The selection of antibodies for targeted therapy of small-cell lung cancer (SCLC) using a human tumour spheroid model to compare the uptake of cluster 1 and cluster w4 antibodies.

Spheroids of a small-cell lung cancer (SCLC) cell line POC were used to evaluate the uptake and penetration of two antibodies recognising different SCLC antigens. Spheroids approximately 300-400 microns in diameter were incubated with 1 microgram ml-1 125I-labelled NY.3D11, an antibody which reacts with the cluster 1 group antigen (neural cell adhesion molecule; NCAM) and [125I]SWA11, which binds to the cluster w4 antigen. The rate of uptake of both antibodies was similar; an initially rapid phase was seen during the first 8 h and maximum uptake occurred by 24 h. The mean uptake per spheroid at 24 h was 0.97 ng for [125I]NY.3D11 and 0.45 ng for [125I]SWA11. An objective measurement of antibody penetration into spheroids was developed using a computerised image analysis of immunostained sections of spheroids. The concentration of antibody and incubation times were varied. Both antibodies penetrated the spheroids to a depth of 50 microns after 30 min. This increased to about 100 microns after 4 h incubation with 1 or 100 micrograms ml-1 SWA11. The results with 1 microgram ml-1 NY.3D11 were similar, but in the presence of 100 micrograms ml-1 NY.3D11 penetration into the spheroid was deep and diffuse. These results demonstrate a major concentration-dependent difference in the uptake and penetration of cluster 1 and cluster w4 antibodies in this spheroid model and they have implications for the selection of antibodies for targeted therapy of SCLC.     

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.     

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.     

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.     

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).     

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.     

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.     

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab'')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab'')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab'')2 gave highest tissue values of 125I, followed by cationic OC125F(ab'')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab'')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab'')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.     

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.     

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.     

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.     

Immunoscintigraphy of small-cell lung cancer: a study using technetium and indium labelled anti-carcinoembryonic antigen monoclonal antibody preparations.

Immunoscintigraphy with radiolabelled anti-carcinoembryonic antigen monoclonal antibody was performed on 21 patients with active small-cell lung cancer. Patients received either In-111-labelled Mab F6 F(ab'')2 fragments or Tc-99m-labelled BW 431/26 intact antibody. Tumour was imaged in 13 patients (62%). Of 38 known sites of disease, 18 sites were positively detected. Serum CEA levels were known in 19 patients and were abnormally elevated in three (tumour being detected in all three patients). Eight of 15 patients with normal serum CEA had positive imaging. Using the In-111-labelled antibody seven out of ten patients (nine out of 18 sites) gave positive scans; with the Tc-99m-labelled antibody these were obtained in nine out of 18 patients (nine out of 20 sites).     

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.     

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.     

小细胞肺癌抗独特型疫苗功能的试验研究

目的探讨小细胞肺癌(SCLC)抗独特型抗体3F6和其单链抗体(3F6ScFv)诱导体液和细胞免疫应答的能力,以证明其作为抗SCLC疫苗的可行性。方法3F6和3F6ScFv(Ab2)免疫BALB/c小鼠获得抗血清,以ELISA和Westernblot方法分别检测抗血清中Ab3结合SCLC细胞膜表面特异抗原(NCI H128抗原)的能力,用竞争Westernblot检测Ab3与2F7竞争结合NCI H128抗原的能力。以迟发型超敏反应(DTH反应)和小鼠脾脏淋巴细胞增殖实验检测3F6及3F6ScFv诱发细胞免疫应答的潜能。结果ELISA和Westernblot方法均证明,3F6和3F6ScFv免疫同系小鼠所产生的Ab3能特异的与NCI H128抗原相结合,与对照血清相比,差异有统计学意义(P<0.001),且有很强的与2F7(Ab1)竞争结合靶抗原的能力。在DTH反应中,3F6和3F6ScFv所致小鼠足垫肿胀的程度均明显高于对照组(P<0.001)。小鼠脾脏淋巴细胞增殖反应试验证明,用3F6和3F6ScFv免疫的小鼠脾脏淋巴细胞对靶细胞的再次刺激有明显的增殖反应,与阴性肿瘤细胞对照组和阴性抗体对照组相比,差异有统计学意义(P<0.05)。结论抗独特型抗体3F6和3F6ScFv均有模拟SCLC细胞膜表面特异抗原的能力,成功诱导了相应的体液和细胞免疫应答,可作为抗SCLC疫苗进行深入研究。     

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.     

