Gm Boxer

PHAGE LIBRARIES FOR GENERATION OF CLINICALLY USEFUL ANTIBODIES

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

A Mouse Model for Calculating the Absorbed Beta-Particle Dose from 131 I- and 90 Y-Labeled Immunoconjugates, Including a Method for Dealing with Heterogeneity in Kidney and Tumor

Abstract Flynn, A. A., Green, A. J., Pedley, R. B., Boxer, G. M., Boden, R. and Begent, R. H. J. A Mouse Model for Calculating the Absorbed Beta-Particle Dose from 131I- and 90Y-Labeled Immunoconjugates, Including a Method for Dealing with Heterogeneity in Kidney and Tumor. Radiat. Res. 156, 28–35 (2001). Conventional internal radiation dosimetry methods assume that the β-particle energy is absorbed uniformly and completely in the source organ and that the radioactivity is distributed uniformly in the source. However, in mice, a considerable proportion of the β-particle energy can escape the source organ, resulting in large cross-organ doses. Furthermore, the distribution of radioactivity is generally heterogeneous in kidney and tumor. Therefore, a model was developed to account for cross-organ doses and for the effects of heterogeneity in kidney and tumor in mice for two of the most important radionuclides used in therapy, 131I and 90Y. Most mouse organs were modeled as single-compartment ellipsoids or cylinders, while heterogeneity in kidney and in tumor was addressed by using two compartments to represent the cortex and the medulla and viable and necrotic cells, respectively. The dimensions of these models were taken from previous studies, with the exception of kidney and tumor, which were defined using radioluminography and mosaics of high-power microscopy images. The absorbed fractions in each compartment were calculated using β-particle point dose kernels. The self-organ dose was significantly higher for 131I compared to 90Y in all compartments, but a considerable amount of β-particle energy was shown to escape the source organ for both radionuclides, with as much as 85% and 36% escaping the marrow for 90Y and 131I, respectively. The cortex was found to occupy a greater proportion of the total kidney volume than the medulla, and consequently the self-dose was higher in the cortex. In addition, the thickness of the viable shell in the tumor increased with tumor size, as did the self-dose fractions in both necrotic and viable areas. This dosimetry model improves dose estimates in mice and gives a conceptual basis for considering dosimetry in humans.

Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer

Abstract Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues. Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue. We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA). Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic. Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries. MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery. Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer. We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFα) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

Microdistribution of targeted, fluorescently labeled anti carcinoembryonic antigen antibody in metastatic colorectal cancer: Implications for radioimmunotherapy

Purpose: Most radioimmunotherapy studies on radiolabeled antibody distribution are based on autoradiographic and radioluminographic data, which provide a lack of detailed information due to low resolution. We used fluorescently labeled anti–carcinoembryonic antigen (CEA) antibody (A5B7) to investigate quantitatively the kinetics and microdistribution of antibody in a clinically relevant orthotopic colorectal cancer model (LS174T) using high-resolution digital microscopy. Experimental Design: Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to morphologic images of the same or adjacent tumor sections. Results: Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with tumor size at 10 minutes and 1 hour (P ≤ 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were positively related to tumor size (P ≤ 0.001). Conclusion: The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.

Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

Characterisation and radioimmunotherapy of L19-SIP, an anti-angiogenic antibody against the extra domain B of fibronectin, in colorectal tumour models

Angiogenesis is a characteristic feature of tumours and other disorders. The human monoclonal antibody L19- SIP targets the extra domain B of fibronectin, a marker of angiogenesis expressed in a range of tumours. The aim of this study was to investigate whole body distribution, tumour localisation and the potential of radioimmunotherapy with the L19-small immunoprotein (SIP) in colorectal tumours. Two colorectal tumour models with highly different morphologies, the SW1222 and LS174T xenografts, were used in this study. Localisation and retention of the L19-SIP antibody at tumour vessels was demonstrated using immunohistochemistry and Cy3-labelled L19-SIP. Whole body biodistribution studies in both tumour models were carried out with 125I-labelled L19-SIP. Finally, 131I-labelled antibody was used to investigate the potential of radioimmunotherapy in SW1222 tumours. Using immunohistochemistry, we confirmed extra domain B expression in the tumour vasculature. Immunofluorescence demonstrated localisation and retention of injected Cy3-labelled L19-SIP at the abluminal side of tumour vessels. Biodistribution studies using a 125I-labelled antibody showed selective tumour uptake in both models. Higher recorded values for localisation were found in the SW1222 tumours than in the LS174T (7.9 vs 6.6 %ID g−1), with comparable blood clearance for both models. Based on these results, a radioimmunotherapy study was performed in the SW1222 xenograft using 131I-Labelled L19-SIP (55.5 MBq), which showed selective tumour uptake, tumour growth inhibition and improved survival. Radio- and fluorescence-labelled L19-SIP showed selective localisation and retention at vessels of two colorectal xenografts. Furthermore, 131I-L19-SIP shows potential as a novel treatment of colorectal tumours, and provides the foundation to investigate combined therapies in the same tumour models.

