Direct comparison of a radioiodinated intact chimeric anti-CEA MAb with its F(ab')2 fragment in nude mice bearing different human colon cancer xenografts.

Tumour localisation and tumour to normal tissue ratios of a chimeric anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), in intact form and as an F(ab'')2 fragment labelled with 125I and 131I, were compared in groups of nude mice bearing four different colon cancer xenografts, T380, Co112 or LoVo, of human origin, or a rat colon cancer transfected with human CEA cDNA, called ''3G7''. For each tumour, three to four mice per time point were analysed 6, 12, 24, 48 and 96 h after MAb injection. In the different tumours, maximal localisation of intact MAb was obtained at 24 to 48 h, and of F(ab'')2 fragment 12 to 24 h after injection. Among the different tumours, localisation was highest with colon cancer T380, with 64% of the injected dose per gram (% ID/g) for the intact MAb and 57% for its F(ab'')2 fragment, while in the three other tumours, maximal localisation ranged from 14 to 22% ID g-1 for the intact MAb and was about 11% for the F(ab'')2. Tumour to normal tissue ratios of intact MAb increased rapidly until 24 h after injection and remained stable or showed only a minor increase thereafter. In contrast, for the F(ab'')2 fragment, the tumour to normal tissue ratios increased steadily up to 4 days after injection reaching markedly higher values than those obtained with intact MAb. For the four different xenografts, tumour to blood ratios of F(ab'')2 were about 2, 3 and 5 to 16 times higher than those of intact antibodies at 12, 24 and 96 h after injection, respectively.     

Superior localisation and imaging of radiolabelled monoclonal antibody E48 F(ab')2 fragment in xenografts of human squamous cell carcinoma of the head and neck and of the vulva as compared to monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 and its F(ab'')2 fragment, radiolabelled with 131I, were tested for tumour localisation and imaging in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma (HNX-HN) or from a vulva carcinoma (VX-A431). MAb IgG or F(ab'')2 fragments were injected in parallel and at day 1, 2, 3 and 6 or 7, mice were either scanned with a gamma camera or dissected for determination of isotope biodistribution. In HNX-HN bearing mice, E48 IgG as well as F(ab'')2 showed highly specific localisation in tumour tissue. The mean tumour uptake (n = 4) expressed as the percentage of the injected dose per gram of tumour tissue (percentage ID/g) of IgG was 11.9% at day 1 and increased to 14.6% at day 6 whereas percentage ID/g of F(ab'')2 was 7.2% at day 1 and decreased during subsequent days. Tumour to blood ratios (T/B) at day 1 were 1.2 for IgG and 13.6 for F(ab'')2 and reached a maximum at day 6 with values of 6.4 and 54.2 respectively. In VX-A431 bearing mice, only E48 F(ab'')2 showed preferential localisation in tumour tissue. At day 1, Percentage ID/g of IgG was 3.7 and T/B was 0.3, while percentage ID/g of F(ab'')2 was 2.4 and T/B was 3.2. Percentage ID/g decreased after day 1 while T/B increased. In these experiments no preferential localisation of either isotype matched 125I-labelled control IgG or F(ab'')2 was observed. In F(ab'')2 injected HNX-HN bearing mice as well as VX-A431 bearing mice, tumours could be visualised at day 1 and 2 without any appreciable background activity. With MAb IgG this was also possible in HNX-HN bearing mice (but not in VX-A431 bearing mice) but only at day 3 and 6. These findings suggest that the superior tumour to non-tumour ratios render the E48 F(ab'')2 fragment more qualified for specific targeting of radioisotopes to tumour xenografts in this experimental setting.     

Radioimmunotherapy of metastatic colorectal tumours with iodine-131-labelled antibody to carcinoembryonic antigen: phase I/II study with comparative biodistribution of intact and F(ab')2 antibodies.

