ABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Sialic Acids/chemistry , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/metabolism , Amines/chemistry , Animals , Antineoplastic Agents/isolation & purification , Binding Sites , Cell Line , Cysteine , Female , Half-Life , Humans , Mice , Mice, Inbred BALB C , Neoplasms/metabolism , Protein Engineering , Single-Chain Antibodies/genetics , Single-Chain Antibodies/isolation & purification , Substrate SpecificityABSTRACT
The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 nM for the IgG and 10 muM for the Fab), with particularly low but similar dissociation rate constants (0.031-0.095 s(-1)). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.
Subject(s)
Antibodies, Monoclonal/immunology , Mucin-1/immunology , Binding Sites, Antibody , Breast Neoplasms/pathology , Humans , Immunoglobulin Fab Fragments/immunology , Kinetics , Tumor Cells, CulturedABSTRACT
BACKGROUND AND AIMS: The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. MATERIALS AND METHODS: Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. RESULTS: Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. CONCLUSION: PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Carcinoembryonic Antigen/drug effects , Carcinoembryonic Antigen/immunology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/immunology , Aged , Aged, 80 and over , Antibodies, Anti-Idiotypic/blood , Antibodies, Anti-Idiotypic/drug effects , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Murine-Derived , Antibodies, Neoplasm/blood , Antibodies, Neoplasm/drug effects , Antibodies, Neoplasm/immunology , Antigens, Neoplasm/blood , Antigens, Neoplasm/drug effects , Antigens, Neoplasm/immunology , Cancer Vaccines/administration & dosage , Case-Control Studies , Cell Proliferation/drug effects , Cytokines/blood , Cytokines/drug effects , Cytokines/immunology , Dose-Response Relationship, Immunologic , Female , Humans , Hypersensitivity, Delayed/immunology , Immune Sera/drug effects , Immune Sera/immunology , Immunity, Mucosal/drug effects , Injections, Intradermal , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Receptors, Interleukin-2/blood , Receptors, Interleukin-2/drug effects , Receptors, Interleukin-2/immunology , Treatment OutcomeABSTRACT
The use of radiolabeled monoclonal antibodies (MoAbs) has significantly improved the ability to detect tumor antigens, thus improving in vivo tumor diagnosis and treatment. The management of ovarian carcinoma still poses a challenging medical problem. Clinical trials using radioimmunoscintigraphy or a hand-held gamma detection probe intraoperatively were performed in patients with clinical evidence of primary or recurrent ovarian cancer. Immunoscintigraphy of ovarian cancer lesions has been performed mainly with 99mTc, 111In and 123I labeled with HMFG1, HMFG2, OC-125, B72.3, H17E2, OVTL3, MoAb170, Mov18 and other MoAbs. Antibody guided imaging using radioimmunoscintigraphy has demonstrated improved targeting of ovarian cancer, resulting in a highly sensitive and specific method. However, it is not yet known which type of MoAb is the most efficient for radioimmunoscintigraphy. Since these tumors represent a potentially curable disease, radioimmunoscintigraphy could contribute mainly to accurate staging as a supplementary to conventional diagnostic methods, as well as for the localization of active disease after chemotherapy and monitoring for the presence of recurrent disease. Nevertheless, prospective studies in a large number of patients should be undertaken in order to further evaluate the diagnostic contribution of this approach.
Subject(s)
Carcinoma/diagnostic imaging , Germinoma/diagnostic imaging , Ovarian Neoplasms/diagnostic imaging , Radioimmunodetection , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/immunology , Antibody Specificity , Antigens, Neoplasm/analysis , Antigens, Neoplasm/immunology , Biomarkers, Tumor/analysis , Biomarkers, Tumor/immunology , Carcinoma/immunology , Clinical Trials as Topic , Female , Germinoma/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Neoplasm Metastasis/diagnostic imaging , Neoplasm Proteins/analysis , Neoplasm Proteins/immunology , Neoplasm Recurrence, Local/diagnostic imaging , Neoplasm Staging/methods , Ovarian Neoplasms/immunology , RadiopharmaceuticalsABSTRACT
A number of mammalian proteins with suitable biological activities have been considered for use in targeted tumour therapy. Deoxyribonuclease-I (DNase-I), an endonuclease that degrades double-stranded DNA, represents an attractive candidate for tumour targeting since it is normally non-toxic yet could be highly cytotoxic when redirected to the cell nucleus. Our aim was to investigate the cytotoxic potential of mammalian DNase-I and its possible use in tumour-targeting strategies for cancer therapy. A chimeric molecule comprising a scFv reactive against the human placental alkaline phosphatase (hPLAP) and bovine pancreatic DNase-I was designed and investigated. The development of a tightly controlled system for the bacterial expression of DNase-I and its chimera is described. The production and purification of active DNase-I from the soluble cell fraction and significant yields from the insoluble fraction by isolation and refolding are described. The construction, expression, purification and in vitro characterisation of an anti-PLAP scFv-DNase-I chimera is also described. This molecule was shown to possess both antigen-binding and DNA-degrading activity in in vitro assays, thus combining the specific cell-targeting properties of the scFv and the potent, highly catalytic activity of the endonuclease. Furthermore, this chimeric molecule was highly cytotoxic in vitro in cells expressing the PLAP antigen. Targeting mammalian DNase-I provides a novel therapeutic strategy for selective cell killing, with the promise of less systemic toxicity and immunogenicity than currently used immunotoxins.
