Human high-molecular-weight melanoma-associated antigen mimicry by mouse antiidiotypic monoclonal antibody TK7-371.

Since the human high-molecular-weight melanoma-associated antigen (HMW-MAA) represents a useful target to implement active specific immunotherapy with mouse antiidiotypic monoclonal antibody (mAb), the present study is aimed at developing and characterizing mouse antiidiotypic mAbs which bear the mirror image of the determinant defined by the anti-HMW-MAA mAb TP61.5. To this end, a BALB/c mouse was immunized with the syngeneic mAb TP61.5. Screening of the 703 generated hybridomas showed that 13 of them secrete antiidiotypic mAbs which inhibit the binding of the immunizing mAb TP61.5 to melanoma cells by at least 75%. The dose of antiidiotypic mAb required to inhibit by 50% the binding of mAb TP61.5 to melanoma cells ranged between 4 and 200 ng. The 13 antiidiotypic mAbs recognize conformational idiotypes, since they did not react in Western blotting with the heavy and light chain of mAb TP61.5 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Furthermore, the 13 antiidiotypic mAbs did not react with 23 mAbs which recognize 13 determinants of the HMW-MAA distinct from that defined by mAb TP61.5. Analysis of the 13 antiidiotypic mAbs for their ability to elicit cellular and humoral anti-HMW-MAA immunity showed that only mAb TK7-371 elicited a delayed-type hypersensitivity reaction to HMW-MAA-bearing cells in syngeneic hosts and anti-HMW-MAA antibodies in BALB/c mice and in rabbits. Since rabbits express the HMW-MAA, the present results indicate that the antiidiotypic mAb TK7-371 can induce humoral immunity to self HMW-MAA. Therefore, the antiidiotypic mAb TK7-371 may be an efficacious immunogen to implement active specific immunotherapy in patients with melanoma.     

Binding parameters and idiotypic profile of the whole immunoglobulin and Fab' fragments of murine monoclonal antibody to distinct determinants of the human high molecular weight-melanoma associated antigen.

The nonspecific accumulation of radioactivity in bone marrow, liver, and spleen in patients with melanoma injected with radiolabeled whole IgG of anti-high molecular weight-melanoma associated antigen (HMW-MAA) monoclonal antibody (mAb) hampers the application of immunoscintigraphy to visualize melanoma lesions. This nonspecific background can be reduced by utilizing fragments which do not contain the Fc portion of anti-HMW-MAA mAb. Since the in vivo targeting to melanoma lesions of radiolabeled F(ab')2 and Fab' fragments of anti-HMW-MAA mAb is critically dependent on their binding parameters, we have analyzed the effect of fragmentation on the binding characteristics of the anti-HMW-MAA mAbs 225.28, 763.74, and TP41.2. The three mAbs recognize distinct and spatially distant determinants with a heterogeneous distribution on the pool of HMW-MAA molecules synthesized by melanoma cells. The determinant recognized by mAb TP41.2 is detectable on a markedly smaller population of HMW-MAA molecules than those recognized by mAbs 225.28 and 763.74. 125I-labeled F(ab')2 fragments of the three mAbs displayed an immunoreactive fraction similar to that of the whole IgG, while Fab' fragments displayed a lower one. Fragmentation of mAbs 225.28 and TP41.2 to F(ab')2 produced a 2- and 1.5-fold reduction in their association constants but did not cause a significant change in that of mAb 763.74. Cleavage of F(ab')2 fragments to Fab' fragments produced 2-, 40-, and 7-fold reductions in the association constants of mAbs 225.28, 763.74, and TP41.2, respectively. These changes qualitatively fit the predictions of theory for univalent and bivalent mAb binding, since mAbs 763.74 and TP41.2 appear to show bivalent binding to melanoma cells and mAb 225.28 to show univalent binding. The affinity constant of IgG, F(ab')2 and Fab' fragments of mAbs 225.28, 763.74, and TP41.2 displays an inverse relationship with the extent of their time-dependent release from the membrane of melanoma cells. Since no endocytosis of mAb could be detected, the latter results suggest that radioactivity remains bound to melanoma cells in vivo for a longer time following injection of F(ab')2 fragments than following that of Fab' fragments of each of the anti-HMW-MAA mAb tested. Radiolabeled Fab' fragments of mAbs 763.74 and TP41.2 displayed a marked reduction in their reactivity with some of the antiidiotypic mAb tested. The loss of some idiotopes is likely to be caused by changes in the conformation of the molecules associated with the fragmentation of IgG and by damage during the iodination procedure.     

