Specific localization and imaging of amyloid deposits in vivo using 123I-labeled serum amyloid P component.

Highly specific, high-resolution scintigraphic images of amyloid-laden organs in mice with experimentally induced amyloid A protein (AA) amyloidosis were obtained after intravenous injection of 123I-labeled serum amyloid P component (SAP). Interestingly, a much higher proportion (up to 40%) of the injected dose of heterologous human SAP localized to amyloid and was retained there than was the case with isologous mouse SAP, indicating that human SAP binds more avidly to mouse AA fibrils than does mouse SAP. Specificity of SAP localization was established by the failure of the related proteins, human C-reactive protein and Limulus C-reactive protein, to deposit significantly in amyloid and by the absence of human SAP deposition in nonamyloidotic organs. However, only partial correlations were observed between the quantity of SAP localized and two independent estimates, histology and RIA for AA of the amount of amyloid in particular organs. It is not clear which of the three methods used reflects better the extent or clinical significance of the amyloid deposits but in vivo localization of radiolabeled SAP, detectable and quantifiable by gamma camera imaging, is apparently extremely sensitive. These findings establish the use of labeled SAP as a noninvasive in vivo diagnostic probe in experimental amyloidosis, potentially capable of revealing the natural history of the condition, and suggest that it may also be applicable generally as a specific targeting agent for diagnostic and even therapeutic purposes in clinical amyloidosis.

Site-specific polysialylation of an antitumor single-chain Fv fragment.

Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.

Antibody-guided localization of intraperitoneal tumors following intraperitoneal or intravenous antibody administration.

Intraperitoneal tumors from a human cancer cell line (LoVo) were established in nude mice by i.p. inoculation of a single cell suspension. Two preparations of the same monoclonal antibody, radiolabeled with 125I and 131I were injected i.p. and i.v. into the same animals. Localization was assessed by dissection and counting the activity in tumors and normal tissues. Tumor/tissue ratios 1 h after i.p. injection of antibody were approximately 50 times higher than after i.v. administration. This i.p./i.v. advantage fell to around 4 by 8 h and was just greater than 1 by 24 h. This effect was observed with both specific and nonspecific antibody, indicating that it is due to the route of administration. However, the absolute amounts of antibody bound to tumors depended on the specificity of the antibody. Twenty % of the injected dose of specific antibody was bound per gram to tumor 1 to 2 h after i.p. injection, falling to 10%/g by 24 h and remaining at this level up to 5 days after antibody administration. In contrast, less than 10%/g of nonspecific antibody was detected in tumors after 1 h; this fell rapidly to normal organ levels of less than 5%/g by 8 h. This study demonstrates a major advantage when administering radiolabeled monoclonal antibodies i.p. for targeting intraperitoneal tumors.

Enhancement of monoclonal antibody uptake in human colon tumor xenografts following irradiation.

Indium-111-labeled AUA1 tumor-associated monoclonal antibody raised against an antigen of colon adenocarcinoma was used to evaluate the effect of ionizing radiation on antibody uptake by the LoVo adenocarcinoma cell line grown as a xenograft in nude mice. Tumors were exposed to single doses of external X-irradiation of between 400 and 1600 cGy followed, 24 h later, by administration of specific or nonspecific antibody. Animals were sacrificed 3 days after antibody administration. At doses higher than 400 cGy, tumor uptake with both specific and nonspecific antibody was significantly increased. No difference in changes in tumor volume was observed between the groups receiving irradiation and the controls. Specific antibody uptake by tumors was always significantly higher than nonspecific having an approximate 4-fold binding advantage. Vascular permeability and the vascular volume of irradiated and control tumors was measured 24 and 72 h after irradiation, using iodine-125-labeled nonspecific antibody and labelling of the red blood cells in vivo with 99mTcO4. At doses higher than 400 cGy, vascular permeability in the tumor 24 h after irradiation was significantly increased (P less than 0.05), while the vascular volume decreased (P less than 0.001) compared to control values. However at 72 h after irradiation there was no difference between treated and control groups. The results obtained in this study suggest a potential value of external irradiation to increase monoclonal antibody uptake by tumors governed mainly by the increased vascular permeability of the tumor vasculature soon after the irradiation exposure.