Subject(s)
Alkaline Phosphatase/immunology , Antineoplastic Agents/pharmacology , Deoxyribonuclease I/pharmacology , Immunoglobulin Fragments/therapeutic use , Immunotoxins/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , Cattle , HumansABSTRACT
PURPOSE: To determine the long term survival of patients with advanced ovarian cancer treated with radioimmunotherapy following cytoreductive surgery and platinum based chemotherapy. PATIENTS AND METHODS: Eligibility criteria included patients with histological evidence of ovarian cancer stages IC-IV following completion of conventional platinum containing chemotherapy. Of 52 patients entered into the study, 31 had residual disease following standard chemotherapy and 21 patients had achieved complete remission. Treatment consisted of one intraperitoneal administration of 25 mg of monoclonal antibody HMFG1 labelled with 18 mCi/m2 of 90Y. Survival was the primary end-point. RESULTS: In the group of 21 patients who had achieved complete remission following surgery, conventional chemotherapy and intraperitoneal radioimmunotherapy, the median survival has not been reached with a maximum follow-up of 12 years. Survival at greater than 10 years is 78%. CONCLUSION: This study suggests that a substantial proportion of patients who achieve complete remission with conventional therapy can achieve a long-term survival benefit when treated with intraperitoneal radioimmunotherapy using HMFG1 labelled with 90Y.
ABSTRACT
To overcome the disadvantages of bi-specific antibody methodologies in vivo, a novel antibody approach has been designed to improve tumour targeting and effector to target ratio. The technique involves biotinylated anti-CD3 Fab fragments and streptavidinylated anti-tumour monoclonal antibodies (mAbs) that can spontaneously form cross-links. We describe here a method for the direct cross-linking of sulphydryl-conjugated HMFG1 (anti-MUC1 mucin mAb) to streptavidin by sulphosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. Fab fragments generated by papain digestion of the 1452C11 antibody (anti-CD3 mAb without Fc to avoid peripheral activation of T-cells) were biotinylated with NHS-Iminobiotin. MUC1-transfected BALB/c breast cancer cell lines 413BCR and 425CCR and the parental cell line (410.4) were labelled with streptavidinylated mouse anti-MUC1 mucin mAb. BALB/c effector T-cells were separately labelled with biotinylated anti-CD3 Fab fragments (1452C11) and mixed with tumour cells in different effector to target ratios. Percentage of killing was assessed using the 51Cr cytotoxicity assay. Seventy per cent lysis was measured in the case of 413BCR (high MUC1 mucin expressor) and 40% in the case of 425CCR (low expressor) cell line. No lysis was apparent in the MUC1 negative cell line. These results demonstrate that the novel T-cell redirecting approach we have developed can produce effective immune lysis of target cells in vitro.
Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neoplasm/immunology , CD3 Complex/immunology , Immunotherapy , Mucin-1/immunology , Neoplasm Proteins/immunology , Animals , Antibody Specificity , Biotin , Female , Immunoglobulin Fab Fragments/immunology , Mice , Mice, Inbred BALB C , Mucin-1/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Streptavidin , T-Lymphocytes/immunology , TransfectionABSTRACT
Although the management of cancer by exploiting properties distinguishing neoplastic and normal cells has always been an attractive concept, it was the development of hybridoma technology and the resulting tumor-associated monoclonal antibodies (MAbs) that offered new prospects for this strategy. Twenty years later, some of the applications of MAbs in oncology are now part of the everyday diagnosis and treatment, while others are the subject of intensive investigation. We reviewed the current applications of MAbs in the diagnosis and treatment of bladder cancer and attempted to put the issue into perspective, with particular presentation of their therapeutic potential.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Urinary Bladder Neoplasms/diagnosis , Urinary Bladder Neoplasms/therapy , Animals , Biomarkers, Tumor/blood , Biomarkers, Tumor/urine , Humans , Hybridomas/immunology , Immunoconjugates/therapeutic use , Immunohistochemistry , Immunotherapy , Immunotoxins/therapeutic use , Liposomes , Prodrugs/therapeutic use , Radioimmunodetection , Radioimmunotherapy , Urinary Bladder Neoplasms/immunology , Urine/cytologyABSTRACT
The targeting potential of three different monoclonal antibodies (MAbs) was assessed in patients with ovarian cancer. HMFG1, OC-125 and H17E2 labelled with 111In or 123I were evaluated prospectively for their ability to localize ovarian tumour. Forty two patients with ovarian cancer, aged 40-78 years (median = 58 years) were studied using OC-125 (n = 9), HMFG1 (n = 11) and H17E2 (n = 22). Imaging data were compared with the CT and the surgical findings. Presence of tumour was confirmed in 35/42 (83%) patients (8/9 OC-125, 10/11 HMFG1 and 17/22 H17E2) and correlated well with the conventional radiology diagnostic methods. One patient with a negative H17E2 scan and a large abdominal mass detected at laparotomy revealed a PLAP-negative tumour on immunohistochemistry. Scintigraphy revealed the presence of active disease, confirmed by laparotomy/laparoscopy in 6/8 patients considered to be in clinical remission. The sensitivity of the method was high enough and the diagnostic contribution of this approach should be further evaluated.
Subject(s)
Antibodies, Monoclonal , Ovarian Neoplasms/diagnostic imaging , Radioimmunodetection/methods , Adult , Aged , Diagnosis, Differential , Female , Humans , Indium Radioisotopes , Iodine Radioisotopes , Middle Aged , Neoplasm Staging , Ovarian Neoplasms/pathology , Sensitivity and SpecificityABSTRACT
Considerable progress has been made over the past decade in the use of tumour-associated monoclonal antibodies (mAbs) as carriers of cytotoxic agents in the management of several malignancies. In the present study we investigated the tumour localization and biodistribution of the mAb HMFG1 to bladder cancer following intravesical administration. HMFG1, which has been raised against the polymorphic epithelial mucin (PEM), was labelled with 125I and administered intravesically 2 h and 24 h before cystoscopy. During cystoscopy, biopsies were taken from the normal bladder and the malignant lesion. Tissue samples were tested for HMFG1 immunostaining and for radioactivity with the use of a gamma-counter. The mean ratio of tumour to normal uptake for 125I-HMFG1 was 5.81 +/- 1.23, at 2 h and 2.17 +/- 0.42 at 24 h. No radioactivity was detected in the blood. We conclude that HMFG1 could be used intravesically for the successful delivery of a cytotoxic agent.
Subject(s)
Mucin-1/immunology , Radioimmunotherapy , Urinary Bladder Neoplasms/radiotherapy , Administration, Intravesical , Antibodies, Monoclonal/therapeutic use , Humans , Immunohistochemistry , Tissue DistributionABSTRACT
Over the last decade several attempts have been made to generate an active drug from an inactive precursor, by the action of an enzyme present predominantly at the tumour site. The aim was to develop a new, less cytotoxic strategy for the treatment of cancer, by exploiting properties distinguishing neoplastic and normal cells. In fact, monoclonal antibodies were used to carry enzymes at the tumour sites, in a two-step approach, known as Antibody Directed Enzyme Prodrug Therapy (ADEPT). We reviewed the experimental and clinical considerations of this strategy and we presented its problems and limitations. We concluded that ADEPT holds the potential of an effective and relatively non-toxic treatment of cancer and it is expected that the research which is in progress will make ADEPT an important element of the anticancer armament.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Enzymes/administration & dosage , Neoplasms/therapy , Prodrugs/administration & dosage , Alkaline Phosphatase/administration & dosage , Animals , Carboxypeptidases/administration & dosage , Humans , Nitroreductases/administration & dosage , Prodrugs/metabolism , beta-Glucosidase/administration & dosageABSTRACT
We describe a novel version of antibody-directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond beta-glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated beta-glucosidase to a tumour-associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1-beta-glucosidase enhanced the cytotoxic effect of amygdalin by 36-fold. When 2 concentrations of HMFG1-beta-glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb-enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb-enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co-culture of HMFG1-positive and-negative cell lines (HT 1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1-negative) cells in the flask. Our findings indicate that ADEPT is more effective than non-directed enzyme activation of a prodrug and can result in a non-toxic cancer therapy.