Human high molecular weight-melanoma associated antigen mimicry by mouse anti-idiotypic MAb MK2-23. Immunohistochemical analysis of the reactivity of anti-anti-idiotypic MAb with surgically removed melanoma lesions

The mouse anti-id MAb MK2-23 bears the internal image of the antigenic determinant defined by anti-HMW-MAA MAb 763.74. Active specific immunotherapy with anti-id MAb MK2-23 is associated with a statistically significant prolongation of survival in patients with malignant melanoma. Characterization of the immune response elicited by anti-id MAb MK2-23 may contribute to our understanding of the mechanisms underlying the apparent beneficial clinical effects of active specific immunotherapy with anti-id MAb MK2-23. Therefore, 8 HMW-MAA binding anti-anti-id MAbs elicited with MAb MK2-23 were characterized in their reactivity with a large panel of surgically removed benign and malignant tumors and of normal tissues. The 8 anti-anti-id MAbs displayed subtle differences in their immunoperoxidase staining of both benign and malignant lesions and of normal tissues. The diversity in the fine specificity of the 8 anti-anti-id MAbs is likely to reflect the few somatic mutations which occur in the amino-acid sequence of the variable regions of their heavy and light chains in the course of the immune response to MAb MK2-23. The reactivity patterns of the 8 anti-anti-id MAbs with the tissue substrates are similar, although not superimposable upon that of anti-HMW-MAA MAb 763.74 elicited with melanoma cells. This difference may reflect the imperfect mimicry by anti-id MAb MK2-23 of the antigenic determinant defined by anti-HMW-MAA MAb 763.74. © 1995 Wiley-Liss, Inc.     

Immunoscintigraphy of small-cell lung cancer xenografts with anti neural cell adhesion molecule monoclonal antibody, 123C3: improvement of tumour uptake by internalisation.

The efficacy of three murine monoclonal antibodies (MAbs) for immunoscintigraphy of small-cell lung cancer (SCLS) xenografts was studied in a Balb/c nu/nu mouse model. These Mabs, 123C3, 123A8 and MOC191, belong to cluster 1 of anti-SCLC MAbs and bind to the neural cell adhesion molecule (NCAM) with similar affinity. After intraperitoneal injection of these MAbs, labelled with 125I, the highest uptake in tumour tissue was obtained with MAb 123C3. Seven days after the administration of this MAb 13.9% of the injected dose per gram of tumour tissue was retained in the tumour. The corresponding tumour tissue ratios ranged from 3.97 for blood to 31.03 for colon. The imaging results and the tumour uptake were less favourable for the two other MAbs, 123A8 and MOC191 (fractions of injected dose respectively 6.7% and 9.2%), although affinity, biological activity after labelling and uptake in non-tumour tissues were very similar for all three MAbs. These results may be explained by the differences in the interaction between the MAbs and the tumour cells. Mab 123C3 is internalised into tumour cells, whereas both other anti-NCAM Mabs are not. Internalisation into NCI H69 cells was demonstrated in vitro by radioimmunoassay, confocal laser scanning microscopy and electron microscopy. The internalised fraction of MAb 123C3 was 22.3% after 24h, whereas this fraction was only 7.5% for MAb 123A8. Although the internalised radiolabeled Mabs are usually degraded and dehalogenated intracellularly, the retained radioactivity is high. Apparently, intracellular degradation of radiolabelled MAb 123C3 and subsequent secretion of radioactive iodine did not prevent the accumulation of intracellular radioactivity. In conclusion, accumulation and retention of radioactivity in the tumour tissue, due to internalisation of radiolabelled MAbs, may improve the results immunoscintigraphy.     

