John T. Kemshead

Phenotypic analysis of four human medulloblastoma cell lines and transplantable xenografts

An extensive panel of monoclonal antibodies (MAb) and monospecific antisera reactive against neuroectodermal-, neuronal-, glial-, and lymphoid-associated antigens, extracellular matrix, HLA, and cell-surface receptors was used to characterize the phenotype of four continuous, karyotypically distinct medulloblastoma cell lines and transplantable xenografts. All four cell lines demonstrated significant reactivity with anti-neuroectodermal-associatcd MAb. No apparent pattern of reactivity with anti-lymphoid MAb was seen; notably, there was a uniform absence of detectable Thy-1. Review of the complete antibody reactivity profile revealed a dichotomy between lines TE-671 and Daoy and lines D283 Med and D341 Med, which have been previously shown to express neurofilament protein in culture and xenografts, and to exhibit neuroblastic morphological features in biopsy and xenograft tissue sections. TE-671 and Daoy reacted with the MAb directed against tenascin, epidermal growth factor (EGF) receptor, HLA-A,B epitopes, β2-microglobulin and 5/8 of the glioma-associated antigens, but did not react with the anti-neurofilament protein (NFP) MAb. D283 Med and D341 Med expressed NFP but did not react with MAb against tenascin, EGF receptor, HLA-A.B epitopes, β2-microglobulin or 6/8 and 7/8 (respectively) of the glioma-associated antigens. The observed phenotypic differences provide a conceptual framework for investigating basic differences in the biological behavior of medulloblastoma. Moreover, the subdivisions can be evaluated for prospective value in tissue diagnosis, cerebrospinal fluid cytology and antibody-mediated imaging and therapy.

Dosimetry of iodine 131 metaiodobenzylguanidine for treatment of resistant neuroblastoma: results of a UK study

In 1987, the United Kingdom Childrens Cancer Study Group (UKCCSG) set up a multi-centre study to investigate the toxicity of iodine 131 metaiodobenzyl-guanidine (mIBG) in the treatment of resistant neuroblastoma. Since December 1987, 25 children suffering from neuroblastoma have been treated with131I-mIBG at six UK centres. All centres followed standardised physics and clinical protocols to provide consistent toxicity and dosimetry data. These protocols describe the methods employed for both the tracer study using131I-mIBG and the subsequent therapy. Whole-body dosimetry calculations were performed on data from the tracer study. The activity administered for therapy was the amount predicted to deliver a predefined whole-body dose. Estimates of doses delivered to various organs during treatment are given in Table 1.

A monoclonal antibody (ERIC-1), raised against retinoblastoma, that recognizes the neural cell adhesion molecule (NCAM) expressed on brain and tumours arising from the neuroectoderm.

The neural cell adhesion molecules (NCAMs) are a family of closely related cell surface glycoproteins thought to play an important role in embryogenesis and development [1]. They are involved in cell-cell interactions via a homophilic binding site. Extensive studies on NCAM expression in the mouse, rat and chicken [1] and less detailed investigation in the human, show that the expression of the various NCAM isoforms are developmentally and spatially regulated [2, 3]. Four major isoforms have been described in the mouse brain. The two largest isoforms of 180 and 140 kDa. are transmembrane proteins which differ primarily in the length of the cytoplasmic tail associated with them, whereas the 120 kDa. isoform is linked to the membrane via a glycosyl-phosphatidylinositol (GPI) anchor. The smallest isoform (115 kDa.) is believed to be a secretory protein [4]. In the human brain, an isoform of 170 kDa. is present, in addition to those present in mouse brain [5]. NCAM isoforms have also been studied in detail in human skeletal muscle. In this instance, a transmembrane protein of 140 kDa. and two GPI linked isoforms of 125 and 155 kDa. are present. The 115 kDa. secretory protein is also present [3]. Since NCAM plays an important role in cell-cell interactions, it is tempting to speculate that it mayalso be important in disease, especially tumour biology. However, extensive literature searches show that there are very few studies on NCAM expression in tumours, particularly human tumours. This may be a reflection of the fact that few monoclonal antibodies have been described in oncology literature that have been fully characterized and shown to recognize human NCAM. Here, using retinoblastoma tissue as an immunogen, we describe a monoclonal antibody designated ERIC-l, that recognizes human NCAM. This reagent has been extensively characterized and we think that as such should be particularly useful in studies on expression of NCAM on a variety of human tumours.

