Autoradiographic analysis of monoclonal antibody distribution in human colon and breast tumor xenografts

Summary The targeting of monoclonal antibodies to human tumor xenografts in nude mice was investigated by analysis of the cellular distribution of two radioiodinated monoclonal antibodies, B6.2 and B72.3, which recognize different tumor-associated antigens. The time course of distribution of each antibody within Clouser human mammary carcinoma (B6.2 positive, B72.3 negative) and LS174T human colorectal carcinoma (B6.2 positive, B72.3 positive) following i. v. injections was compared using autoradiographic techniques, which were also used to determine the pattern of binding after in vitro incubation with radiolabeled antibody. Both in vivo and in vitro localization of ¹²⁵I-B72.3 in LS174T were characterized by the binding of antibody to antigen-rich mucin globules. In contrast, in vivo localization of B6.2 was restricted to groups of cells in well vascularized regions. Thus, the in vivo accumulations of B6.2 and B72.3 although quantitatively similar showed very different spatial distributions within LS174T tumors. The in vivo binding of B6.2 in Clouser tumors was restricted to small clusters of cells scattered fairly evenly throughout the tumor. There was no evidence for the presence of such antigen-rich foci after in vitro incubation of tumor sections with B6.2 suggesting that heterogeneity of regional uptake may be due to differences in antibody delivery. This type of information may provide a rational basis for the selection of appropriate therapeutic isotopes for radioimmunotherapy studies using these and other tumor models.     

Monoclonal antibodies against carcinoembryonic antigen (CEA) discriminate between CEA produced by gastrointestinal tumors and CEA produced by other tumors

Carcinoembryonic antigen (CEA) is a common tumor marker. It is not specific since many tumor types express it. We report here the use of a series of monoclonal anti-CEA antibodies with apparent restricted activity. These antibodies reacted histochemically with CEA produced by tumors originating in the gastrointestinal tract, but they did not react with CEA-secreting tumors of nongastrointestinal origin. We have used this phenomenon to accurately diagnose the tumor of origin in 20 patients who had a CEA-producing metastatic tumor, but in whom the primary origin was clinically not yet discovered. The use of such antibodies for diagnostic purposes may be an adjunct to other methods in evaluating tumors of unknown origin.     

Complementation of expression of carcinoembryonic antigen and tumor associated glycoprotein-72 (TAG-72) in human colon adenocarcinomas.

Enzyme-labeled monoclonal antibodies (MAbs) were used in an immunohistochemical, dual-staining study of 10 colon adenocarcinomas. MAbs B72.3 and COL-4, reactive with the high molecular weight tumor-associated glycoprotein-72 (TAG-72) antigen and carcinoembryonic antigen (CEA), respectively, were labeled with horseradish peroxidase or alkaline phosphatase. Dual staining using the two MAbs on a single tissue section (formalin-fixed, paraffin-embedded) showed that greater numbers of carcinoma cells could be detected by using the combination of the two MAbs than could be detected by use of either MAb alone. In many tumors, some carcinoma cells reacted with MAb B72.3, some reacted with MAb COL-4, and some cells reacted with both MAbs. Only 1 of 10 carcinomas showed greater than 75% reactive cells when stained with each MAb individually. In 9 of 10 cases, however, greater than 75% of cells reacted when the combination of MAbs was used. Cell surface and cytoplasmic patterns of reactivity were observed with both MAbs while some pools of extracellular mucin were composed of both TAG-72 and CEA. This study supports the rationale for the use of a combination of anti-TAG-72 and anti-CEA MAbs for in vitro immunologic detection and potential in vivo immunodiagnostic and immunotherapeutic applications for these MAbs in colon adenocarcinoma patients.     

The use of a panel of monoclonal antibodies in the evaluation of cytologic specimens from the central nervous system

A panel of monoclonal antibodies was tested immunohistochemically to determine the utility of such reagents in distinguishing among metastatic carcinoma, lymphoma, leukemia and primary brain tumors. The monoclonal antibodies used were: (1) a cocktail comprised of three anti-glial fibrillary acidic protein antibodies (alpha-GFAP); (2) UJ13A, a panneuroectodermal antibody; (3) B72.3, which recognizes a carcinoma-distinctive tumor-associated glycoprotein complex; and (4) 2D1, a pan-leukocyte antibody. Fifty-three specimens (21 cerebrospinal fluids, 1 ventricular fluid, 2 brain cyst fluids, 12 needle washings, 15 imprints, 1 subdural fluid and 1 post-shunt fluid) were obtained from 21 gliomas, 2 meningiomas, 1 pineoblastoma, 11 metastatic tumors, 3 lymphomas, 1 leukemia and 14 cases without tumor. alpha-GFAP stained all 21 gliomas and 5 of 5 cases containing reactive brain fragments. UJ13A had a reactivity pattern similar to that of alpha-GFAP, but also stained the meningiomas, pineoblastoma, oat-cell carcinoma and embryonal rhabdomyosarcoma. B72.3 stained all adenocarcinomas and the large-cell carcinoma. 2D1 stained lymphoma and leukemia, all inflammatory cells and 4 of 12 glioblastomas. Although no single antibody was diagnostic of a specific tumor type, this panel accurately differentiated among most primary brain tumors, metastases, leukemias and lymphomas.     