Network of idiotypic and anti-idiotypic antibodies related to the ovarian carcinoma-associated folate binding protein.

The present paper describes the generation and characterization of anti-idiotypic monoclonal antibodies (Ab2 MAbs) produced against the MOv19 MAb (Ab1 MAb), which shows a restricted reactivity with human ovarian carcinoma. The Ab2 MAbs were produced by immunizing mice with MOv19 conjugated to syngeneic mouse red blood cells (MRBC). After the somatic hybridization, five Ab2 MAbs, designated Id19.1, Id19.2, Id19.3, Id19.4 and Id19.5, were selected by a sandwich assay on the basis of their specific reactivity to the MOv19 MAb, but not to the isotype-matched MAb used as a control. A complete cross-inhibition between these 5 MAbs was observed, indicating that they recognize overlapping idiotopes on MOv19. The Ab2/antigen relationship of two Ab2 MAbs (Id19.1 and Id19.2) was investigated by competition experiments on a relevant tumor target cell line. We showed that both Ab2 MAbs were able to interfere with the antigen binding capacity of 125I-MOv19. Moreover, Id19.1 was able partially to inhibit the binding of the purified radiolabelled antigen recognized by the MOv19 MAb (125I-CaMOv19) on insolubilized MOv19. To investigate further whether the Ab2 MAb is the "internal image" of CaMOv19, Id19.1 was used as immunogen with the aim to induce an anti-CaMOv19 response. The antisera tested by indirect solid-phase radioimmunoassay (RIA) on the Ab2 MAb and the irrelevant isotype-matched control MAb revealed an anti-anti-idiotypic response (Ab3). However, no Ab3 antibodies with Ab1-like specificity (Ab1') were found in the immune sera, as assessed by testing the antisera both in indirect immunofluorescence (IF) and solid-phase RIA on CaMOv19-positive target cell lines.     

Improved tumor targeting of rhenium-186-labeled anti-human high-m.w. melanoma-associated antigen monoclonal antibody 763.74 following purification with anti-idiotypic monoclonal antibody MK2–23

Because of its high-energy beta emissions and imageable gamma emissions, ¹⁸⁶Re represents an attractive isotope to radiolabel monoclonal antibodies (MAbs) recognizing human tumor-associated antigens (TAAs) for radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) of patients with malignant diseases. Application of¹⁸⁶Re, however, is hindered by the frequent denaturation of MAbs following exposure to the strong reducing conditions employed in the labeling procedures. To overcome this problem, we have utilized a direct labeling procedure and combined it with affinity chromatography over columns of immobilized anti-idiotypic (anti-id) MAbs which recognize idiotopes in the antigen-combining site of the radiolabeled MAb. The validity of the procedure was demonstrated with the anti-high-m.w. melanoma-associated antigen (HMW-MAA) MAb 763.74 and its corresponding anti-id MAb MK2–23. Utilizing this approach, MAb 763.74 was labeled to specific activities of 2.8 ± 0.6 mCi/mg with ¹⁸⁶Re. Furthermore, its immunoreactivity, which was reduced to about 30% following labeling with ¹⁸⁶Re, was improved to about 50% by affinity chromatography over columns of anti-id MAb MK2–23. The improvement in immunoreactivity of ¹⁸⁶Re MAb 763.74 resulted in a significant (p < 0.05) increase in targeting to human melanoma tumors grown in nude mice and an increase in sensitivity of RIS. Our results suggest that direct labeling of anti-TAA MAb with ¹⁸⁶Re coupled with purification over affinity columns of anti-id MAb may facilitate the application of ¹⁸⁶Re to RIS and RIT. Int. J. Cancer 78:486–490, 1998. © 1998 Wiley-Liss, Inc.     