Studies in animal tumour models of colorectal cancer suggest that F(ab'')2 antibody fragments to carcinoembryonic antigen (CEA) labelled with iodine-131 give superior therapy compared with intact anti-CEA antibody. The purpose of this study was to investigate this hypothesis in patients. Ten patients received intact A5B7 IgG1 mouse monoclonal antibody (MAb) to CEA and nine patients received the F(ab'')2 fragment of the same antibody. The biodistribution for each molecule was compared using quantitative single-photon emission computerised tomographic (SPECT) gamma-camera imaging. Tumour responses were seen in both groups and myelosuppression was the limiting toxicity. F(ab'')2 localised more rapidly than intact antibody in tumour, giving a mean percentage injected activity per kg at 4.25 h after injection of 8.2% for F(ab'')2 compared with 4.4% for intact antibody (P < 0.05). No significant difference in antibody clearance from, or cumulative dose per unit administered activity (cGy MBq-1) to, tumour was seen. Distribution in blood was similar for both the intact and fragment antibody. These findings are consistent with more rapid penetration of the smaller F(ab'')2 into tumour masses. More efficient early uptake will give higher maximum dose rates to the tumour which is valuable for radioimmunotherapy (RIT) when low dose rates may limit effectiveness of treatment. F(ab'')2 fragments may provide a substantially enhanced method of delivering RIT.     

In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab'')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab'')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab'')2 to monovalent F(ab'') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab'')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab'')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab''). In normal mice, M26.1 F(ab'')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab'')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab'')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab'')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab'')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).     

Phase I study of intravenously applied bispecific antibody in renal cell cancer patients receiving subcutaneous interleukin 2.

In a phase I trial the toxicity and immunomodulatory effects of combined treatment with intravenous (i.v.) bispecific monoclonal antibody BIS-1 and subcutaneous (s.c.) interleukin 2 (IL-2) was studied in renal cell cancer patients. BIS-1 combines a specificity against CD3 on T lymphocytes with a specificity against a 40 kDa pancarcinoma-associated antigen, EGP-2. Patients received BIS-1 F(ab'')2 fragments intravenously at doses of 1, 3 and 5 micrograms kg-1 body weight during a concomitantly given standard s.c. IL-2 treatment. For each dose, four patients were treated with a 2 h BIS-1 infusion in the second and fourth week of IL-2 therapy. Acute BIS-1 F(ab'')2-related toxicity with symptoms of chills, peripheral vasoconstriction and temporary dyspnoea was observed in 2/4 and 5/5 patients at the 3 and 5 micrograms kg-1 dose level respectively. The maximum tolerated dose (MTD) of BIS-1 F(ab'')2 was 5 micrograms kg-1. Elevated plasma levels of tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were detected at the MTD. Flow cytometric analysis showed a dose-dependent binding of BIS-1 F(ab'')2 to circulating T lymphocytes. Peripheral blood mononuclear cells (PBMCs), isolated after treatment with 3 and 5 micrograms kg-1 BIS-1, showed increased specific cytolytic capacity against EGP-2+ tumour cells as tested in an ex vivo performed assay. Maximal killing capacity of the PBMCs, as assessed by adding excess BIS-1 to the assay, was shown to be decreased after BIS-1 infusion at 5 micrograms kg-1 BIS-1 F(ab'')2. A BIS-1 F(ab'')2 dose-dependent disappearance of circulating mononuclear cells from the peripheral blood was observed. Within the circulating CD3+ CD8+ lymphocyte population. LFA-1 alpha-bright and HLA-DR+ T-cell numbers decreased preferentially. It is concluded that i.v. BIS-1 F(ab'')2, when combined with s.c. IL-2, has a MTD of 5 micrograms kg-1. The treatment endows the T lymphocytes with a specific anti-EGP-2-directed cytotoxic potential.     

The potential for enhanced tumour localisation by poly(ethylene glycol) modification of anti-CEA antibody.