Application of genetically-engineered anti-CEA antibodies for potential immunotherapy of colorectal cancer.

Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors, and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for "humanization" of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.     

Comparative biodistribution and antibody-dependent cellular cytotoxicity of native and heavy chain chimeric antibody.

We have recently chimerized the heavy chain of the pan-carcinoma monoclonal antibody (mAb) B72.3. Studies were undertaken to compare the IgG1 chimeric antibody, B72.3-1-3 with native murine B72.3 (nB72.3). Using fluorescence-activated cell sorting analysis, B72.3-1-3 demonstrated specific binding to fresh LS174T tumor cells. Biodistribution of 131I B72.3-1-3 was similar to 131I nB72.3 in nude mice bearing LS174T xenografts. Peak radiolocalization indices were noted on day 6 for B72.3-1-3 and day 8 for nB72.3. Both antibodies were capable of imaging LS174T tumors by radioimmunoscintigraphy. Antibody-dependent cellular cytotoxicity of LS174T by human peripheral blood lymphocytes was tested in 8h 51Cr release assays. With either no antibody or nB72.3, lymphocytes were not capable of killing LS174T cells. However, B72.3-1-3 at a concentration of 5 and 50 micrograms/ml mediated significant lysis of tumor cells by human lymphocytes. These results suggest that chimeric antibodies retain their binding properties to tumor cells and display biodistribution patterns similar to their unmodified counterparts. Such modifications may reduce the deleterious human antimouse antibody response to murine mAbs as well as augment antibody-dependent cellular cytotoxicity of tumor cells by human effectors.     

An improved method of direct labeling monoclonal antibodies with 99mTc

An improved method of direct labeling MAbs with ^99mTc is described. Two murine monoclonal antibodies, designated Lym-1 and B72.3, have been successfully labeled with ^99mTc in 0.1 M borate buffer at pH 9.3. The choice of buffer and pH was essential for obtaining a radiolabeling yield ⩾98%. In vitro studies demonstrated that the radiolabeled antibodies were stable and retained their immunoreactivity. Imaging and biodistribution studies using Raji and LS174T human tumor-bearing nude mice demonstrated a significant tumor uptake at 24-h post-injection of ^99mTc-labeled MAbs. This improved labeling method showed better stability than those of previously published methods and resulted in significant improvement in the uptake of antibody in tumor. External images at 24 h post-injection revealed clearly visible tumors demonstrating the benefit of this method for tumor immunoscintigraphy.     

Technetium labeling of monoclonal antibodies with functionalized BATOs: 2. TcCl(DMG)3CPITC labeling of B72.3 and NP-4 whole antibodies and NP-4 F(ab')2.

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive 2-carboxy-4-phenyl isothiocyanate (CPITC). The 99Tc complexes, where the dioxime was either dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO), were prepared and characterized. The 99mTc complex TcCl(DMG)3CPITC was prepared from a freeze-dried kit and used to label B72.3 (anti-TAG.72) and NP-4 (anti-CEA) whole antibodies, and the NP-4 F(ab')2 fragment. SDS-PAGE electrophoresis indicated that the labeling reagent was strongly bound to antibody. The labeled antibodies displayed high binding to affinity columns and good tumor uptake in GW39 tumor-bearing mice.     

Cancer imaging with CEA antibodies: historical and current perspectives.

This article reviews the history and status of cancer imaging with radiolabeled antibodies against carcinoembryonic antigen (CEA). Although CEA and many other cancer-associated antigens are not distinct for neoplasia, the quantitative increase of these markers in malignant tissues provides a sufficient differential for selective antibody targeting. Animal studies with xenografted human tumors provided the first evidence of the prospects of this technology, followed by initial clinical success with purified goat whole IgG antibodies to CEA, labeled with 131I and with the use of dual-isotope subtraction methods. Subsequently, improved and earlier imaging could be accomplished with monoclonal antibody fragments, which then would permit the use of shorter-lived radionuclides, such as 111In, 123I, and 99mTc. The preferred use of a monoclonal anti-CEA IgG Fab' fragment, labeled with 99mTc by a recently developed, simple and rapid kit, has enabled the detection of small lesions, including those in the liver, within 4 h of injection. By means of SPECT imaging, a high sensitivity and specificity for RAID could be achieved.     