Complementation of anti-CEA and anti-TAG-72 monoclonal antibodies in reactivity to human gastric adenocarcinomas

Monoclonal antibody (MAb) B72.3 and MAb COL-4 are reactive with the high-molecular-weight (Mr greater than 10(6] tumor-associated glycoprotein (TAG)-72, and the Mr 180,000 carcino-embryonic antigen (CEA), respectively. Antibody competition radioimmunoassays (RIAs) using 125I-MAb B72.3 or 125I-COL-4 have demonstrated that each MAb also recognizes a distinct antigenic determinant. Solid-phase RIAs using MAbs B72.3 and COL-4, however, demonstrated similar reactivity for each MAb with gastric carcinomas versus normal gastric mucosa. Tissue sections from all of 17 gastric adenocarcinomas also reacted with both MAb B72.3 and MAb COL-4 when immunoperoxidase techniques were used. Double-staining techniques using both MAbs on the same section were performed on formalin-fixed, paraffin-embedded gastric tissue sections using the combination of avidin-biotin peroxidase complex and avidin-biotin alkaline phosphatase complex immunohistochemical methods. The double-staining technique revealed that some carcinoma cells react with MAb B72.3, some react with MAb COL-4, and others react with both MAbs. This technique has demonstrated that more carcinoma cells can be detected by both MAbs as compared to the number of stomach carcinoma cells shown to be reactive with either one or the other MAb. These studies thus define the rationale for the use of combinations of MAbs which recognize different tumor-associated antigens as immunological adjuncts for detection and perhaps therapy of gastric carcinoma.     

Generation of bispecific monoclonal antibodies for two phase radioimmunotherapy

A two phase radioimmunotherapy based on bispecific MAbs in which one arm recognises a tumour antigen and the other a radiolabelled chelate, may prove more effective in the treatment of carcinomas than currently available immunotherapies. To establish this system we first showed that penetration into human carcinoma xenografts as well as long term retention of intact MAb outside the carcinoma cells can be obtained. Epitope saturation was not obtained however, despite the large MAb doses injected i.v. for 10 days. We then generated hybridomas producing high avidity anti-metal chelate MAbs (anti-DTPA-Y). These hybridomas were fused with hybridomas producing MAbs against CEA or GIT-mucin, and stable bispecific MAb producing quadromas were obtained. For the anti-GIT-mucin x anti-chelate MAb a purification procedure based on double anti-idiotype affinity chromatography was shown to result in greater than 95% pure bispecific immunoreactive MAb. Comparative in vivo stability studies profiled DTPA-Y as the chelate of choice for in vivo application.     

Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines.

The ability of monoclonal antibody (MAb 108), an immunoglobulin G (IgG)2a against the epidermal growth factor receptor (EGF-R), to interact with lung cancer cell lines was investigated. 125I-EGF bound with high affinity to non-small-cell lung cancer (NSCLC) cells, and MAb 108 inhibited specific binding of nine NSCLC cell lines in a dose-dependent manner (IC50 = 0.3-3 micrograms per ml). 125I-MAb 108 bound with high affinity (kd = 2 nM) to a single class of sites (Bmax = 70,000 per cell) using NSCLC neuroendocrine cell line NCI-H460. Specific 125I-MAb 108 binding was inhibited with high affinity by MAb 108 but not by a control antibody IgG using large-cell carcinoma cell line NCI-H1299. 125I-MAb 108 binding was not internalized at 37 degrees C using NSCLC neuroendocrine cell line NCI-H460 and adenocarcinoma cell line NCI-H23. Also, 1 microgram per ml of MAb 108 but not of a control IgG inhibited the clonal growth of NCI-H23 and squamous cell carcinoma cell line NCI-H157 in vitro. Also, MAb 108 inhibited xenograft formation of cell lines NCI-H460, NCI-H157, and NCI-H727 in nude mice in vivo. After a palpable tumor had formed using NCI-H460 cells, injection of 100 micrograms of MAb 108 (intraperitoneally three times weekly) inhibited xenograft volume in nude mice by approximately 50%. These data suggest that MAb 108 may interact with EGF receptors on lung cancer cell lines and inhibit NSCLC proliferation.     