Attachment of poly(ethylene glycol) (PEG) to proteins can greatly alter their pharmacological properties, including extending the plasma half-life and reducing immunogenicity, both of which are potentially beneficial to tumour targeting. IgG, F(ab'')2 and Fab'' fragments of the anti-CEA antibody A5B7 were chemically modified with PEG (M(r) 5,000), labelled with 125I and their pharmacokinetics compared with the unmodified forms in the LS174T colonic xenograft in nude mice. PEG modification of the intact antibody had little effect on biodistribution, although tumour localisation was slightly reduced. In contrast, similar modification of F(ab'')2 and Fab''A5B7 significantly prolonged plasma half-life and increased radioantibody accumulation in the tumour and to a lesser extent in normal tissues, but reduced tissue to blood ratios. Prior to modification, Fab'' A5B7 (M(r) 50,000) cleared more rapidly from the circulation than F(ab'')2 (M(r) 100,000), but after PEG attachment their biodistributions converged, while the tumour to blood ratios were reduced and resembled that of the intact antibody. The enhanced tumour accumulation, reduced normal tissue to blood ratios and potentially reduced immunogenicity of fragments after PEG attachment may therefore prove superior to either unmodified fragments or intact antibody for antibody-targeted therapy, although the increased plasma half-life may necessitate the use of a clearance mechanism.     

Targeting BCL1 lymphoma with anti-idiotype antibodies: Biodistribution kinetics of directly labeled antibodies and bispecific antibody-targeted bivalent haptens

The mouse BCL₁ lymphoma model has been used for evaluating immunotherapy with anti-idiotype (anti-Id) antibodies, including Id immunisation, IgG therapy and bispecific (Bs) antibody-targeted cytotoxicity. Here, we provide quantitative data on the targeting of small (25 ± 12 mg) intrasplenic BCL₁ tumours, using anti-Id IgG, F(ab')₂ and anti-Id × anti-hapten BsF(ab')₂ covalently labelled with ¹²⁵iodine, as well as noncovalent complexes of BsF(ab')₂ and ¹²⁵ I-labelled bivalent hapten. The results are the following: 1) up to 115% of the injected dose per gram (% ID/g) of spleen can be localised in the first hour, corresponding to approximately 600% ID/g of tumour; 2) localisation is specific for cell-surface Id; 3) optimal doses can overcome circulating Id; 4) circulating Id markedly increases the catabolism of IgG, thus impairing tumour localisation; 5) bivalent reagents are internalised by the target cells; 6) iodine covalently bound to bivalent antibodies [IgG, F(ab')₂]; is rapidly (T_1/2: 6-9 hr) released from the tumour; in contrast, the bivalent hapten is retained for a longer time (T_1/2: 25 hr); and 7) in the absence of bivalent hapten, the monovalent BsF(ab')₂ is not rapidly internalised and dissociates from tumour cell-surface Id. Our results suggest that monovalent anti-Id, lacking Fc, can efficiently be targeted to the BCL₁ tumour surface. For radioimmunotherapy, the intracellular targeting of catabolism-resistant ¹²⁵I-labelled bivalent hapten provides optimal tissue selectivity. Int. J. Cancer 71: 1000-1009, 1997. © 1997 Wiley-Liss Inc.     

Increased tumor localization by monoclonal antibody A7 after F(ab')2 fragmentation in athymic nude mice bearing human pancreatic carcinomas

Much recent research has been directed toward the use of monoclonal antibodies (MoAbs) for the immunodetection of solid tumors. In pancreatic cancer, conventional immunoscintigraphy using intact MoAbs remains disappointing. In this study, ¹²⁵I-labeled F(ab')₂ fragments produced by pepsin digestion of MoAb A7 were injected intravenously into nude mice bearing human pancreatic cancer, HPC-YS, xenografts that have previously been shown to react specifically with MoAb A7. The tumor tissue/blood ratio of ¹²⁵I-labeled F(ab')₂ fragments of MoAb A7 increased with time and was much higher than those for normal tissues. Moreover, the tumor tissue/blood ratio of ¹²⁵I-labeled F(ab')₂ fragments was greater than that of intact MoAb A7, although the F(ab')₂ accumulation was less than that of intact MoAb A7 in the tumor. These results suggest that F(ab')₂ fragments of MoAb A7 may be suitable carriers of radionuclides for immunodetection of human pancreatic cancer. © 1993 Wiley-Liss, Inc.     

