA expression in normal colonic tissue is below the sensitivity of the assays employed. Alternatively, MAb 7F may recognize a "specific" epitope of CEA which was found in colon tumors but not in CEA of normal colonic tissues. At the present time, it is not possible to discern between these two possibilities. Nevertheless, MAb 7F was capable of detecting the differential expression of CEA in colonic carcinomas and, therefore, may be useful for the immunodiagnosis, radioimaging, and immunotherapy of colon cancer.     

Evaluation of chimpanzee antiserum to human carcinoembryonic antigen

An adult chimpanzee (Pan troglodyte) with an endogenous circulating carcinoembryonic antigen (CEA) level of 60 ng/ml was immunized s.c. with human CEA. After 1 year of immunizations, the anti-human CEA antibody titer had plateaued. This chimpanzee antiserum demonstrated high avidity specific recognition of human CEA and showed ionic strength effects for CEA recognition similar to those previously described for goat and baboon anti-CEA antisera. Radioimmunoassay of 93 human plasma samples for CEA content using chimpanzee anti-CEA versus Roche goat anti-CEA antisera gave essentially identical results (R = 0.985). Endogenous CEA in chimpanzee blood was very poorly identified by chimpanzee anti-human CEA antisera compared to Roche goat antisera. Column chromatography of human and chimpanzee CEA in the presence of chimpanzee anti-CEA antibody showed only reactivity for the human CEA. In addition, chimpanzee antiserum had only minimal blocking effect on the binding of either goat or baboon antiserum to human CEA. We conclude from these studies that chimpanzee anti-human CEA antiserum recognized a determinant(s) on human CEA which was different from these recognized by goat or baboon antiserum to human CEA and this determinant(s) was poorly represented on chimpanzee CEA. In contrast, the human CEA determinant(s) recognized by baboon and goat anti-CEA antiserums were readily detected on chimpanzee (CEA).     

Radioimmunotherapy of human colonic cancer xenografts with 90Y labeled monoclonal antibodies to carcinoembryonic antigen

Monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA) or alpha-fetoprotein (AFP) were conjugated with diethylenetriaminepentaacetic acid and radiolabeled with 90Y at a specific activity of 4.0-6.0 mCi/mg. Approximately 50% of the radiolabeled anti-CEA antibody (90Y-labeled NP-2) bound to an immunoadsorbent containing CEA while analysis by high performance liquid chromatography revealed that 95-98% of the 90Y was associated with immunoglobulin. Less than 5% of the 90Y dissociated from either MAb after incubation in plasma for 48 h at 37 degrees C. After injection into nude mice, 98% of the circulating radioactivity remained associated with antibody and no loss of immunoreactivity was observed at 3 days. To evaluate 90Y-labeled NP-2 as a therapeutic agent, varied doses (10-100 microCi) were administered as a single i.v. injection into groups of nude mice bearing s.c. implants (0.3-0.4 g) of a CEA-producing human colonic cancer xenograft, GW-39. At the 10-microCi dose, no inhibition of tumor growth was observed. After 28 days, tumor growth was inhibited by as much as 77% in mice treated with 50 microCi of 90Y-labeled NP-2 as compared to tumor growth in control animals given 90Y-labeled anti-AFP. Doses higher than 50 microCi (75 and 100 microCi) were toxic to most of the animals, killing them within 2-3 weeks after administration. Marked suppression of circulating leukocytes was observed with 20 and 50 microCi by 1-2 weeks postinjection, but they returned to normal levels 3-4 weeks later. These studies show that treatment with 90Y-labeled MAbs against CEA can produce significant antitumor effects. However, toxicity to the bone marrow may limit the therapeutic efficacy of systemically administered 90Y-labeled MAbs.     