Intravesical administration of radiolabeled antitumor monoclonal antibody in bladder carcinoma.

Tumor associated AUA1 monoclonal antibody and 11.4.1. nonspecific monoclonal antibody, which does not react with human tissues, were radiolabeled with 111In and administered intravesically to 23 patients undergoing cystoscopy for bladder carcinoma. The antibody solution remained in the bladder for 1 h and then was washed out prior to cystoscopy. Tumor and nontumor samples were obtained during cystoscopy and were counted in a gamma counter. Conventional and immunoperoxidase staining with both antibodies were also performed. AUA1 reacted with all bladder carcinomas while 11.4.1. was negative in all cases. The mean uptake of AUA1 at 2, 24, and 48 h after the instillation (expressed as 10(3) x percentage of injected dose/g of tissue) was: 6.12 +/- 5.50 (SD), 1.70 +/- 2.57, 0.30 +/- 0.17 in the tumors and 0.32 +/- 0.50, 0.22 +/- 0.30, 0 in the nontumor areas, and for 11.4.1. it was: 0.075 +/- 0.075, 0.025 +/- 0.025 in the tumors and 0.30 +/- 0.42, 0.15 +/- 0.26 in the nontumor areas. The uptake of AUA1 by the tumors correlated with the tumor grade. There was no radioactivity in the blood at 2 h, and at 1, 2, and 3 days after the instillation. Our results indicate that intravesical administration of radiolabeled monoclonal antibody AUA1 targets selectively to tumor tissue without any significant normal tissue uptake. This finding might allow the development of a nontoxic and specific therapeutic approach for superficial bladder carcinoma.

Enhancement by gamma-interferon of in vivo tumor radiolocalization by a monoclonal antibody against HLA-DR antigen.

Athymic nu/nu (nude) mice bearing s.c. human breast tumors were treated systemically with recombinant human gamma-interferon. These tumors were phenotypically negative for HLA-DR prior to therapy, but after 4 days of treatment, 80% of the cells expressed this antigen in vivo as assessed by immunoperoxidase (F. R. Balkwill et al., Eur. J. Cancer Clin. Oncol., in press, 1986). A radioiodine-labeled murine monoclonal antibody (TAL-1B5) against HLA-DR specifically localized to the tumors in recombinant human gamma-interferon-treated but not in control mice. An isotype-identical murine monoclonal antibody that did not react with control or recombinant human gamma-interferon-treated tumors did not show any specific localization. These results demonstrate that specific localization to tumors of radio-labeled monoclonal antibodies to HLA-DR can be facilitated by systemic therapy with gamma-interferon.

Uptake and distribution of specific and control monoclonal antibodies in subcutaneous xenografts following intratumor injection.

Nude mice bearing s.c. xenografts of the human colon adenocarcinoma HT29 were given intratumor injections of a mixture of 125I-labeled specific antibody (AUA1) and 131I-labeled control antibody (HMFG1), or with the labels reversed. After dissection at 1 and 4 h postadministration, both specific and control antibodies had 47-63% of the injected dose (% ID) in the tumor. By 24 h, the tumor contained 43 +/- 11% ID of AUA1 which persisted at around this level for 5 days and remained at nearly 20% ID at 18 days. In contrast, the HMFG1 activity was 23 +/- 9% ID at 24 h, which continued to fall and was less than 5% ID by 7 days. Normal organ levels were less than 2% ID/g for both antibodies, with HMFG1 being higher than AUA1 at all times, resulting in specificity indices greater than 20 by 5 days. Autoradiography of tumors removed 2 h postinjection of 125I-labeled AUA1 or HMFG1 showed high levels of antibody at the injection site. At 48 h and 7 days postinjection, the specific antibody was bound to the surface of tumor cells in islands remote from the injection site, whereas the control antibody was found only in the stroma and blood vessels, or as diffuse nonspecific uptake. These data indicate that intratumor injection of radiolabeled monoclonal antibodies may achieve high radiation doses in accessible tumors without systemic irradiation.