Comparative radioimmunotherapy using intact or F(ab')2 fragments of 131I anti-CEA antibody in a colonic xenograft model.

The therapeutic efficacy of intact and F(ab'')2 fragments of a 131I anti-CEA antibody were compared in an established LS174T colonic xenograft model in nude mice. A single IV dose of either 0.5 mCi (18.5 MBq) intact or 1.0 mCi (37 MBq) F(ab'')2 fragments significantly delayed tumour growth, and increased survival time to the same extent. Biodistribution studies showed that the more rapid clearance of the fragments from the circulation improved the tumour: normal tissue ratios found for the intact antibody, but reduced the duration and therefore absolute amount of radioantibody localisation (% injected dose/gram) at the tumour site. The tumours received a similar accumulated beta radiation dose, with 4,065 cGy from 0.5 mCi intact antibody and 4,500 cGy from 1.0 mCi F(ab'')2 fragments. The dose rate to the tumour was initially higher for the fragments, but fell off more rapidly as clearance occurred. However, the rapid circulatory clearance resulted in a radiation dose of only 995 cGy to the blood, compared with 2,300 cGy for the intact antibody. This suggests that twice the radiation dose could be delivered to the tumour in the form of fragments for the same blood dose from the intact antibody. Fractionating the 1.0 mCi dose of F(ab'')2 into three doses of 0.33 mCi (12.2 MBq), given on days 1, 3 and 5, significantly reduced the therapeutic effect of the treatment. The clinical relevance of these findings is discussed.     

Immunomodulatory effects of intravenous BIS-1 F(ab')2 administration in renal cell cancer patients.

We report the immunomodulatory effects of an intravenous treatment with F(ab'')2 fragments of the bispecific monoclonal antibody BIS-1 during subcutaneous recombinant interleukin 2 (rIL-2) therapy of renal cell cancer (RCC) patients. BIS-1 is directed against both the CD3 antigen on T cells and the EGP-2 molecule on carcinoma cells and some normal epithelia. The amount of BIS-1 F(ab'')2 bound to peripheral blood lymphocytes (PBLs) increased dose-dependently. This occupation degree was highest at the end of the 2 h infusion and rapidly decreased subsequently. During the first hour of BIS-1 F(ab'')2 infusion the number of PBLs decreased slowly. This was followed by an increase in serum tumour necrosis factor alpha (TNF-alpha) concentrations and a rapid decrease in the numbers of peripheral blood lymphocytes, monocytes and eosinophils. In our view, the most likely explanation for the observed decrease in occupation degree of BIS-1 F(ab'')2 and the rise in TNF-alpha levels is based on the assumption that BIS-1-carrying T cells leave the circulation. The CD3 antigens on these extravasated T cells become cross-linked by EGP-2 antigens, inducing TNF-alpha secretion. This results in an enhanced decrease in the numbers of PBLs, monocytes and eosinophils. These preliminary results suggest that BIS-1 F(ab'')2 treatment during IL-2 therapy may induce local T-cell activation.     

Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')₂ fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv 18 (overexpressed by ovarian carcinoma cells) as part of a phase l/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10⁶?10⁹) of bsAbtargeted T lymphocytes plus lowdose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10⁹ targeted T lymphocytes, 2 mg soluble bsAb, and lowdose IL-2. Using enzymelinked immunosorbent assays (ELISA), human antimouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (I) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18⁺ or CD3⁺ cells by absorption of human antimouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18⁺ ovarian carcinoma cell line lgrov-1 by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.     

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.     

Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab' fragments.

The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab'')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab'' fragments in nude mice bearing human colorectal tumour xenografts. The cross-linkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy beta-emitter. We have also compared the above antibody forms conjugated to both 131I and 90Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37 degrees C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab'')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab'')2 and DFM and elevated splenic uptake of DFM labelled with 90Y was observed. Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 10(5) Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab'')2 radiolabelled with 131I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 131I and 90Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for 131I-labelled antibodies. Because of the faster clearance, it should be possible to administer a higher total dose of 90Y-labelled TFM than IgG, which is attractive for RIT. Both A5B7 DFM and TFM, therefore, show favourable properties compared with their parent antibody forms.     

Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

Polyethylene glycol (PEG) modification of a chimeric Fab'' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab'' to F(ab'')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab'' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.     

Galactosylated streptavidin for improved clearance of biotinylated intact and F(ab')2 fragments of an anti-tumour antibody.

Persistence of high levels of radiolabelled antibody in the circulation is a major limitation of radioimmunotherapy. Biotinylation of the radiolabelled anti-tumour antibody followed by administration of streptavidin is known to give much improved tumour to blood ratios as the radioantibody is complexed and subsequently cleared via the reticuloendothelial system, although prolonged splenic uptake is a problem. We have investigated the effect on the clearance pattern and tumour localisation of a 125I-labelled biotinylated anti-CEA antibody (A5B7) after administration of a galactosylated form of streptavidin (gal-streptavidin) in nude mice bearing a human colon carcinoma xenograft. Fifteen minutes to 1 h after gal-streptavidin administration the complexes were cleared via the liver alone (as opposed to liver and spleen after native streptavidin). Twenty-four hours after administration of gal-streptavidin, the tumour to blood ratio for biotinylated A5B7 IgG increased from 2.9 to 13.2 and for biotinylated F(ab'')2 fragments an increase from 4.9 to 33.2 was achieved. The reduction in tumour accumulation of F(ab'')2 24 h after injection of the clearing agent was less than that seen with intact antibody. Injection of asialofetuin inhibited clearance, confirming that removal of the gal-streptavidin-biotinylated antibody complexes from the blood was via the asialoglycoprotein receptor on liver hepatocytes. Therefore, galactosylation of the streptavidin clearing agent allows rapid removal of radiolabelled biotinylated antibodies via the liver asialoglycoprotein receptor, as opposed to the reticuloendothelial system.     

Altered biodistribution of an antibody--enzyme conjugate modified with polyethylene glycol.

Polyethylene glycol modification of the antibody--enzyme conjugate, F(ab'')2-A5B7-CPG2, extends its duration in the circulation of nude mice bearing human colonic cancer xenografts (LS174T). Increased concentration of modified conjugate is achieved in the tumour, but residual non-specific enzyme concentrations in normal tissue and blood demonstrate the fundamental requirement to remove or inactivate non-specifically held enzyme in this system.     

Biodistribution of a radiolabelled monoclonal antibody NY3D11 recognizing the neural cell adhesion molecule in tumour xenografts and patients with small-cell lung cancer.

The neural cell adhesion molecule (NCAM) is highly expressed on the surface of small-cell-lung cancer (SCLC) cells. We have produced a monoclonal antibody, NY3D11, that binds to NCAM to investigate whether this antigen could be used to develop antibody-directed therapy for SCLC. 125I-labelled IgG and F(ab'')2 fragments of NY3D11 localized selectively in human SCLC xenografts grown in nude mice. The human biodistribution of 131I-labelled NY3D11 after intravenous administration was investigated by gamma-camera imaging in six patients with SCLC. Three patients received IgG and three received F(ab'')2. No evidence of localization to primary tumours or metastases was seen and antibody accumulated rapidly in the liver and bone marrow. The probable explanation for this distribution is that NY3D11 reacted with soluble NCAM or natural killer cells that possess the CD56 (NCAM) antigen.     

Biodistribution of iodine-125 and indium-111 labeled OV-TL 3 intact antibodies and F(ab')2 fragments in tumor-bearing athymic mice.