Antiidiotype antibodies in cancer patients receiving monoclonal antibody to carcinoembryonic antigen

The initial 10 patients of a Phase I clinical trial involving multiple injections of murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, NP-2, were studied for the presence in their sera of antiidiotypic antibody. Most patients had advanced gastrointestinal adenocarcinoma and received 1 mg/m2 monoclonal antibody three times weekly, or once a week, resulting in five to 13 injections over 12 to 240 days. Antiidiotype antibody was detected with a blocking radioimmunoassay using [125I]NP-2-F(ab')2 binding to CEA-coated microwells and [125I]NP-4-F(ab')2 as a control antibody. Five out of 10 patients demonstrated 65-96% inhibition of NP-2 binding at 1/20 dilution of serum compared to NP-2 binding in the presence of pretreatment sera. The inhibitory activity was preserved after adsorption over a polyclonal mouse IgG immunoadsorbent whereas exposure to a NP-2 affinity column completely depleted the activity. Specificity testing, including the blocking effect of patient sera on the control antibody NP-4, and interference by the possible presence of circulating NP-2, circulating CEA, and human anti-CEA activity, confirmed that the inhibition observed was specific for NP-2 and was caused by an agent with CEA-like characteristics. Longitudinal studies demonstrated that elevated titers of antiidiotypic antibody appeared later in the course of immunization than did antibody against mouse immunoglobulin. These studies indicate that patients can be sensitized to the idiotype (anti-combining site and/or combining site-related) of monoclonal antibodies to CEA following multiple infusions.     

Immunohistochemical analysis of pulmonary and pleural neoplasms using a monoclonal antibody (47D10) which reacts with nonspecific cross-reacting antigen

A series of 251 human pulmonary carcinomas were analyzed immunohistochemically for the antigens recognized by a new monoclonal antibody (MAb) 47D10. These antigens are part of a complex family of substances similar to, yet distinct from carcinoembryonic antigen (CEA), and are termed 'nonspecific cross-reacting antigens' (NCAs). The NCA epitope recognized by the MAb 47D10 is expressed on the cell surface and has previously been shown to be distinct from epitopes detected by several anti-CEA MAbs, as well as by MAbs 19-9 and Du-PAN-2. The NCA epitope recognized by MAb 47D10 is well preserved in formalin-fixed and paraffin-embedded tissues. Using immunohistochemistry, this epitope has been shown to have a limited biodistribution in normal tissues, and to be expressed by adenocarcinomas arising in the pancreas, colon, breast, ovary, prostate and lung. The frequency and pattern of NCA expression in human pulmonary neoplasms was found to correlate with the known distribution of CEA: and was often present in the non-small-cell carcinomas. In addition, the expression of CEA relative to NCA was evaluated in a select group of non-small-cell carcinoma cases using several anti-CEA MAbs, to directly compare the expression of CEA to NCA. In general, the NCA reaction pattern is more intense and expressed on more cells within the tumors than that of CEA expression.     

Mapping epitope characteristics on carcinoembryonic antigen

A method of epitope analysis is described in which the binding of one monoclonal antibody (MAb) to radiolabeled carcinoembryonic antigen (CEA) competes with the subsequent binding of an immobilised second MAb. From the degree of blocking obtained, we have identified both structurally related and independent epitopes on CEA. Using this technique to study fifteen MAbs, we have been able to recognise at least 6 unrelated epitopes of the CEA glycoprotein. Further characterisation of these epitopes was accomplished by means of immunohistochemistry. Of the fifteen MAbs, 6 were specific for CEA and reacted with at least 3 unrelated regions of the glycoprotein. Of the remaining 9 MAbs, 2 cross-reacted with erythrocytes, 5 with components of liver and 7 with polymorphonuclear neutrophils. Cross-reactions with liver were varied showing differential antibody specificity for bile canaliculi, Kupffer cells and bile duct epithelium. A high degree of correlation between epitope relatedness and immunohistochemical specificity was found. Two CEA-specific and 4 cross-reactive MAbs were also shown to react with ion-sensitive sites on the CEA glycoprotein.     

Successful radioimmunotherapy for lung metastasis of human colonic cancer in nude mice.