Reduced blood accumulation of biotinylated monoclonal antibody A7 after the subsequent administration of avidin

Clear immunoscintigraphy with radiolabeled monoclonal antibodies (MAbs) requires a high tumor tissue/blood ratio of radioactivity. In this study, we attempted to obtain a high tumor tissue/blood ratio by the active removal of radiolabeled MAb from the circulation, using the avidin–biotin system. Biotinylated ¹²⁵I-labeled MAb A7 was injected intravenously into nude mice bearing a human colon cancer (WiDr) xenograft. Avidin was injected 24 h later. The tumor tissue/blood ratio of radioactivity was almost four times that of controls. These results suggest that biotinylated ¹²⁵I-labeled MAb A7 and avidin are potentially useful for the rapid immunodetection of human colon cancer.     

Comparison of the pharmacokinetics, biodistribution and dosimetry of monoclonal antibodies OC125, OV-TL 3, and 139H2 as IgG and F(ab')2 fragments in experimental ovarian cancer.

Monoclonal antibody (MAb) 139H2 was previously shown to localise specifically into ovarian cancer xenografts in nude mice. MAb 139H2 was compared with MAbs OC125 and OV-TL 3, all reactive with ovarian carcinomas, for the binding characteristics as IgG and F(ab')2 fragments with the use of the OVCAR-3 cell line grown in vitro and as s.c. xenografts. Immunoperoxidase staining of OVCAR-3 tissue sections with MAbs OC125 and 139H2 was heterogeneous, whereas MAb OV-TL 3 showed homogeneity. No differences in binding were observed between IgG and F(ab')2. The avidity expressed as apparent affinity constants of MAbs OC125, OV-TL 3 and 139H2 for OVAR-3 cells were 1 x 10(9) M-1, 1 x 10(9) M-1, and 1 x 10(8) M-1, while the number of antigenic determinants were 5 x 10(6), 1 x 10(6) and 7 x 10(6), respectively. In OVCAR-3 bearing nude mice the blood half-lives of the MAbs as IgG and F(ab')2 were approximately 50 h and 6 h, respectively. Maximum tumour uptake for the whole MAbs OC125, OV-TL 3, 139H2 and a control MAb 2C7 was 8.5%, 17.7%, 11.1% and 2.5% of the injected dose g-1, reached at 72 h after injection. For the respective F(ab')2 fragments, the maximum values were 5.2%, 10.0%, 5.5% and 1.9% of the injected dose g-1, reached between 6 h and 15 h. Tumour to non-tumour ratios were more favourable for the F(ab')2 fragments as compared to those for MAbs as IgG. Biodistribution in mice bearing a control tumour confirmed the specificity of tumour localisation of MAbs OC125, OV-TL 3 and 139H2. After injection of a tracer dose of 10 microCi of radiolabelled MAbs OC125, OV-TL 3 and 139H2 as IgG, tumours received 38 cGy, and 9 cGy. In our OVCAR-3 model, a ranking in efficiency in tumour localisation would indicate MAb OV-TL 3 as most favourable MAb, but cross-reactivity with subpopulations of human white blood cells might hamper its clinical use. Dosimetric data indicate a 4-fold higher radiation absorbed dose to tumours for IgG compared with F(ab')2 fragments.     

Predicted and observed effects of antibody affinity and antigen density on monoclonal antibody uptake in solid tumors.

