A comparative study of PDGFR inhibition with imatinib on radiolabeled antibody targeting and clearance in two pathologically distinct models of colon adenocarcinoma

The potential of radioimmunotherapy to selectively kill tumour cells is well established. However, optimisation is required with regards to increasing tumour localisation of antibodies. We used the PDGF-receptor inhibitor imatinib mesylate to improve tumour-specific antibody localisation in two models of colorectal adenocarcinoma and correlated antibody localisation with changes to tumour microvasculature. Mice bearing human colorectal xenografts (LS174T or SW1222) were treated with imatinib prior to administration of radiolabeled anti-CEA antibodies (¹²⁵I-A5B7). Whole tumour and regional localisation of radiolabeled antibodies were measured. Microvessel density and pericyte coverage were quantified in whole tumours and correlated with ¹²⁵I-A5B7 localisation. Imatinib increased uptake of ¹²⁵I-A5B7 in LS174T but not SW1222 tumours after 48?h (p?<?0.05). Imatinib reduced microvessel density in both models (p?<?0.05) but reduced pericyte attachment to endothelial cells only in SW1222 xenografts (p?<?0.05). Imatinib increases antibody distribution in LS174T tumours but not SW1222 tumours, and this correlated to changes in tumour microvessels. Accelerated clearance of radiolabeled antibody from normal tissues in both models resulted in enhanced tumour to normal tissue ratios. This improvement in tumour/normal tissue ratio has potential clinical benefit from a therapy and imaging perspective, and merits further investigation.     

Radioimmunotherapy of DU-145 tumours in nude mice--a pilot study with E4, a novel monoclonal antibody against prostate cancer

The anti-tumour effect of the 131I-labelled antiprostate monoclonal antibody (MAb) E4 was studied in an experimental model with 41 nude mice, subcutaneously xenografted with a human prostate cancer cell line (DU-145). The mice were divided into four study groups, i.e. one receiving single and another repeated injections of the radiolabelled MAb. A third group was injected with non-labelled MAb, and the fourth served as an untreated control group. The tumour volumes increased similarly in all groups during the 27-day observation period. The tumour tissue was morphologically disintegrated in the group that received repeated radioimmunotherapy (RIT). The tumours from this group contained large fluid-filled cystic parts and demonstrated pronounced cellular and subcellular polymorphism in the remaining viable tumour tissue. The untreated control tumours and single therapy tumours remained solid. The proportion of the total tumour volume that consisted of viable tumour cells, as determined by morphometric techniques, was significantly lower in the 131I-E4-treated groups. The use of 131I-labelled E4 MAb has thus demonstrated a promising therapeutic potential.     

Radioimmunotherapy of human hepatocellular carcinoma xenografts with 131I-labelled antiferritin antibody

The effects of 131-labelled antiferritin polyclonal antibody for the treatment of established hepatocellular carcinoma (HC-04) in athymic nude mice were evaluated. 131I-labelled antiferritin antibody localised specifically to a subcutaneous tumour with a mean of 8.1% of the infused dose per gram of tumour at 24 h after infusion when the experiment was started 15 days after inoculation and with a mean of about 6.5% of the infused dose per gram of tumour when the experiment was started 30 days after tumour transplantation. The concentrations of 131I-antiferritin antibody in tumour delivered a mean of 1994 cGy to tumour following infusion of 500 microCi of radiolabelled antiferritin antibody in the early group and a mean of 1600 cGy in the late group. Treatment with 500 microCi led to regression of the tumour in 55% of animals in the early group and 44% in the late group. In contrast, unlabelled antiferritin and 131I-labelled IgG failed to exert any significant effect on tumour growth. The transplanted tumours in the early groups of animals had relatively higher concentration of ferritin than those in the late group. There was accelerated inhibition of tumour growth and prolonged survival in animals in the early group compared with those in the late group.     

In vitro characterization of 211 At-labeled antibody A33--a potential therapeutic agent against metastatic colorectal carcinoma