Preexisting human anti-murine immunoglobulin reactivity due to polyclonal rheumatoid factors.

We report that sera from healthy controls, patients with ovarian or lung cancer, and patients with rheumatoid arthritis all contain IgM polyclonal rheumatoid factors which recognize antigenic determinants on murine and to a greater extent human immunoglobulin IgG. The major part of this reactivity is directed against conserved, shared antigenic determinants present on both human and murine IgG. Such antigenicity resides in the protein and not the carbohydrate moiety of IgG, since deglycosylation of the target murine monoclonal antibody did not result in any loss of antibody binding. Studies comparing the binding of polyclonal and monoclonal rheumatoid factors (from patients with rheumatoid arthritis and mixed essential cryoglobulinaemia, respectively) to murine and human IgG show that the antigenic determinants recognized by polyclonal rheumatoid factors are present on both whereas the antigenic determinant recognized by the monoclonal rheumatoid factors is present only on human IgG. Furthermore, patients with rheumatoid arthritis display an elevated human IgM anti-murine immunoglobulin response similar to that seen in cancer patients who have received murine monoclonal antibody therapy. We therefore conclude that, where possible, F(ab')2 fragments of murine monoclonal antibodies should be used for in vivo tumor localization studies to avoid possible immune complex formation, and that patients with rheumatoid arthritis should be considered to be possibly at higher risk of developing immune complex disease, were these rheumatoid factors to bind to the administered murine antibodies in vivo.

Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms.

Tumor-associated monoclonal antibodies were radiolabeled with 125I and 131I and given i.v. in pairs to 19 patients 1-26 days prior to surgical excision of primary and metastatic breast, ovarian, and gastrointestinal tumors. For individual patients each monoclonal antibody was designated as specific or nonspecific according to prior immunoperoxidase staining results on the appropriate target neoplastic tissues. Quantitation of antibody uptake was performed on resected normal and neoplastic tissues. Although good tumor:non-tumor ratios were obtained with the specific antibodies (maximal tumor:blood ratio, 35.8:1 at 12 days postadministration), the absolute amount of radiolabel detected in tumors was small (mean value of 0.015% of total injected amount per g of tumor occurring 1 day postadministration). Furthermore, both specific and nonspecific antibodies accumulated in normal lymph nodes to a significant extent (mean value of 0.0026% of total injected amount per g of tissue occurring 1 day postadministration). Knowledge of such data is essential prior to considering therapeutic uses of radiolabeled monoclonal antibodies.

Radiolabeled antibody combined with external radiotherapy for the treatment of head and neck cancer: reconstruction of a theoretical phantom of the larynx for radiation dose calculation to local tissues.

We propose to use radiolabeled antibodies in combination with external beam radiotherapy to improve locoregional control of head and neck cancer. In this case radiation toxicity to mucosa may become a dose-limiting factor and a calculation of the possible compensatory decrease to the external beam radiotherapy would be needed. For this purpose, the following theoretical phantom of a representative organ of this anatomic region, the larynx, was reconstructed and local dosimetric data were derived for a selection of beta-emitting isotopes. The phantom was reconstructed as cylindrical concentric tubes using the established values of an outer diameter of 38 mm and a height of 44 mm. Published mean adult larynx weight (28 g) and cartilage weight (14.7 g) were used. Mean mucosa weight from 5 mucosa samples of our patients was calculated to be 2.0 +/- 0.4 (SD) g. The remaining weight was apportioned to a fat/muscle compartment (11.3 g). The specific gravity of cartilage (1.10 g/cm3), mucosa (1.04 g/cm3), and fat/muscle (1.04 g/cm3) were used to cross-check the volume/mass disparity of the theoretical tubular tissue shells. The established maximum glottic diameter of 24 mm was used to calculate the central air column volume. Mean laryngeal tumor volume from 8 representative laryngeal tumors was 4.4 +/- 3.1 cm3. Tissue compartment thickness was 660 microns for mucosa, 3100 microns for muscle/fat, and 3320 microns for cartilage. These values allowed the calculation of dose absorbed fractions for a number of theoretical radioimmunoconjugates by extending the established calculation of absorbed fractions for spheres of known diameter to absorbed fractions of tissue planes (annuli) of known thickness. We calculated a Deq for the respective tissues in the larynx for 131I-, 186Re-, 188Re-, 67Cu-, 90Y-, and 153Sm-labeled HMFG1. Compensatory decrease to the external radiotherapy dose is 1.1 Gy for each injection of the radioimmunoconjugate we propose to use (131I-HMFG1). This would be best implemented through the modification of the external radiotherapy fractions falling within 2 effective half-lives of this radioconjugate in the mucosa.