The uptake and binding of monoclonal antibodies (MAbs) in solid tumors after a bolus i.v. injection are described using a compartmental pharmacokinetic model. The model assumes that MAb permeates into tumor unidirectionally from plasma across capillaries and clears from tumor by interstitial fluid flow and that interstitial antibody-antigen interactions are characterized by the Langmuir isotherm for reversible, saturable binding. Typical values for plasma clearance and tumor capillary permeability of a MAb and for interstitial fluid flow and interstitial volume fraction of a solid tumor were used to simulate the uptake of MAbs at various values of the binding affinity or antigen density for a range of MAb doses. The model indicates that at low doses, an increase in binding affinity may lead to an increase in MAb uptake. On the other hand, at doses approaching saturation of antigen or when uptake is permeation limited, an increase in the binding affinity from moderate to high affinity will have only a small effect on increasing MAb uptake. The model also predicts that an increase in antigen density will greatly increase MAb uptake when uptake is not permeation limited. Our experiments on MAb uptake in melanoma tumors in athymic mice after injection of 20 micrograms MAb (initial plasma concentration, about 120 nM) are consistent with these model-based conclusions. Two MAbs differing in affinity by more than 2 orders of magnitude (3.8 x 10(8) M-1 and 5 x 10(10) M-1) but with similar in vivo antigen densities in M21 melanoma attained similar concentrations in the tumor. Two MAbs of similar affinity but having a 3-fold difference in in vivo antigen density in SK-MEL-2 melanoma showed that the MAb targeted to the more highly expressed antigen attained a higher MAb concentration. We also discuss the model predictions in relation to other experiments reported in the literature. The theoretical and experimental findings suggest that, for high dose applications, efforts to increase MAb uptake in a tumor should emphasize the identification of an abundantly expressed antigen on tumor cells more than the selection of a very high affinity MAb.     

Characterisation of a humanised bispecific monoclonal antibody for cancer therapy.

A humanised bispecific monoclonal antibody (bsMAb) with binding specificity for carcinoembryonic antigen (CEA) on one arm and a radiolabelled chelate (DTPA-90Y) on the other arm was generated by consecutively transfecting the humanised genes of an anti-CEA MAb and the chimerised genes of an anti-chelate MAb into eucaryotic BHK cells using the calcium-phosphate coprecipitation technique. The antibodies secreted were of IgG3 isotype with a shortened hinge region (delta gamma 3) and light chains. Double transfectomas were screened for the secretion of bsMAbs using a double determinant enzyme-linked immunosorbent assay (ELISA) based on solid phase attached HSA-benzyl-DTPA and an anti-idiotypic MAb selective for the CEA-specific arm. After purification on two immunoaffinity chromatography columns, the humanised bsMAbs were characterised by SDS-PAGE and a quantitative binding assay in antigen excess. The purification procedure resulted in 95% reactive bispecific MAb. This humanised bsMAb may be employed in two phase radioimmunotherapy, binding to the tumour via the anti-CEA arm and localising a radiolabelled chelate with the other arm, without inducing a strong immune response observed sometimes with murine MAbs.     

Chimeric anti-N-glycolyl-ganglioside and its anti-idiotypic MAbs: immunodominance of their variable regions

P3 monoclonal antibody (MAb) is a murine IgM that specifically recognizes N-glycolyl (NeuGc)-gangliosides and sulfatides. It also reacts with antigens expressed in human breast tumors and melanoma. In syngeneic model, P3 MAb is able to elicit a strong anti-idiotypic (Ab2) antibody response, even in the absence of adjuvants or carrier proteins. 1E10 MAb is an anti-idiotypic antibody specific for P3 MAb that has demonstrated anti-tumoral effects in syngeneic and allogeneic animals. Here we report the construction of the human IgG(1) chimeric P3 and 1E10 antibodies, and the evaluation of the maintenance of the main properties of the murine MAbs. Chimeric P3 antibody specifically reacted with GM3(NeuGc) and GM2(NeuGc) gangliosides, and not with their acetylated variants. Also, it strongly recognized the anti-idiotypic 1E10 MAb. Chimeric 1E10 antibody specifically reacted with P3 MAb. Upon immunization of Balb/c mice with both chimeric antibodies, we were able to demonstrate the immunodominance of their variable regions. The anti-idiotypic response induced by both antibodies was strong and in most of the mice was even significantly higher than the anti-isotypic response, despite the fact that 70% of the chimeric molecule is xenogenic with respect to the animal model.     