The monoclonal antibody OV-TL 3, directed against an ovarian carcinoma-associated antigenic determinant, was tested as a vehicle for radioimmunolocalization of ovarian carcinomas in athymic mice bearing NIH:OVCAR-3 xenografts. The biodistribution of intact. OV-TL 3 was compared with the distribution of OC 125. Tumor uptake with OV-TL 3 was significantly higher than with OC 125, and almost 7 times higher than with a non-specific control antibody (OV-TL 19). Administration of a mixture of intact OV-TL 3 and OC 125 did not improve tumor uptake in comparison with OV-TL 3 alone. Subsequently, intact OV-TL 3 and its F(ab')2 fragments were labeled with either 111In or 125I. The highest tumor uptake was obtained with 111In-labeled intact OV-TL 3 (14.7% ID/g, 48 hr p.i.). For both antibody forms uptake of 111In in liver, spleen and kidneys was very high. Furthermore, 111In cleared more slowly from most tissues than 125I. As a result, tumor/tissue ratios with 111In-labeled OV-TL 3 were lower than with 125I-labeled OV-TL 3. The highest tumor/tissue ratios (6.9 to 53) were obtained with 125I-labeled OV-TL 3 F(ab')2 fragments, 48 hr post injection. 111In-labeled OV-TL 3 F(ab')2 has already been shown to be a clinically useful label for the detection of ovarian cancer. The results of our comparative animal study suggest that these clinical results may even be improved by using 123I-labeled OV-TL 3 F(ab')2.     

Localization of radiolabelled F(ab')2 fragments of monoclonal antibodies in nude mice bearing intraperitoneally growing human ovarian cancer xenografts

The biodistribution and pharmacokinetics of 2 monoclonal antibodies (MAbs) specific for ovarian carcinoma, OC125 and OV-TL3, were studied in nude mice bearing intraperitoneally (i.p.) growing human ovarian carcinoma xenografts of NIH:OVCAR-3. The ovarian carcinoma xenografts grew as non-adherent cells in ascites and as solid implants in the peritoneal cavity of injected mice. The biodistribution and pharmacokinetics were determined by measurement of radioactivity in tumor masses, ascites, blood and other tissues after intravenous (i.v.) and i.p. injection of radioiodinated F(ab')2 fragments of MAbs. The specificity of the observed tumor localization was then evaluated by comparing the uptake of the anti-ovarian carcinoma antibodies OC125 and OV-TL3 with the uptake of a radioiodinated non-ovarian carcinoma-specific MAb A2C6. The results of the study indicate that uptake of the anti-ovarian carcinoma antibodies was highest in the non-adherent tumor cells in the ascites after i.p. injection. The observed uptake was 85% injected dose/g for OV-TL3 and 22% injected dose/g for OC125. This compares to the observed antibody uptake of 9% injected dose/g for OV-TL3 and less than 1% injected dose/g for OC125 in solid tumor masses after i.p. injection. After i.v. injection, uptake of OC125 and OV-TL3 was less than 3% injected dose/g, both for nonadherent tumor cells and for solid tumor masses. The data support the conclusion that OV-TL3 is superior to OC125 and that i.p. administration of radiolabelled MAb F(ab')2 fragments is superior to their i.v. administration for immunotherapy of ovarian carcinoma.     

The value of the human milk fat globule membrane antigen HMFG2 in epithelial ovarian cancer monitoring: comparison with CA125.

We assayed serum HMFG2 in serial samples from 215 primary epithelial ovarian cancer patients using an ''in-house'' single determinant ELISA, 45% of patients with stage I, 54% with stage II, 61% with stage III and 75% with stage IV disease had elevated serum HMFG2. Post-operative levels were significantly related with residual tumour volume (P < 0.005), and fell in the majority of responders, although the association with response to first-line chemotherapy was not significant. HMFG2 had a sensitivity of 50% specificity of 83%, accuracy of 61%, PVP of 86% and PVN of 45% for disease at second-look laparotomy. Serial levels gave a lead time to clinical relapse in 47% of patients who responded to therapy, including one patient with negative CA125 levels. HMFG, paralleled CA125 in many respects, although it was elevated in fewer patients. In a stepwise discriminant analysis, HMFG2 added to the discrimination of CA125 (r = 0.183, P < 0.005), although additional accurate information was only given in patients with advanced poorly differentiated serous cystadenocarcinoma. Given that HMFG2 is expressed in few patients who are CA125 negative it is unlikely that it will have a significant clinical impact upon patient management.