Pharmacokinetics, biodistribution, and dosimetry of specific and control radiolabeled monoclonal antibodies in patients with primary head and neck squamous cell carcinoma.

The pharmacokinetics, biodistribution, and dosimetry of an IgG1 radiolabeled anti-mucin mAb (HMFG1) and an isotype-matched control (4D513) were studied in 29 patients with primary head and neck squamous cell carcinoma. Patients were given injections at 3 fixed time points prior to surgery, i.e., 24 (n = 12), 48 (n = 9), or 72 (n = 8) h. They were subsequently classified into two groups based on their immunohistochemical positivity for polymorphic epithelial mucin. Fourteen patients (48%) were positive, 5 of which were studied with both antibodies; and 15 patients were negative (52%), 7 of which were studied with both antibodies. There was no significant difference in serum pharmacokinetics and cumulative urinary clearance of the two antibodies. There was no significant difference in overall normal tissue uptake of specific and control antibody; however, when each component of the normal tissue category was analyzed individually, there was a significantly increased uptake of HMFG1 in mucosa as compared to control antibody. Immunohistochemical studies revealed positive staining of mucosa with HMFG1. There was significantly increased uptake of specific antibody in antigen-positive tumors as compared to uptake of control antibody (P < 0.02). A tendency for less label loss over time from positive tumors as compared to control was documented. Absolute antibody uptake and tumor/normal tissue ratios demonstrated significant overlap in individual patients from each category depending on the specific ratio (e.g., tumor/adipose tissue) or time point studied; hence arbitrary cutoff values could not be recommended as indicators of specific uptake. Specificity and localization indices were the most reproducible indicators of specific localization. Areas under the curve were calculated for all tissues, and local dosimetry for the two beta-emitting isotopes 131I and 90Y is presented. The Deq values for antigen-positive tumors were 2.9 cGy/mCi for 131I and 9.0 cGy/mCi injected for 90Y. For antigen-negative tumors these values were significantly lower at 0.83 and 2.4 cGy/mCi of 131I and 90Y, respectively. Bone marrow Deq was calculated to be 0.87 cGy/mCi of 131I-HMFG1 injected. Because the purpose of our ongoing research is to assess the therapeutic potential of the combination of radiolabeled antibody and external radiotherapy, detailed dose calculation to local dose-limiting tissues is required. Deq to mucosa was calculated to be 1.1 and 3.8 cGy/mCi of injected 131I and 90Y, respectively. We conclude that a 9-10-Gy dose increment may be achieved in two administrations of 150 mCi of 131I-HMFG1 during a course of external radiotherapy. This may lead to improved control of local disease in patients with head and neck cancer.

Development of humoral immune responses against a macrocyclic chelating agent (DOTA) in cancer patients receiving radioimmunoconjugates for imaging and therapy.