Different behaviour of mouse-human chimeric antibody F(ab')2 fragments of IgG1, IgG2 and IgG4 sub-class in vivo.

Mouse-human chimeric monoclonal antibodies (MAbs) of 3 different human IgG sub-classes directed against carcinoembryonic antigen (CEA) have been produced in SP-0 cells transfected with genomic chimeric DNA. F(ab')2 fragments were obtained by pepsin digestion of the purified chimeric MAbs of human IgG1, IgG2 and IgG4 sub-class and of parental mouse MAb IgG1. The 4 F(ab')2 fragments exhibit similar molecular weight by SDS-PAGE. They were labelled with 125I or 131I and high binding (80 to 87%) to purified unsolubilized CEA was observed. In vivo, double labelling experiments indicate that the longest biological half-life and the highest tumour-localization capacity is obtained with F(ab')2 from chimeric MAb of human IgG2 sub-class, whereas F(ab')2 from chimeric MAb IgG4 give very low values for these 2 parameters. F(ab')2 from chimeric MAb IgG1 and from parental mouse MAb yield intermediate results in vivo. Our findings should help to select the appropriate human IgG sub-class to produce chimeric or reshaped MAb F(ab')2 to be used for tumour detection by immunoscintigraphy and for radioimmunotherapy.     

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.     

Comparison of monoclonal antibodies 17-1A and 323/A3: the influence of the affinity on tumour uptake and efficacy of radioimmunotherapy in human ovarian cancer xenografts.

The low-affinity monoclonal antibody (MAb) chimeric 17-1A(c-17-1A) and the high-affinity MAb mouse 323/A3 (m-323/A3) were used to study the effect of the MAb affinity on the tumour uptake and efficacy of radioimmunotherapy in nude mice bearing subcutaneously the human ovarian cancer xenografts FMa, OVCAR-3 and Ov.Pe. Both MAbs are directed against the same pancarcinoma glycoprotein. In vitro, the number of binding sites on tumour cells at 4 degrees C was similar for both MAbs, but m-323/A3 had an approximately 5-fold higher affinity (1.3-3.0x10(9) M-1) than c-17-1A (3.0-5.4x10(8) M-1). This difference in affinity was more extreme at 37 degrees C, when no binding of c-17-1A could be observed. MAb m-323/A3 completely blocked binding of c-17-1A to tumour cells, whereas the reverse was not observed. Immunohistochemistry showed a similar but more intense staining pattern of m-323/A3 in human ovarian cancer xenografts than of c-17-1A. In vivo, the blood clearance in non-tumour-bearing nude mice was similar for both MAbs with terminal half-lives of 71.4 h for m-323/A3 and 62.7 h for c-17-1A. MAb m-323/A3 targeted better to tumour tissue, but was more heterogeneously distributed than c-17-1A. The cumulative absorbed radiation dose delivered by m-323/A3 to tumour tissue was 2.5- to 4.7-fold higher than that delivered by c-17-1A. When mice were treated with equivalent radiation doses of 131(I)m-323/A3 and 131(I)c-17-1A, based on a correction for the immunoreactivity of the radiolabelled MAbs, m-323/A3 induced a better growth inhibition in two of the three xenografts. When the radiation doses were adjusted to obtain a similar amount of radiation in the tumour c-17-1A was more effective in tumour growth inhibition in all three xenografts.     

Monoclonal antibody Po66 uptake by human lung tumours implanted in nude mice: effect of co-administration with doxorubicin.