Comparative localization of glioma-reactive monoclonal antibodies in vivo in an athymic mouse human glioma xenograft model

Radioiodinated murine monoclonal antibodies (Mabs) 81C6, Me 1-14, C12, D12, and E9, made against or reactive with human gliomas but not normal brain, and Mab UJ13A, a pan-neuroectodermal Mab reactive with normal human glial and neural cells, were evaluated in paired label studies in the D-54 MG subcutaneous human glioma xenograft model system in nude mice. Following intravenous injection in the tail vein of mice bearing 200-400 mm3 tumors, specific localization of Mabs to tumor over time (6 h-9 days) was evaluated by tissue counting; each Mab demonstrated a unique localization profile. The comparison of localization indices (LI), determined as a ratio of tissue level of Mab to control immunoglobulin with simultaneous correction for blood levels of each, showed Mabs 81C6 and Me 1-14 to steadily accumulate in glioma xenografts, maintaining LI from 5-20 at 7-9 days after Mab injection. Mab UJ13A peaked at day 1, maintaining this level through day 2, and declining thereafter. Mabs D12 and C12 peaked at days 3 and 4, respectively, and E9 maintained an LI of greater than 3 from days 3-9. Percent injected dose localized/g of tumor varied from a peak high of 16% (81C6) to a low of 5% (Me 1-14 and UJ13A). Immunoperoxidase histochemistry, performed with each Mab on a battery of primary human brain neoplasms, revealed that Mabs 81C6 and E9, which demonstrated the highest levels of percent injected dose localized/g of tumor over time, reacted with antigens expressed in the extracellular matrix. This finding suggests that extracellular matrix localization of antigen represents a biologically significant factor affecting localization and/or binding in the xenograft model used. The demonstration of significant localization, varied kinetics and patterns of localization of this localizing Mab panel warrants their continued investigation as potential imaging and therapeutic agents for human trials.

A pilot study of monoclonal antibody targeted radiotherapy in the treatment of central nervous system leukaemia in children.

Summary. A pilot study was performed to investigate the toxicity, pharmacokinetics and therapeutic effect of intrathecally administered radiolabelled monoclonal antibody (MAb) in patients with meningeal acute lymphoblastic leukaemia (ALL). Six children aged 3–16, in second or subsequent central nervous system (CNS) relapse of ALL, received between 629 and 1480 MBq of131Iodine conjugated to either MAb HD37 (CD19, n= 2) or WCMH15.14 (CD10, n= 4). Conjugate was administered as a single injection either via an Ommaya reservoir (n= 4) or by lumbar puncture (n= 2). Acute toxicity was manifest by headache (n= 4), nausea and vomiting (n= 4) and pyrexia (n= 2). All acute symptoms resolved within 72 h. Transient myelosuppression occurred in three patients. Pharmacokinetic studies included investigation of whole body, blood and CSF clearance of isotope. 131I was seen to clear from the CSF by biexponential kinetics. Five patients responded to therapy. In four, the CSF became clear of blast cells at both 2 and 4 weeks following antibody injection, but evidence of relapse was seen at 6 weeks. The fifth patient, with blast cells present on a cytospin preparation, responded to therapy over an 8‐week period but relapsed at 12 weeks. This study demonstrates the potential of targeted radiotherapy in CNS ALL, but further studies are necessary to increase the length of remission.

Pharmacokinetics and dose estimates following intrathecal administration of 131I-monoclonal antibodies for the treatment of central nervous system malignancies

PURPOSE Treatment of malignant disease in the central nervous system (CNS) with systemic radiolabeled monoclonal antibodies (MoAbs) is compromised by poor penetration into the cerebrospinal fluid (CSF), limited diffusion into solid tumors, and the generation of anti-mouse antibodies. To attempt to avoid these problems we have treated patients with diffuse neoplastic meningitis with radioimmunoconjugates injected directly into the intrathecal space. METHODS AND MATERIALS Tumor-specific MoAbs were conjugated to Iodine-131 (131I) (629-3331 MBq) by the Iodogen technique, and administered via an intraventricular reservoir. A clinical response rate of approximately 33% was achieved, with better results in more radiosensitive tumors. Here, we present detailed pharmacodynamic data on patients receiving this intracompartmental targeted therapy. RESULTS Elimination from the ventricular CSF appeared biphasic, with more rapid clearance occurring in the first 24 h. Radioimmunoconjugate entered the subarachnoid space and subsequently the vascular compartment. From this information, the areas under the effective activity curves for ventricular CSF, blood, and subarachnoid CSF were calculated to permit dosimetry. Critical organ doses were calculated using conventional medical internal radiation dose (MIRD) formalism. Where available, S-values were taken from standard tables. To calculate the doses to CSF, brain, and spinal cord, S-values were evaluated using the models described in the text. CONCLUSION A marked advantage could be demonstrated for the dose delivered to tumor cells within the CSF as compared to other neural elements.