The development of stable immunoconjugates by the advent of macrocyclic metal chelating agents (DOTA) has enabled us to study the ability of 111In-DOTA-labeled monoclonal antibodies to detect tumor lesions in a pilot radioimmunolocalization study, as well as to evaluate the kinetics, toxicity, and efficacy of i.p. administered 90Y-DOTA-labeled murine monoclonal antibody in a Phase I/II clinical trial of advanced ovarian cancer. The development of serum sickness-like reactions in three of six treated patients, in the absence of previous monoclonal antibody administration, led us to study the potential immunogenicity of the new chelate. Six patients with ovarian cancer received 25 mg of HMFG1 monoclonal antibody coupled with 90Y-DOTA (doses of radioactivity, 15 to 25 mCi), administered i.p. Eight patients with various malignant tumors received low doses (220 micrograms to 1 mg) of monoclonal antibodies, labeled with 111In-DOTA, i.v. for imaging studies. Using a solid-phase enzyme-linked immunosorbent assay method, the immunogenicity of DOTA was evaluated. Serial dilutions of patients' sera, before and after imaging or therapy with DOTA-coupled monoclonal antibodies, as well as sera from patients who did not receive DOTA-coupled antibody, were screened on enzyme-linked immunosorbent assay plates coated with human serum albumin (HSA), HSA-2-iminothiolane, and HSA-2-iminothiolane-benzyl-DOTA. All patients treated with i.p. monoclonal antibody developed anti-DOTA antibodies. Four of eight patients who received i.v. "imaging" doses of DOTA-coupled monoclonal antibody developed antibodies against DOTA. The levels of anti-DOTA response correlated with the amount of injected radioimmunoconjugate (r = 0.889, P less than 0.001). None of the patients who received DOTA-coupled antibody had detectable antibodies against the macrocycle before immunoconjugate administration. We then addressed further the restriction of the immune response against the macrocycle. We found that there was no or very low response against the aromatic ring attached to DOTA. Most, if not all, of the immune response is directed against the DOTA ring structure. Affinity purification of anti-DOTA antibody from serum enabled quantitation of these antibodies in the serum of patients. An inverse, statistically significant correlation was observed between the percentage of binding inhibition of a patient's serum to DOTA, by HSA-2-iminothiolane-DOTA (100 micrograms/ml) and the level of anti-DOTA immunoglobulin in the serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody.

Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment.

Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms.

Human anti-mouse immunoglobulin immune responses were studied in ten patients, eight with ovarian cancer and two with grade IV gliomas, diagnosed and treated with radiolabeled (123I, 131I) murine monoclonal antibodies. It was found that serum from these patients before treatment and from 18 control healthy individuals contained detectable antibodies to antigenic determinants on the Fc but not the F(ab')2 portion of mouse immunoglobulin. No change in this reactivity occurred after the initial (imaging) dose of monoclonal antibodies. However, repeated administration of mouse immunoglobulins for therapy resulted in an elevated immune response directed against determinants on both Fc and F(ab')2 regions of mouse immunoglobulin. This response contained increased levels of immunoglobulin M as well as immunoglobulin G and showed a marked prozone effect in our enzyme linked immunosorbent assay system. None of the immunized patients developed a detectable antiidiotypic response.

Antibody guided targeting of non-small cell lung cancer using 111In-labeled HMFG1 F(ab')2 fragments.

Immunoscintigraphy using F(ab')2 fragments of tumor-associated monoclonal antibody HMFG1 was performed in 14 patients with primary and metastatic non-small cell carcinoma of lung cancer. The antibody was conjugated with diethylenetriamine pentaacetic acid and labeled with 111In. Quality control studies showed efficient incorporation of 111In onto antibody (5 mCi/mg), no significant loss of immunoreactivity, and in vitro and in vivo stability. The optimal time for imaging was between 48 and 72 h. Following i.v. administration, serum activity fell rapidly (t1/2a = 2.5 +/- 1.3 (SD) h; t1/2b = 42 +/- 4.5 h). The majority of the radioactivity was associated with the plasma and not with the blood cells. All patients had a significant concentration of 111In in the liver (approximately 20% of the injected dose, 48 h postadministration). No toxicity was encountered. No human antimurine-IgG antibody was detected in any of the patients within 4 months of follow-up, even in patients receiving two administrations of F(ab')2 fragments. Localization of all primary lesions and the majority (80%) of metastatic lesions was achieved. Seven of 14 patients were also studied using a 111In-labeled nonspecific antibody (Fab')2 fragment (4C4). In three patients the specificity index was higher than the other four (P less than 0.05). We conclude that although successful targeting of 111In-labeled (Fab')2 fragments of HMFG1 can be achieved in patients with non-small cell carcinoma of lung, observable tumor localization can also be achieved using a nonspecific antibody. Based on these findings, we recommend that in order to demonstrate specific radioimmunolocalization, patients with lung and possibly other tumor types should be studied using both specific and nonspecific antibodies.