Tumor uptake of intravenously administered radiolabeled antibodies in ovarian carcinoma patients in relation to antigen expression and other tumor characteristics

To study factors that possibly influence the heterogeneous tumor uptake of radiolabeled antibodies, tissues from 34 ovarian-carcinoma patients were obtained 2 to 8 days after i.v. injection with radiolabeled murine OV-TL 3 or chimeric MOv 18 (cMOv18). The tumor uptake and the ratio of tumor to normal tissue (T/NT) were studied in relation to the histopathological classification, prior treatment, site of tumor, time interval, antigen expression, volume percentage of (malignant) epithelium in the tumor tissue, and the size of the tumor. The results of immuno-scintigraphy were also included. In addition, autoradiography using storage phosphor technology was performed on tissue sections from patients injected with iodine-labeled cMOv18. Tumor uptake varied largely, not only between patients, but also between tumor deposits within the same patient. Uptake of OV-TL 3 F(ab')₂ was higher than of cMOv18 F(ab')₂, but the T/NT ratios were similar. The antibody uptake was positively correlated with the pattern of antigen expression and inversely correlated with the time interval between injection and surgery. No correlation was observed with any of the other factors studied. The visibility with immunoscintigraphy was related to the size of the detected lesion, but not to the other factors studied. Autoradiography showed that antibodies preferentially localized in areas with cancer cells, which were immunohisto-chemically positive for MOv18. In areas with weak antigen expression, autoradiography revealed less activity. The antigen expression by the tumor is an important factor for estimation of the tumor uptake of radiolabeled antibodies. © 1995 Wiley-Liss, Inc.     

Radiolocalization of human small cell lung cancer and antigen-positive normal tissues using monoclonal antibody LS2D617

The murine monoclonal antibody LS2D617, which reacts with an antigen associated with human small cell lung carcinoma (SCLC), was tested in preclinical models to assess its potential for specific targeting of tumors in human SCLC cancer patients. LS2D617 detects a cell antigen on the surface of cultured SCLC and neuroblastoma cell lines. Scatchard analysis of the binding of LS2D617 to NCIH69 SCLC cells indicates an affinity constant of about 1 x 10(8) M-1 and an epitope expression level of approximately 2 x 10(6) antigenic sites/cell. Molecular weight analysis of the target antigen and antibody competition experiments showed that LS2D617 should be classified as a SCLC Cluster 1 antibody (i.e., reacts with the neural cell adhesion molecule). LS2D617 was labeled with 111In and tested for biodistribution (4, 24, 48, 72, and 96 h postinjection) in nude mice bearing the human SCLC NCIH69 tumor. Tumor values peaked at about 35% injected dose/g (Day 3) compared with about 8% injected dose/g for an irrelevant IgG1 antibody while normal tissue accumulation for both antibodies was about 2-8% injected dose/g. Immunohistochemical studies demonstrated that LS2D617 reacts with the central nervous system, peripheral nerves, endocrine tissues, and heart tissue of rabbits as it does in human tissues. The ability of LS2D617 to accumulate in vivo in normal tissues that express the specific target antigen was tested in rabbits. Rabbits given i.v. injections of 111In-LS2D617 or control labeled antibody were sacrificed at 48 h and tissues were examined by gamma well counting, autoradiography, and immunohistochemical staining for murine immunoglobulin. Specific uptake was seen in all sites defined as antigen positive by immunohistology (i.e., heart, liver bile duct, peripheral nerves, pituitary, adrenal), excepting the central nervous system (brain and spinal cord) which was inaccessible to antibody because of the blood brain barrier. The use of preclinical in vivo targeting models to assess tumor as well as antigen-positive normal tissue targeting should aid in the strategy of antibody-based therapeutic intervention of human cancer by providing insight into the potential for tumor targeting and normal tissue toxicity that may be encountered in the clinic.     

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.     

Internalization and intracellular processing of an anti B-cell lymphoma monoclonal antibody,ll2

The successful clinical experience with antibody LL2 (an IgG_2a, anti-B-cell lymphoma antibody) in radipimmunodetection and radioimmunotherapy suggests that this antibody may have potential as a carrier of cytotoxic agents. The internalization, cellular trafficking, and catabolism of this antibody in target human Burkitt lymphoma cells (Raji) were investigated. Internalization of intact antibody as well as of the F(ab')₂ and Fab' fragments was detected by an FITC-labeled anti-mouse second antibody probe, and evaluated by fluorescence microscopy. Internalization of intact IgG (or the fragments) was observed as early as 5 min after incubation at 37°C. Initially, the internalized antibodies were present as micro-particles inside the cell membrane, and were translocated to the lysosomal compartment within 2 hr. The anatomic location of the internalized antibody, before translocation to the lysosomal compartment, was deduced by comparing the fluorescence images obtained with the antibody to those obtained with fluorescent probes with known cellular distribution in a co-internalization study. A Golgi-like compartment was found to be involved in the translocation of the antibody. Cellular catabolism of the bound antibody was studied by using ¹²⁵I-labeled antibody on the target cells. At 21 h, 40% of the radioactivity was released into the supernatant as degraded fragments. The observation suggested that the antibody was degraded mainly in the lysosomes, since the degradation was significantly inhibited in the presence of lysosomal inhibitors such as ammonium chloride or leupeptin. Subcellular fractionation of Raji cells after the binding of ¹²⁵I-lbeled LL2 indicated that the antibody was translocated to lysosomes as evidenced by SDS-PAGE. The rate of internalization (K) of LL2, and the re-expression of the antigen were determined. The rapid Internalization of LL2 and the reexpression of the antigen suggest that this antibody may have potential as a therapeutic immunoconjugate, since it could deliver a higher accumulation of cytotoxic agents into lymphoma cells.