The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabelled monoclonal antibodies. In this work, the effect of doxorubicin on the biodistribution of Po66, an MAb directed against an intracellular antigen, was studied in nude mice grafted with the human non-small-cell lung carcinoma cell line SK-MES-1. After injection on day 0 of 125I-labelled Po66, tumour radioactivity increased up to days 3-5, and then remained unchanged to day 14. The combined administration of 125I-labelled Po66 with 8 mg kg-1 doxorubicin, in two doses separated by 7 days, doubled the radioactivity retained by the tumour. Histological and historadiographic analysis showed, however, that the drug induced cellular damage. In the absence of doxorubicin, the accumulation of Po66 was restricted to some necrotic areas, whereas with doxorubicin the necrosis was more extensive and the antibody more evenly distributed. These results suggest that chemotherapy and immunoradiotherapy combined would enhance tumour uptake of radioisotope and promote more homogenous distribution of the radiolabelled MAb. This would promote eradication of the remaining drug-resistant cells in tumours.     

Anti-idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon-carcinoma xenografts as efficiently as AB1

BALB/c mice were immunized with anti-idiotypic monoclonal (MAb) antibody (anti-Id or Ab2) directed against an ABI Mab anti-carcinoembryonic (CEA) in order to obtain AB3 MAbs (anti-anti-Id). AB3 MAbs were shown to recognise the primary antigen (CEA) and one of them was tested extensively in vitro and in vivo. This AB3 MAb was shown to bind specifically to CEA on frozen sections of a human colon carcinoma by immunoper-oxidase. Scatchard plot analyses showed that the affinity of this AB3 was of the same order of magnitude as the ABI. In vivo experiments, in nude mice bearing CEA-producing human colon-carcinoma xenografts showed that up to 30% of the intravenously injected dose of ¹²⁵I-labelled AB3 were localized per gram of tumour tissue. Furthermore, calculation of the ratios of AB3 concentration in the tumour over those in normal organs such as lung, liver, kidney, spleen and bone gave relatively high values similar to results obtained with ABI. All together our results show that AB3 can localize as efficiently and specifically in the tumour as ABI, despite the fact that the mice from which it was derived were immunized with a mouse MAb (AB2) and had never been exposed to CEA. © 1994 Wiley-Liss, Inc.     

Pilot trial of murine monoclonal antibodies in patients with advanced melanoma

We have performed a pilot trial with two murine monoclonal antibodies (MAbs) directed against two surface membrane antigens, p97 (MAb 96.5) and a proteoglycan antigen (MAb 48.7) primarily expressed in human melanoma. Five patients with disseminated melanoma were studied, all of whom had multiple cutaneous metastases. Four patients received 212 mg each of antibodies 96.5 and 48.7, and one patient received 424 mg of antibody 96.5 alone. MAbs were administered in escalating doses over ten days in four patients and over six days in one patient. There was no clear treatment-related toxicity. Immunohistologic studies on biopsies taken two to 240 hours after treatment showed extensive binding of murine immunoglobulin to melanoma cells, but not to normal cells in the same section. The intensity of antibody binding was uniform across the diameter of the tumor nodules. In two patients, no murine immunoglobulin was detected in biopsies taken ten days after the last treatment. The mean initial elimination half-life (T1/2) of infused MAbs was 40.5 hours in two patients who received a combination of both antibodies and 53.0 hours in a third patient who received only antibody 96.5; none of these patients had previously been exposed to mouse immunoglobulin. The elimination T1/2 was 21 hours in a fourth patient, who three months previously had tumor imaging with 2-mg radiolabeled antigen-binding fragments (Fab) prepared from antibody 48.7. Serum from this patient appeared to contain anti-idiotypic antibodies which specifically bound Fabs of antibody 48.7. Three other patients also developed human anti-mouse antibodies. There were no objective tumor regressions, and no histologic changes were noted on biopsy.