Antibody-guided irradiation of advanced ovarian cancer with intraperitoneally administered radiolabeled monoclonal antibodies.

Twenty-four patients with persistent epithelial ovarian cancer after chemotherapy with or without external beam irradiation, were treated with intraperitoneally administered 131I-labeled monoclonal antibodies HMFG1, HMFG2, AUA1, H17E2, directed against tumor-associated antigens. Acute side effects were mild abdominal pain, pyrexia, diarrhea, and moderate reversible pancytopenia. One patient developed a subphrenic abscess requiring surgical drainage. Eight patients with large volume disease, ie, greater than 2 cm tumor diameter, did not respond to antibody-guided irradiation and died of progressive disease within 9 months of treatment. Sixteen patients had small-volume (less than 2 cm) disease at the time of treatment with radiolabeled antibody. Seven patients failed to respond, and of nine initial responders, four patients remain alive and free from disease 6 months to 3 years from treatment. Analysis of the data on relapse indicated that doses greater than 140 mCi were more effective than lower doses. We conclude that the intraperitoneal administration of 140 mCi or more of 131I-labeled tumor-associated monoclonal antibodies represents a new and potentially effective form of therapy for patients with small-volume stage III ovarian cancer.

CAMPATH-1H monoclonal antibody in therapy for previously treated low-grade non-Hodgkin's lymphomas: a phase II multicenter study. European Study Group of CAMPATH-1H Treatment in Low-Grade Non-Hodgkin's Lymphoma.

PURPOSE: CAMPATH-1H is a human immunoglobulin G1 (IgG1) anti-CD52 monoclonal antibody (MAb) that binds to nearly all B-cell and T-cell lymphomas. We report here the results of a multicenter phase II trial of CAMPATH-1H in patients with advanced, low-grade non-Hodgkin's lymphoma (NHL) who were previously treated with chemotherapy. PATIENTS AND METHODS: Fifty patients who had relapsed (n=25) after or were resistant (n = 25) to chemotherapy were treated with CAMPATH-1H 30 mg administered as a 2-hour intravenous (i.v.) infusion three times weekly for a maximum period of 12 weeks. RESULTS: Six patients (14%) with B-cell lymphomas achieved a partial remission (PR). Patients with mycosis fungoides appeared to respond more frequently (50%; four of eight patients, which included two complete remissions [CRs]). Lymphoma cells were rapidly eliminated from blood in 16 of 17 patients (94%). CR in the bone marrow was obtained in 32% of the patients. Lymphoma skin lesions disappeared completely in four of 10 patients and partial regression was obtained in three patients. Lymphadenopathy and splenomegaly were normalized in only 5% and 15% of patients, respectively. Lymphopenia (< 0.5 x 10(9)/L) occurred in all patients. World Health Organization (WHO) grade IV neutropenia occurred in 14 patients (28%). Opportunistic infections were diagnosed in seven patients and nine patients had bacterial septicemia. Death related to infectious complications occurred in three patients. CONCLUSION: CAMPATH-1H had a significant but limited activity in patients with advanced, heavily pretreated NHL. The most pronounced effects were noted in the blood and bone marrow and in patients with mycosis fungoides. The risk for serious infectious complications needs to be considered for severely ill patients who are evaluated for CAMPATH-1H treatment.