The role of monoclonal antibodies in brain tumour diagnosis and cerebrospinal fluid (CSF) cytology

SummaryThis paper reviews the diagnostic role of monoclonal antibody immunohistochemistry in a series of 189 brain tumour biopsies and 22 cases of neoplastic meningitis. The diagnostic monoclonal antibody panel, which includes markers for glial, neural, epithelial and lymphoid differentiation antigens, was used to test a wide variety of cerebral and spinal tumours by indirect immunofluorescence and immunoperoxidase techniques on unfixed frozen sections. Gliomas, meningiomas, schwannomas, medulloblastomas, choroid plexus tumours, cerebral lymphomas and metastatic carcinomas could all be reliably differentiated by means of their characterisctic antigenics profiles, as defined by their patterns of reactivity with the antibody panel. Confident diagnosis was possible even in very poorly differentiated tumours and in biopsies distorted by surgical squeeze artefact, where paucity of morphological clues made diagnosis by conventional histological methods difficult or impossible. It was estimated that use of the antibody panel was responsible for, or made a significant contribution towards the final diagnosis in approximately 20% of cases.The monoclonal reagents were also found to be of great value in the detection and characterisation of neoplastic cells in CSF specimens from patients with malignant meningitis. Malignant cells were detected in 73% of cases and characterised in 16% of cases by routine cytological techniques. Employing monoclonal immunocytology however, these figures were improved to 95% and 95% respectively. Our findings suggest that patients with neoplastic meningtitis can be spared prolonged investigation and inappropriate management by the early detection and characterisation of malignant cells in CSF using panels of monoclonal antibodies.

Radioimmunolocalisation of human brain tumours: Biodistribution of radiolabelled monoclonal antibody UJ13A

Monoclonal antibody UJ13A, radiolabelled with 131I, was intravenously administered to patients with primary brain tumours. The antigen recognised by UJ13A is present on most neuroectodermally derived tissue. The ratio of uptake in tumour to normal brain, assessed by scintigraphy, improved with time. Maximal tumour uptake occurred between 4 and 48 h. Dynamic and static scintigrams indicated some early sequestration of radiolabelled antibody by the liver. Tumours were surgically resected in seven patients at various intervals after antibody administration showing tissue to blood ratios increasing with time in all parts of the lesion (viable and necrotic tumour, cyst fluid), and in normal brain. The highest tissue to blood ratio in viable tumour was 5.1 at 16 days after injection. In tissues resected 2–3 days after injection there was relatively greater uptake in the viable tumour compared to necrotic tumour and cyst fluid. In contrast, tissues resected later (6–16 days) showed greater uptake in ischaemic tissue than viable tumour, suggesting diffusion was an important factor influencing tumour uptake. The amount of radioactivity per gram of tumour tissue was less then 0.005% of the injected dose. Future studies are needed using different antibodies, antibody fragments and additional methods of optimising delivery.

Neoplastic Meningitis from a Pineal Tumour Treated by Antibody-guided Irradiation via the Intrathecal Route

A monoclonal antibody (UJ181.4) was labelled with 131I and given intrathecally to a patient who was critically ill with neoplastic meningitis due to a disseminated pineoblastoma. The target antigen had first been demonstrated on tumour cells by immunocytological testing of cerebrospinal fluid (CSF). As a preparation for therapy, a test dose of 131I-UJ181.4 antibody was given simultaneously with a control antibody labelled with 125I. Good evidence of in vivo immunolocalization was obtained by external gamma counting and by analysis of the radiolabels on centrifuged CSF cells. Specificity ratios of between 10:1 and 20:1 were obtained by these methods. A single relatively small therapeutic dose of 131-I (870 MBq) given by the intrathecal route, resulted in a marked clinical improvement and sustained remission for 22 months.

Dosimetry of intrathecal iodine131 monoclonal antibody in cases of neoplastic meningitis

Radioiodinated monoclonal antibodies (MCA) were administered by the lumbar route into the cerebrospinal fluid (CSF) of four patients with malignant leptomeningeal disease. Evidence suggesting uptake of131I-MCA by tumour sites was seen in scintigrams. Dosimetry calculations were carried out, assuming that a proportion of the administered radionuclide was bound as a thin layer on the CSF surfaces of the meninges. The percentage injected dose and the clearance curves for the head and four spinal segments were obtained by scintigraphy after administration of tracer amounts of131I-MCA (7–18 MBq). Although radioisotope levels in the central nervous system (CNS) fell, as determined by both external scintillation counting and direct CSF sampling, a marked difference in the measurements developed with respect to time. The ratio of these two measurements reached a maximum of 49:1, 7 days after monoclonal antibody administration. Patients subsequently received therapeutic amounts (870–1600 MBq) of131I-MCAs, resulting in clinical remissions and prolonged survival. The mean absorbed radiation dose was estimated as 3.9 cGy·MBq−1 to the thoraco-lumbar region of the spine and 0.51 cGy·MBq−1 to the outer surface of the brain. The maximal dose delivered to the surface of the CNS in the region of the spine and brain was 5800 and 600 cGy, respectively.