The humanized antibody A33 binds to the A33 antigen, expressed in 95% of primary and metastatic colorectal carcinomas. The restricted pattern of expression in normal tissue makes this antigen a possible target for radioimmunotherapy of colorectal micrometastases. In this study, the A33 antibody was labeled with the therapeutic nuclide (211)At using N-succinimidyl para-(tri-methylstannyl)benzoate (SPMB). The in vitro characteristics of the (211)At-benzoate-A33 conjugate ((211)At-A33) were investigated and found to be similar to those of (125)I-benzoate-A33 ((125)I-A33) in different assays. Both conjugates bound with high affinity to SW1222 cells (K(d) = 1.7 +/- 0.2 nM, and 1.8 +/- 0.1 nM for (211)At-A33 and (125)I-A33, respectively), and both showed good intracellular retention (70% of the radioactivity was still cell associated after 20 hours). The cytotoxic effect of (211)At-A33 was also confirmed. After incubation with (211)At-A33, SW1222 cells had a survival of approximately 0.3% when exposed to some 150 decays per cell (DPC). The cytotoxic effect was found to be dose-dependent, as cells exposed to only 56 DPC had a survival of approximately 5%. The (211)At-A33 conjugate shows promise as a potential radioimmunotherapy agent for treatment of micrometastases originating from colorectal carcinoma.     

Morphoregulatory activities of E-cadherin and beta-1 integrins in colorectal tumour cells.

The cadherin family of adhesion molecules are prime mediators of cell-cell interactions while the integrins predominantly mediate cell-matrix and to a lesser extent cell-cell binding specificity. We have recently shown that a human colon carcinoma cell line (SW1222) organizes into glandular structures, with well defined polarity when cultured in three-dimensional type I collagen gel. The current study indicates that SW1222 cells display high levels of E-cadherin (E-cd, epithelial cadherin) by western blotting and immunohistochemical staining. A monoclonal antibody (HECD-1) specific for human E-cd blocks cell-cell adhesion (100%) and inhibits (up to 75%) the glandular differentiation of SW1222 cells growing in collagen gel. Furthermore the anti-beta 1 integrin monoclonal antibody (mAb13) inhibits the glandular differentiation of SW1222 cells (61%) and their cellular binding to type I collagen (60%). However, no significant inhibition of cell-cell adhesion was demonstrated using mAb13 nor the anti-carcinoembryonic antigen monoclonal antibody (PR3B10). These results are consistent with E-cd being a cell-cell adhesion molecule expressed by SW1222 cells. These data indicate that E-cd and beta 1 integrins mediate cell-cell and cell-collagen interactions required for the induction and maintenance of the glandular differentiation of colorectal tumour cells. Thus the down-regulation or loss of E-cd and beta 1 integrins seen in poorly differentiated colorectal tumours may represent one of the abnormalities underlying their progression towards an undifferentiated phenotype in vivo.     

Combretastatin A-4-phosphate effectively increases tumor retention of the therapeutic antibody, 131I-A5B7, even at doses that are sub-optimal for vascular shut-down

Radioimmunotherapy using 131I-A5B7, an anti-CEA antibody, in combination with the vascular disrupting agent, combretastatin A4-phosphate (CA-4-P, 200 mg/kg), has produced tumor cures in SW1222 colorectal xenografts. CA-4-P causes acute tumor blood vessel shutdown, which can be monitored in clinical trials using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). The purpose of this study was to determine the magnitude of the anti-vascular effect of CA-4-P in the SW1222 tumor, at 200 mg/kg and at lower, more clinically relevant doses, using conventional assays; relate effects to changes in DCE-MRI parameters and determine the corresponding effects on tumor retention of 131I-A5B7. The tumor vascular effects of 30, 100 and 200 mg/kg CA-4-P were determined, at 4- and 24-h post-treatment, using DCE-MRI, uptake of Hoechst 33342 for tumor vascular volume and conventional histology for necrosis. The effect of CA-4-P on tumor and normal tissue 131I-A5B7 retention was also determined. A significant reduction in tumor DCE-MRI kinetic parameters, the initial area under the contrast agent concentration time curve (IAUGC) and the transfer constant (Ktrans), was demonstrated at 4 h after CA-4-P, for all dose levels. These effects persisted for at least 24 h for the 200 mg/kg group but not for lower doses. A similar pattern was seen for vascular volume and necrosis. Despite this dose response, all three dose levels increased tumor retention of radio labeled antibody to a similar degree. These results demonstrate that moderate tumor blood flow reduction following antibody administration is sufficient to improve tumor antibody retention. This is encouraging for the combination of CA-4-P and 131I-A5B7 in clinical trials.     

Microvascular biodistribution of L19-SIP in angiogenesis targeting strategies

Introduction

Various strategies using L19-mediated fibronectin targeting have become useful clinical tools in anti-tumour therapy and diagnostics. The aim of our study was to characterise the microvascular biodistribution and binding process during tumour angiogenesis and after anti-angiogenic therapy.