Expression of immunoglobulin heavy chain-ricin A chain fusions in mammalian cells.

Mammalian cell lines were transfected with antibody heavy (H) chain-ricin A chain gene fusions in attempts to assemble a recombinant immunotoxin. We found that a light chain-secreting mouse plasmacytoma cell line can be transfected stably with such a chimaeric gene, but only if the ricin A chain portion is disarmed by genetic means prior to transfection; if not, stable transfection appears to select for genetic inactivation of the transfected gene. Co-expression of an antibody heavy chain-ricin A chain fusion with light chain in non-lymphoid cells results in cell death. We conclude that the ricin A chain moiety retains biological activity precluding the expression of biologically active antibody-ricin A chain fusion proteins in mammalian cells.

Exploiting altered glycosylation patterns in cancer: progress and challenges in diagnosis and therapy.

In this review, we describe the changes in glycosylation and expression of mucins, in particular, polymorphic epithelial mucin (PEM), the product of the MUC1 gene in tumours and normal tissues. In addition, some of the areas where exploitation of altered glycosylation patterns in tumour mucins can be used for the better understanding of the disease process and be applied for in vivo diagnosis and therapy are addressed.

Targeting of tumours with murine and reshaped human monoclonal antibodies against placental alkaline phosphatase: immunolocalisation, pharmacokinetics and immune response.

Anti-tumour monoclonal murine and humanised (reshaped human) antibodies (H17E2 and Hu2PLAP, respectively) against placental alkaline phosphatase (PLAP), radioactively labelled with indium-111 (111In) and iodine-123 (123I), were evaluated for their ability to localise mainly testicular and ovarian tumours in sequential pilot studies of the Hammersmith Oncology Group. 33 patients with active primary and/or metastatic testicular cancer were studied with the [111In]- or [123I]H17E2 antibody. 8 patients with testicular cancer were studied with the same antibody after being rendered free of disease after induction chemotherapy and surgical resection of residual tumour. 3 additional patients, 2 with ovarian cancer and 1 with testicular seminoma, were studied with [111In]H17E2 via a macrocyclic chelating agent (DOTA). 7 patients; 5 with ovarian cancer, 1 with breast cancer, and 1 with gastric cancer, received the reshaped human Hu2PLAP antibody [111In]DOTA labelled. One of these was imaged twice, with H17E2- and Hu2PLAP-DOTA-111In, respectively. In the initial 33 patients with active primary and/or metastatic testicular cancer, the presence of tumour was confirmed and correlated well with conventional radiological diagnostic methods, and in addition, the antibody scan revealed the presence of active disease in 2 patients with negative conventional imaging, but elevated serum tumour markers. In the 8 patients with complete remission (CR), imaging studies with the radiolabelled antibody did not show any localisation. The best images were obtained at 24 and 48 h after the [123I]- and [111In]H17E2, respectively. None of these patients developed human anti-mouse antibody responses (HAMA). Successful imaging with the reshaped human antibody, Hu2PLAP-DOTA-111In, was seen in 3 patients with PLAP-positive tumours (2 ovarian and 1 gastric cancer). The 3 negative patients were 1 in complete remission, 1 with PLAP-negative tumour and 1 who cleared the Hu2PLAP antibody immediately after infusion due to the presence of anti-chelating agent (anti-DOTA) antibodies from a previous H17E2-DOTA-111In scan. One patient with PLAP-negative breast carcinoma had a false-positive scan with Hu2PLAP, showing localisation to the pleural effusion. Antibody pharmacokinetics showed a mean t1/2 beta = 73.1 +/- 30.2 h (n = 5) for Hu2PLAP versus t1/2 beta = 27.2 +/- 5.9 h (n = 3) for H17E2 (P < 0.05). 2 patients receiving Hu2PLAP were excluded due to the rapid clearance of the radiolabel as a result of the presence of high HAMA and anti-chelate antibody levels, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)