A comparison of monoclonal antibodies against distinct colon tumor-associated antigens in immunohistochemistry and in tumor localization

Monoclonal antibodies to colon/ovary tumor antigen (COTA), carcinoembryonic antigen (CEA), colon-specific antigen (CSA), and colon-specific antigen “protein” (CSAp) were evaluated for specificity, reactivity with normal tissues, and tumor localizations using athymic rats bearing xenografted human colon tumors. Radioiodine labeled anti-CSA and anti-COTA retained immunoreactivity and effectively localized the tumors; anti-CSAp retained immunoreactivity, but localized less effectively; and anti-CEA lost most of its immunoreactivity and localized poorly. Of the antibodies tested, anti-COTA showed potential for human colorectal tumor radiolocalization.     

Monoclonal antibodies

The hybridoma technology developed by K?hler and Milstein has initiated a new era in biological sciences. In the last decade the possibility of generating limited amounts of monoclonal antibodies of predefined specificity has become a routine method in many laboratories throughout the world. The constant quality of various antibody preparations from 1 hybridoma cell line represents another important advantage of this method. Apart from the use for several purposes, e.g. HLA-typing, differentiation of lymphocyte subpopulations and blood group antigens, monoclonal antibodies play an important role in the determination of various tumor markers. In most modern immunoassays monoclonal antibodies are used. Furthermore, there is nowadays a limited experience concerning the in vivo use of monoclonal antibodies in malignant disease. Radiolabelled antibody immunodetection has been applied e.g. in colorectal and testicular tumors for the detection of tumor metastases. The therapeutic use of monoclonal antibodies has been reported in some patients with tumors of the hemopoietic system. The production of new murine and human monoclonal antibodies against various tumor types is subject of current investigations. The aim of these efforts is the development of monoclonal antibodies suitable for in vitro tumor diagnosis and application in vivo.     

Applications of monoclonal antibodies in clinical cytology as exemplified by studies with monoclonal antibody B72.3. The George N. Papanicolaou award lecture

The monoclonal antibody (MAb) B72.3, reactive with a high-molecular-weight, glycoprotein, tumor-associated antigen, designated TAG-72, has been previously shown to be reactive with formalin-fixed, paraffin-embedded tissue sections of adenocarcinomas of the ovary, colon and breast, but not a variety of normal adult tissues. It has demonstrated utility as an immunocytochemical adjunct for the diagnosis of carcinoma in cell blocks and cytocentrifuge preparations of human serous effusions, with selective reactivity for tumor cells (particularly adenocarcinoma) over reactive mesothelium. Using the avidin-biotin complex (ABC) method of immunoperoxidase staining and formalin-fixed, paraffin-embedded cell suspensions, MAb B72.3 detected tumor cells in effusions from all of 21 patients with adenocarcinoma of the breast. No reactivity was demonstrated in any cell type in benign effusions from 41 patients. In contrast, MAb B72.3 showed no reactivity to leukemic or lymphomatous effusions, or to mesothelial cells from malignant effusions. MAb B72.3 also detected adenocarcinoma cells in effusion specimens from 12 of 12 patients with adenocarcinoma of the lung and 16 of 16 patients with adenocarcinoma of the ovary. MAb B72.3 has recently been used with fine needle aspiration (FNA) biopsy specimens and the corresponding surgically excised tumors to determine cellular reactivity. Using the ABC immunoperoxidase method, fine needle aspirates and corresponding surgically excised tumors were analyzed for TAG-72 expression. Positive staining with MAb B72.3 was observed in needle aspirates of 27 of 27 adenocarcinomas and adenosquamous carcinomas of the lung, 17 of 21 adenocarcinomas of the breast, 6 of 6 adenocarcinomas of the colon and in carcinomas from other body sites. In contrast, 21 small-cell carcinomas of the lung, 13 malignant melanomas, 2 lymphomas and 2 sarcomas did not stain with the antibody. Benign lesions from the breast, lung, pancreas, parotid and thyroid also showed no staining. In many patients, tumor-bearing tissue had also been resected and was available for comparative examination with MAb B72.3. In more than 90% of these patients, the staining patterns of the tumor cells in the aspirates were found to be predictive of the patterns of antibody reactivity in the comparable surgically resected tumors. From these studies, it is concluded that MAb B72.3 defines a tumor-associated antigen that is expressed in neoplastic cells versus benign cells, that is most selectively expressed in carcinomas and that may be used as a novel adjunct for the diagnosis of neoplasms in effusions and in fine needle aspiration biopsies.     