Materials and methods

SF126 glioma and F9 teratocarcinoma cells were implanted into dorsal skin fold chambers (SF126: n = 4; F9: n = 6). Using fluorescence and confocal intravital microscopy the biodistribution process was assessed at t = 0 h, t = 4 h and t = 24 h after intravenous application of Cy3-L19-SIP. Sunitinib treatment was applied for six days and microscopy was performed 2 and 6 days after treatment initiation. Analysed parameters included: vascular and interstitial binding, preferential binding sites of L19-SIP, microvascular blood flow rate, microvascular permeability. Histological analysis included CD31 and DAPI.

Results

L19-SIP showed a specific and time-dependent neovascular binding with a secondary extravasation process reaching optimal vascular/interstitial binding ratio 4 hours after iv administration (F9: L19-SIP: vascular binding: 74.6 ± 14.5; interstitial binding: 46.8 ± 12.1; control vascular: 22,2 ± 16.6). Angiogenic sprouts were preferred binding sites (F9: L19-SIP: 188 ± 15.5; RTV: 90.6 ± 13.5). Anti-angiogenic therapy increased microvascular hemodynamics (SF126: Su: 106.6 ± 13.3 μl/sec; Untreated: 19.7 ± 9.1 μl/sec) and induced increased L19-SIP accumulation (SF 126: t24; Su: 92.6 ± 2.7; Untreated: 71.9 ± 5.9) in therapy resistant tumour vessels.

Conclusion

L19-SIP shows a time and blood-flow dependent microvascular biodistribution process with angiogenic sprouts as preferential binding sites followed by secondary extravasation of the antibody. Microvascular biodistribution is enhanced in anti-angiogenic-therapy resistant tumour vessels.     

Predicting response to radioimmunotherapy from the tumor microenvironment of colorectal carcinomas

Solid tumors have a heterogeneous pathophysiology, which directly affects antibody-targeted therapies. Here, we consider the influence of selected tumor parameters on radioimmunotherapy, by comparing the gross biodistribution, microdistribution, and therapeutic efficacy of either radiolabeled or fluorescently labeled antibodies (A5B7 anti-carcinoembryonic antigen antibody and a nonspecific control) after i.v. injection in two contrasting human colorectal xenografts in MF1 nude mice. The LS174T is moderately/poorly differentiated, whereas SW1222 has a well-differentiated glandular structure. Biodistribution studies (1.8 MBq (131)I-labeled A5B7, four mice per group) showed similar gross tumor uptake at 48 h in the two models (25.1% and 24.0% injected dose per gram, respectively). However, in therapy studies (six mice per group), LS174T required a 3-fold increase in dose (18 versus 6 MBq) to equal SW1222 growth inhibition ( approximately 55 versus approximately 60 days, respectively). To investigate the basis of this discrepancy, high-resolution multifluorescence microscopy was used to study antibody localization in relation to tumor parameters (5 min, 1 and 24 h, four mice per time point). Three-dimensional microvascular corrosion casting and transmission electron microscopy showed further structural differences between xenografts. Vascular supply, overall antigen distribution, and tumor structure varied greatly between models, and were principally responsible for major differences in antibody localization and subsequent therapeutic efficacy. The study shows that multiparameter, high-resolution imaging of both therapeutic and tumor microenvironment is required to comprehend complex antibody-tumor interactions, and to determine which tumor regions are being successfully treated. This will inform the design of optimized clinical trials of single and combined agents, and aid individual patient selection for antibody-targeted therapies.     

Bispecific antibody pretargeting of tumor neovasculature for improved systemic radiotherapy of solid tumors.

PURPOSE: Extra domain B (ED-B) fibronectin is a specific tumor matrix marker for targeting angiogenesis in solid tumors. In this study, the radiotherapeutic potential of the directly radioiodinated divalent anti-ED-B antibody fragment, L19 small immunoprotein (L19-SIP; 75,000 Da), was compared with a pretargeting approach using the bispecific antibody AP39xm679 (bsMAb; 75,000 Da). EXPERIMENTAL DESIGN: The bsMAb was prepared by coupling an anti-ED-B single-chain Fv (AP39) to the Fab' of the murine antibody m679, which binds to the small peptidic hapten histamine-succinyl-glycine (HSG). As an effector molecule for the pretargeting approach, the 111In-labeled HSG-DOTA complex was injected 25 or 41 hours after the bsMAb. The kinetics of both the iodinated bsMAb and the pretargeted 111In-labeled HSG hapten were investigated in mice bearing human glioblastoma xenografts (U251) and compared with the kinetics and tumor accumulation of radioiodinated L19-SIP. 111In and 125I were used as surrogate marker for the therapeutic radioisotopes 90Y/177Lu and 131I, respectively. RESULTS: Tumor uptake of the pretargeted 111In-labeled peptide was significantly higher than 125I-L19-SIP over 7 days. At the calculated maximally tolerated dose for each agent (with the kidney being the dose-limiting organ for pretargeting and the bone marrow for direct targeting), a mouse tumor dose of 146 Gy could be given by pretargeting versus 45 Gy delivered by the direct approach. CONCLUSIONS: These data suggest that pretargeting of ED-B with AP39xm679 and subsequent injection of the 90Y-hapten-peptide would improve the therapeutic efficacy in solid tumors by >3-fold compared with directly radiolabeled 131I-L19-SIP.     

Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.     

Growth factor requirements of human colorectal tumour cells: Relations to cellular differentiation

Human colorectal tumour lines that exhibit different degrees of differentiation were studied to define their growth requirements. The poorly differentiated cell lines SW620, SW480, SW48 and SW837 proliferated in Dulbecco's modified Eagle's medium without exogenously added growth factors. In contrast, the moderately differentiated cell lines SW1222, HT29, PC/JW and LS174T proliferated only in medium supplemented with growth factor. SW1222 and HT29 cells required transferrin for growth, which was improved by other growth-promoting factors including epidermal growth factor (SW1222) and sodium selenite (HT29). PC/JW and LS174T required both insulin and transferrin for optimal growth. The tumour cell lines could be passaged continuously in serum-free medium supplemented with growth factor and in some cases they grew better than in serum-supplemented medium. The serum-free growth conditions should prove useful for studies on differentiation in colorectal cell lines and their interactions with growth factors.     

Immunoscintigraphy of human lung squamous cell carcinoma using an iodine-131 labelled monoclonal antibody (Po66)

Monoclonal antibody (McAb) Po66 has been obtained by immunisation of mice against a human lung squamous cell carcinoma. The in vitro reactivity of the antibody with cancer cells and its ability to localise in human lung cancer xenografts growing in nude mice have been reported earlier. Presented here is the first clinical evaluation of the antibody for scintigraphic detection of tumours. Thirty-three patients with histologically confirmed primary non-small cell lung carcinoma were investigated. Twenty-seven of them were explored at the preoperative stage and six at 6 months after surgery. Biodistribution results were obtained from seven operated patients by combining injections of 131I-radiolabelled Po66 and of 125I-labelled unrelated immunoglobulin. The localisation index was three times higher for this specific antibody. Immunoscintigraphy detected 78% of primary tumours and 100% of recurrences. In this short series of patients, immunoscintigraphy proved helpful in the assessment of tumour spread in four patients by visualising localisations in the mediastinum or the contralateral lung which the CT scan had failed to demonstrate. Immunoscintigraphy was also more efficient than plain chest X-ray for the detection of local tumour recurrences.     

Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab' fragments

We have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab')2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab')2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397-1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab')2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM.     

Tumour targeting of humanised cross-linked divalent-Fab' antibody fragments: a clinical phase I/II study

Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab' cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab' maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab')(2). Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab' maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab' maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab')(2) used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab' maleimide over murine versions of this antibody suggesting that humanised divalent-Fab' maleimide should be a useful vehicle for repeated therapies.     

Inhibition of growth and enhancement of differentiation of colorectal carcinoma cell lines by MAb MR6 and IL-4

We have previously shown that expression of gp 200-MR6, a molecule that is functionally associated with the interleukin-4 receptor (IL-4R), is lost from breast carcinoma cells as malignancy increases. Here we have analysed a series of colorectal carcinoma cell lines and show a similar decrease with increasing malignancy. Moreover, analysis of the HRA-19 cell line, which can exhibit a poorly or a well-differentiated phenotype according to culture conditions, shows that gp200-MR6 is weakly expressed on the former but strongly expressed on the latter. Functional analysis using either IL-4 or monoclonal antibody (MAb) MR6 and the well-differentiated cell line SW1222 revealed that MAb MR6 acts as an agonist for IL-4, with both reagents causing a dose-dependent inhibition of cell division, but greatly enhancing the glandular differentiation of SW1222 in three-dimensional collagen gels. These observations suggest that the gp200-MR6 molecule may act as the product of a tumour suppressor gene and that its loss may be a primary event in tumourigenesis. Int. J. Cancer 71:605-611, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Localisation of monoclonal antibodies reacting with different epitopes on carcinoembryonic antigen (CEA)--implications for targeted therapy.