The potential for radioimmunotherapy as an adjuvant treatment for early disseminated colonic cancer was investigated in an experimental lung metastasis model. Nude mice receiving intravenous injection with a suspension of human colonic cancer cells (GW-39) developed multiple (10-100) tumor nodules throughout the lungs, and more than 50% of the animals died of extensive tumor involvement within 5-10 weeks. Groups of eight or nine animals bearing 7-day-old tumor transplants were treated with a single intravenous injection of radioiodinated agents: either 0.15 or 0.30 mCi of whole IgG of the NP-4 murine monoclonal antibody (MAb) against carcinoembryonic antigen (CEA) or 0.15 or 0.30 mCi of whole IgG of Immu-31, an anti-alpha-fetoprotein (anti-AFP) MAb. Treatment of animals with 0.15 or 0.30 mCi of 131I-labeled NP-4 IgG 7 days after injection of tumor cells resulted in survival for 23 weeks after tumor implantation in four of eight and seven of nine animals, respectively. Microscopic examination revealed that over 90% of the lung tumor colonies had no evidence of surviving cells. Animals treated with 0.30 mCi of anti-AFP, an irrelevant MAb, survived 4 weeks longer than controls. Toxicity was evident in four of the 17 animals given 0.30 mCi of NP-4 IgG (specific) or anti-AFP IgG (irrelevant) MAb. These animals died within 1-3 weeks after radioantibody injection, suggesting that death was related to the radiation dose. None of the animals given 0.15 mCi of 131I-MAb died within this period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of tumor targeting of mouse monoclonal and goat polyclonal antibodies to carcinoembryonic antigen in the GW-39 human tumor-hamster host model

We have evaluated 4 radioiodinated mouse monoclonal anticarcinoembryonic antigen antibodies (MAbs) by using the GW-39 human colorectal tumor xenograft transplanted i.m. in immunocompetent hamsters to determine whether there were any differences in their tumor localization properties. Additional comparisons were made to affinity-purified goat anticarcinoembryonic antigen antibody. Statistically significant differences were found in the percentage/g of tumor uptake and tumor/nontumor ratios among the antibodies, so that the antibodies could be ranked according to their tumor localization properties (NP-2 greater than NP-4 = goat antibody greater than NP-1 greater than NP-3). Although statistical differences were found, tumor/nontumor values generally were not distinguished by a factor of more than 1.5, suggesting that these differences may not be biologically significant. F(ab')2 fragments of NP-2 were found to be superior to NP-4 F(ab')2 fragments, giving tumor/liver and tumor/blood ratios of 16 and 11.5, respectively, within 3 days, in comparison to 5.4 and 3.8 for NP-4 F(ab')2 fragments. Mixtures of all of the MAbs or a mixture of NP-2 and NP-4 did not improve tumor localization, in comparison to NP-2 alone. These studies suggest that mixtures of these anticarcinoembryonic antigen MAbs may not afford better tumor imaging than the use of a certain single antitumor MAb.     

Polyreactivity of monoclonal antibodies made against human erythrocyte membranes with various pathogenic bacteria

Glycophorins comprise the major sialoglycoproteins of the human erythrocyte membrane. Several years ago we described a murine monoclonal antibody (MAb), designated 124,D-7 (IgM), developed by in vitro immunization with human erythrocyte membranes as antigen. We found the MAb reacted with a neuraminidase-dependent epitope on glycophorin A. Recent findings using ELISA with various bacteria as coating antigens have demonstrated strong cross-reactions of MAb 124,D-7 with some bacteria like Legionella and no reaction with bacteria such as Streptococcus pneumoniae. A second MAb, 130,E-4 (IgM), generated by the in vitro immunization technique, agglutinated human red cells irrespective of blood groups. This MAb showed strong cross-reactions with bacteria different from those being positive with MAb 124,D-7. The broad cross-reactivities of the two MAbs suggested that they are polyreactive antibodies. Sequencing of the V(H) and V(L) genes of MAb 124,D-7 showed germ-like sequences characteristic of polyreactive antibodies. The nucleotide sequences of the V(H) and V(L) genes of MAb 124,D-7 matched sequences coding for antibodies against CD34 and cross-reacting streptococcal antibodies. For Legionella pneumophila, the main interacting band on immunoblots was identified as the major outer membrane protein by mass spectrometry after separation by isoelectric focusing followed by SDS-PAGE. Flow cytometry showed that the epitope for MAb 124,D-7 was not displayed on live L. pneumophila but became exposed after heat treatment. Studies with one of the control MAbs, 145,F-2, directed against phosphorylcholine, which is known to be present on erythrocytes and some bacteria, showed that the epitope is deeply buried in the human erythrocyte membrane as neither neuraminidase nor papain exposed the epitope. The positive control MAb 3/1 directed against an epitope on LPS of L. pneumophila revealed weak cross-reactions with Pseudomonas aeruginosa.