Isolation of anti-carcinoembryonic antigen (CEA), specific immunoglobulins and their use in immunocytochemical and enzyme-immunoassays (ELISA)

Anti-carcinoembryonic (CEA) polyclonal antibodies in sheep and rabbits were raised using purified CEA from acid extracts of human colon adenocarcinoma. CEA was purified by gel filtration on Sepharose 4B CL and chromatography on DEAE-Sephadex A50. The antiserum was adsorbed with human serum and perchloric acid extract from normal colon. Anti-CEA IgG was purified from monospecific antiserum by ion-exchange chromatography and its specificity was tested on cryostat sections from colon adenocarcinoma by the indirect immunoperoxidase technique. The specific reaction was compared with that obtained by using a similar technique and two CEA specific monoclonal antibodies. An anti-CEA IgG peroxidase conjugate was obtained allowing to establish a "sandwich" ELISA-CEA system with two antibodies. CEA determinations were made in a group of 15 normal controls (mean value 4.8 +/- 0.4 ng/ml) and in 30 colorectal tumor patients (mean value 26.6 +/- 2.15 ng/ml). The anti-CEA antibodies are proven useful in immunocytochemical and ELISA techniques and may be further used in radioimaging of tumors.     

Syngeneic anti-idiotypic antibodies eliminate excess radiolabeled idiotypes at experimental radioimmunolocalization

Significant improvements in tumor/nontumor ratio can be achieved by injections of nonlabeled anti-idiotypic monoclonal antibodies (MAbs) during radioimmunolocalization and radioimmunotherapy using MAbs to target experimental tumors. The in vivo effects of an anti-idiotypic MAb (αH7) against a radioiodinated, high affinity, low dissociation rate, monoclonal antiplacental alkaline phosphatase antibody (H7) was investigated. Following in vivo injection of the anti-idiotypic MAb, the radioactivity in experimental tumors was found to decrease only 25% while the reduction of corresponding radioactivity in nontumor tissues amounted to 65–85%, compared to the group receiving no anti-idiotypic MAbs. These results indicate that it is possible to partially clear the circulation and nontumor tissues from excess of radiolabeled idiotypic antibody, without significant decrease in specific tumor localization, increasing the tumor/ nontumor ratio three- to fourfold. Circulating nontumor targeting radiolabeled antibodies is one of the major limiting factors in radioimmunotherapy today. Injection of anti-idiotypic MAbs could selectively significantly reduce the radiation dose to radiosensitive tissues, i.e., bone marrow and intestine, thus improving efficiency in radioimmunoscintigraphy and radioimmunotherapy.     

Evaluation and clinical relevance of patient immune responses to intravenous therapy with murine monoclonal antibodies conjugated to adriamycin

A retrospective study was performed in order to examine the clinical relevance of human anti-murine antibodies (HAMA) to concurrent clinical events in 21 patients receiving intravenous therapy with cocktails of murine monoclonal antibodies conjugated to Adriamycin. In vivo tumor localization of the murine antibodies was also evaluated. Serum levels of HAMA, human-murine immune complexes (HMIC), and murine antibodies were measured using an automated fluorescence immunoassay. Immunohistochemical staining was performed on frozen sections of tumor biopsies from eight of the patients to examine the in vivo binding of the murine antibodies. The patients were divided into low, intermediate, and high antibody dose groups. The incidence of allergic symptoms (80%) and HAMA correlation (75%) were highest in the low dose group. Specific IgM HAMA was the most highly correlated with allergic reactions, being present in 61.5% of the allergic patients. Thirteen of the 21 patients studied (61.9%) developed allergic symptoms after one or more doses of the murine monoclonal antibody conjugates. The percentages of total antibody doses in the patients' sera at varying intervals post-infusion varied widely from patient to patient for any given time point and dose, suggesting complex factors in the distribution and clearance of the murine antibodies. All eight of the patients biopsied during or post-therapy exhibited tumor localization of the murine monoclonal antibodies. Six of the eight had concurrent HAMA in their sera. Thus, the presence of HAMA did not prevent in vivo localization of the murine antibodies in the target tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies

Summary The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes ¹²⁵I and ¹³¹I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with ¹³¹I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with ¹²⁵I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.     