Antibody targeting has potential for selective delivery of cancer therapy. However, there is a wide variation in the degree of antibody localisation in individual patients with colorectal adenocarcinoma. Colorectal adenocarcinomas are composed of glandular structures separated from fibrovascular stroma by a basal lamina which may represent a significant barrier to extravasated antibody. Basement membrane-associated CEA epitopes may be more accessible to antibodies than those which are cytoplasmic or lumenal. We have investigated by immunohistochemistry and in vivo localisation, the extent to which distribution of antigen epitopes influences targeting. Two monoclonal antibodies (A5B7 and EA77) recognising non-overlapping CEA epitopes were reacted immunohistochemically with samples of 39 tumours. Intensity and site of reaction were assessed for basement membrane, cytoplasmic or lumenal surface association. 125I-labelled antibodies were injected into nude mice bearing LS174T tumour. Per cent injected activity per gram was measured in tumour and normal tissues, 24, 72 and 168 h later. Tissues reacted immunohistochemically for CEA were autoradiographed to assess the relationship of injected antibody to target antigen. Immunohistochemistry showed that A5B7 antibody favours basement membrane aspects of malignant glands; in contrast, EA77 concentrated generally on lumenal surfaces. In vivo localisation showed that per cent inj.act g-1 in tumour for A5B7 reached 36.5% at 24 h. EA77 localised to a lesser extent (9.1% at 24 h), despite a longer circulatory half-life. Autoradiography combined with immunohistochemistry showed A5B7 reacting with antigen close to vasculature after 24 h, slowly penetrating deeper parts of the tumour by 72 h. In contrast, EA77 was confined mainly to fibrovascular stroma, showing little labelling of antigen-positive tumour cells. Localisation differences between A5B7 and EA77 may partly be due to accessibility of epitopes on tumour cells.     

Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

Background:

Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.

Methods:

In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.

Results:

These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

Conclusion:

The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.     

Radioimmunotherapy of transplanted small cell lung cancer with 131I-labelled monoclonal antibody

Monoclonal antibody TFS-4 has previously been shown to react selectively with human small cell lung cancer (SCLC). We evaluated the use of 131I-labelled TFS-4 for the treatment of established human SCLC transplanted in nude mice. The specific accumulation of the antibody in the transplanted tumour was recorded by both scintigraphic and biodistribution studies. Administration of 200 microCi 131I-labelled TFS-4 inhibited tumour growth when compared with the same radiation dose of the control monoclonal antibody. The therapeutic effect was dose-dependent and complete disappearance of the tumour was observed transiently in one out of the three animals following the administration of 500 microCi 131I-labelled TFS-4.     

Orthotopic xenografts of human melanoma and colonic and ovarian carcinoma in sheep to evaluate radioimmunotherapy.

Extrapolation to humans from experimental radioimmunotherapy in nude mouse xenograft models is confounded by large relative tumour size and small volume of distribution in mice allowing tumour uptake of radiolabelled antibodies unattainable in patients. Our large animal model of human tumours in cyclosporin-immunosuppressed sheep demonstrated tumour uptake of targeted radiolabelled monoclonal antibodies comparable with uptakes reported in clinical trials. Sheep immunosuppression with daily intravenous cyclosporin augmented by oral ketoconazole maintained trough blood levels of cyclosporin within the range 1000-1500 ng ml(-1). Human tumour cells were transplanted orthotopically by inoculation of 10(7) cells: SKMEL melanoma subcutaneously; LS174T and HT29 colon carcinoma into bowel, peritoneum and liver; and JAM ovarian carcinoma into ovary and peritoneum. Tumour xenografts grew at all sites within 3 weeks of inoculation, preserving characteristic morphology without evidence of necrosis or host rejection. Lymphatic metastasis was demonstrated in regional nodes draining xenografts of melanoma and ovarian carcinoma. Colonic LS1 74T xenografts produced mucin and carcinoembryonic antigen (CEA). The anti-CEA IgG1 monoclonal antibody A5B7 was radiolabelled with iodine-131 and administered intravenously to sheep. Peak uptake at 5 days in orthotopic human tumour transplants in gut was 0.027% DI g(-1) (percentage of injected dose per gram) and 0.034% DI g(-1) in hepatic metastases with tumour to blood ratios of 2-2.5. Non-specific tumour uptake in melanoma was 0.003% DI g(-1). Uptake of radiolabelled monoclonal antibody in human tumours in our large animal model is comparable with that observed in patients and may be more realistic than nude mice xenografts for prediction of clinical efficacy of radioimmunotherapy.