A comparison of image registration techniques for the correlation of radiolabelled antibody distribution with tumour morphology

Image registration is a powerful tool for correlating functional images with images of anatomical structure. This facilitates more accurate quantitation of regional radiopharmaceutical uptake. Similarly, registration of images of radiolabelled antibody distribution, in tissue sections, with the equivalent histological images allows the comparison and measurement of radiopharmaceutical distribution with morphological structure. The images used were obtained by storage phosphor plate technology, for the radiopharmaceutical distribution, and by digitization of the stained histological sections. Here we compare four fully automatic registration techniques and one manual technique in terms of their spatial accuracy. We have found that there was no difference in accuracy between cross-correlation, minimization of variance and mutual information. These techniques were more accurate than principal axes and the manual technique. However, minimization of variance and mutual information were more time-consuming than the other methods. Consequently, cross-correlation is the method of choice for automatic registration of large numbers of these image pairs.

Effectiveness of radiolabelled antibodies for radio-immunotherapy in a colorectal xenograft model: a comparative study using the linear--quadratic formulation.

Purpose : To develop a model that relates the pattern of dose delivery during radio-immunotherapy to biological effect. This model was used to assess the efficacy of a range of antibodies labelled with 131 I, 186 Re and 90 Y. Materials and methods : Pharmacokinetic data were obtained by injecting tumour-bearing nude mice with radiolabelled antibody. The dose-rate in bone marrow and tumour was then given by a two-compartment model description of the pharmacokinetics combined with the radionuclide properties. Response characteristics of tumour and marrow were defined in terms of radiosensitivity, repair capacity and proliferation, and the biological effect was assessed using the linear-quadratic formulation. Results : Tumour-specific antibodies with intermediate molecular weight and clearance from the circulation delivered the most effective doses to tumour due to their rapid uptake and prolonged retention in tumour coupled with efficient clearance from blood. Matching the radionuclide with antibody pharmacokinetics and tumour type further increased this effect. Conclusions : The model improves conceptual understanding of the relationship of parameters affecting therapy and makes it possible to optimize radio-immunotherapy by selecting the most effective antibody and radionuclide according to tumour biology.PURPOSE To develop a model that relates the pattern of dose delivery during radio-immunotherapy to biological effect. This model was used to assess the efficacy of a range of antibodies labelled with 131I, 186Re and 90Y. MATERIALS AND METHODS Pharmacokinetic data were obtained by injecting tumour-bearing nude mice with radiolabelled antibody. The dose-rate in bone marrow and tumour was then given by a two-compartment model description of the pharmacokinetics combined with the radionuclide properties. Response characteristics of tumour and marrow were defined in terms of radiosensitivity, repair capacity and proliferation, and the biological effect was assessed using the linear quadratic formulation. RESULTS Tumour-specific antibodies with intermediate molecular weight and clearance from the circulation delivered the most effective doses to tumour due to their rapid uptake and prolonged retention in tumour coupled with efficient clearance from blood. Matching the radionuclide with antibody pharmacokinetics and tumour type further increased this effect. CONCLUSIONS The model improves conceptual understanding of the relationship of parameters affecting therapy and makes it possible to optimize radio-immunotherapy by selecting the most effective antibody and radionuclide according to tumour biology.

The effect of serum CEA on the distribution and clearance of anti-CEA antibody in a pancreatic tumour xenograft model.

A human pancreatic adenocarcinoma was used to develop two histologically distinct xenograft lines, one associated with high levels (180-2000 ng ml-1) and one with low levels (greater than 2.0 less than 8.0 ng ml-1) of serum carcinoembryonic antigen (CEA). A strong correlation was found between tumour size and both circulating and tumour CEA levels in the former group, and also correlation at the 5% level between tumour size and serum CEA in the latter. Administration of either monoclonal or polyclonal 125I-anti-CEA antibody led to the formation of intravascular antigen-antibody immune complexes in mice with high CEA levels, and these were rapidly cleared by the liver, deiodination commencing within the first hour. Blood activity was reduced to 20% of the injected dose by 15 min, and by 24 h the radioactivity in all tissues except muscle was significantly below that found in either the low CEA group or in mice without tumours. No difference in radio-antibody clearance pattern was found between mice without tumours and the group with low levels of serum CEA. In spite of higher levels of CEA within the tumour in mice with elevated serum CEA, the rapid clearance of antigen-antibody complexes reduced tumour localisation to one quarter of that seen in mice with low serum, and correspondingly low tumour, CEA levels. Gamma-camera imaging confirmed these results. Possible implications to patient selection and treatment are discussed.

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.