A new conjugating agent for radioiodination of proteins: low in vivo deiodination of a radiolabeled antibody in a tumor model

A new radioiodinating agent, N-(p-[125I]iodophenyl)maleimide, has been synthesized for its potential utility in the radioiodination of monoclonal antibodies and other proteins. The efficiency of incorporation of 125I in the B72.3 antibody by iodine monochloride iodination or by maleimide conjugation was 19% and 43%, respectively. The thyroid uptake following intraperitoneal administration of the two 125I-labeled antibody preparations was evaluated in nude mice implanted with LS174T colon carcinoma xenografts. The iodine monochloride preparation showed substantially greater uptake in the thyroid with values of 2.1% ID at 6 h after injection and reaching a maximum of 4.3% ID after 6 days. In contrast, the maleimide preparation showed a uniformly low uptake in thyroid of 0.1%-0.2% ID. The results of these preliminary studies demonstrate that the N-(p-[125I]iodophenyl)maleimide-labeled monoclonal antibodies showed markedly less (less than 10-fold) uptake of radioiodine in the thyroid, indicating significantly less in vivo deiodination than iodine monochloride-labeled monoclonal antibodies, while retaining some tumor localization in vivo. Studies are in progress to optimize N-(p-[125I]iodophenyl)maleimide radioiodination conditions and to improve the tumor localization.     

Immunoscintigraphy of colorectal carcinoma with an anti-CEA monoclonal antibody: a critical review

An anti-CEA monoclonal antibody (FO23C5), belonging to the IgG_1a class, was used to perform several preclinical and clinical studies on radioimmunoscintigraphy (RIS) in patients with colorectal carcinoma. Preliminary screening on different tissues showed high specificity of this antibody for gastric and colorectal carcinomas. A pilot study on 51 patients with 64 localizations of colorectal carcinoma was realized, followed by a multicenter validation study in which, among 509 patients bearing CEA secreting tumors, 254 patients had primary or relapsed colorectal tumors. High sensitivity and specificity values were obtained by these studies and many “unsuspected” localizations were recorded. In order to better define the clinical utility of this approach, a prospective trial was run on 59 patients previously submitted to surgery for colorectal carcinoma and with suspected local relapses. A comparative evaluation of RIS, CT scan, US and MRI was performed. RIS and MRI have the highest value of accuracy (86%) followed by CT scan (68%) and US (54%). No adverse reactions were noticed in any of the patients examined.     

Tumor radioimmunolocation: differential antibody retention by antigenic normal tissue and tumor

On the assumption that a high specificity would be required for in vivo tumor location and drug targeting, much labor has been expended to produce monoclonal antibodies directed to antigens found primarily on tumors. However, for at least one tumor-associated antigen, stage-specific embryonic antigen 1 (SSEA-1), the concentration of the target antigen need not be higher in the tumor than that found in normal tissues for successful use of the corresponding monoclonal antibody in in vivo tumor location; rather, accessibility and antibody metabolism are more important.     

Combined radioimmunotherapy and chemotherapy of breast tumors with Y-90-labeled anti-Her2 and anti-CEA antibodies with taxol

Because breast cancer cells often express either Her2/neu or carcinoembryonic antigen (CEA) or both, these tumor markers are good targets for radioimmunotherapy using Y-90-labeled antibodies. We performed studies on nude mice bearing xenografts from MCF7, a cell line that has low Her2 and CEA expression, to more accurately reflect the more usual situation in breast cancer. Although uptake of In-111 anti-CEA into tumors was lower than that for In-111-labeled anti-Her2, radioimmunotherapy (RIT) with Y-90 anti-CEA was equivalent to that of Y-90 anti-Her2. When either Y-90 antibody was combined with a split-dose treatment with Taxol, the antitumor effect was greater than with either agent alone. When Y-90 anti-CEA was combined with a single dose of Taxol, the results were equivalent to the split-dose regimen. RIT plus cold Herceptin had no additional effects on tumor size reduction over RIT alone. When animals were first treated with Y-90 anti-Her2 and imaged 1-2 weeks later with In-111 anti-CEA or anti-Her2, tumor uptake was higher for anti-CEA and improved over tumor uptake with no prior RIT. These results suggest that a split dose of RIT with anti-Her2 antibody followed by anti-CEA antibody would be more effective than a single dose of either. This prediction was partially confirmed in a controlled study comparing single- vs split-dose anti-Her2 RIT followed by either anti-Her2 or anti-CEA RIT. These studies suggest that combined RIT and Taxol therapy are suitable in breast cancers expressing either low amounts of Her2 or CEA, thus expanding the number of eligible patients for combined therapies. They further suggest that split-dose RIT using different combinations of Y-90-labeled antibodies is effective in antitumor therapy.