Subject(s)
Amygdalin/administration & dosage , Antibodies, Monoclonal/administration & dosage , Prodrugs/administration & dosage , Urinary Bladder Neoplasms/drug therapy , beta-Glucosidase/administration & dosage , Antibody Specificity , Antigens, Neoplasm , Cell Survival , Enzyme-Linked Immunosorbent Assay , Humans , Tumor Cells, Cultured , Urinary Bladder Neoplasms/immunologyABSTRACT
We aimed to study in detail the in vivo stoichiometry of the individual elements of the 2-step streptavidin based approach to tumour targeting, in a nude mouse xenograft model, by the administration of a first step consisting of biotinylated anti-tumour specific antibody and a second step consisting of streptavidin. This process was undertaken to identify the optimum conditions for radiotherapeutic tumour targeting using this approach. Antibody was biotinylated to various degrees (1-25 biotins per antibody). Protein stoichiometry of the 2 steps was studied over a range of 2 logs. Both steps, i.e., the biotinylated-antibody (1st step) and streptavidin (2nd step) were radiolabelled (125I and 131I, respectively). A 24-hr interval between 1st and 2nd step was studied, animals being killed 24 hr after the 2nd step. Streptavidin excess led to a decrease in levels of monobiotinylated-antibody in the circulation and in the tumour. Biotinylated-antibody excess led to an increase in circulating levels of streptavidin, a decrease in renal uptake of streptavidin and increased targeting of streptavidin to tumour. At a constant protein molar ratio of biotinylated antibody to streptavidin of 10:1, increasing biotinylation density resulted in an increase in circulating levels, increase in tumour uptake, decrease in renal uptake and increase in liver uptake of streptavidin. As early as 24 hr, the tumour-to-blood ratios of streptavidin already exceeded 1 (max 1.27). Compared with antibody tumour-to-blood ratios, they were better by a factor of between 2 and 3. Tumour-to-normal tissue ratios of radiolabelled streptavidin (with the exception of liver) were also significantly improved when polybiotinylated-antibody was administered first. We have thus shown that the 2-step biotinylated antibody/streptavidin approach can lead to a significant increase in absolute amounts of activity in the tumour under appropriate stoichiometric conditions. This was accompanied by high levels of circulating streptavidin and relatively favourable tumour-to-blood and normal tissue ratios of streptavidin.
Subject(s)
Antibodies, Neoplasm/administration & dosage , Neoplasms, Experimental/radiotherapy , Radioimmunotherapy , Streptavidin/administration & dosage , Animals , Biotinylation , Enzyme-Linked Immunosorbent Assay , Female , Humans , Mice , Mice, Nude , Quality Control , Streptavidin/pharmacokinetics , Transplantation, HeterologousABSTRACT
Integrins are cell-surface glycoproteins found in different forms on all cells except erythrocytes. Integrins bind to cell adhesion molecules and to proteins found in the extracellular matrix. A tripeptidic sequence Arg-Gly-Asp (RGD) is often the primary site of recognition by integrins which are expressed on tumour cells and are responsible for tumour invasion and metastasis. A synthetic decapeptide designated alpha P2 containing two RGD sequences radiolabelled with technetium-99m was used to image malignant melanoma in vivo. Fourteen patients previously diagnosed with metastatic melanoma underwent gamma camera imaging 20-180 min following intravenous administration of the radiolabelled synthetic decapeptide alpha P2. Six out of eight (6/8) of the lymph node metastases (75%) and all other neoplastic sites (11 sites) were successfully imaged, with the exception of three sites in the mediastinal area which were not positively imaged. In two cases there was false positive uptake in the rounded pigmented areolar/nipple area. In three cases (seven sites) the peptide scan confirmed the absence of disease in suspected lesions (true-negative). The synthetic peptide was rapidly removed from the circulation by filtration through the kidneys and excretion in the urine. No toxicity or adverse events were recorded. Radiolabelled alpha P2 peptide, which binds specifically to adhesion molecules on tumours, can be used for the in vivo detection of neoplastic metastases.
Subject(s)
Melanoma/diagnostic imaging , Oligopeptides , Radiopharmaceuticals , Adult , Aged , Aged, 80 and over , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/secondary , Female , Gamma Cameras , Humans , Integrins , Lymphatic Metastasis/diagnostic imaging , Male , Middle Aged , Oligopeptides/adverse effects , Oligopeptides/pharmacokinetics , Radionuclide Imaging , Radiopharmaceuticals/adverse effects , Radiopharmaceuticals/pharmacokinetics , Technetium/adverse effects , Technetium/pharmacokinetics , Tissue DistributionABSTRACT
The ability to effectively define disease status in ovarian cancer after initial therapy or to selectively screen high-risk populations remains a major challenge for in vivo monoclonal antibody (mAb)-targeted approaches. Antitumour murine mAbs (HMFG1, HMFG2, H317, and H17E2) and the reshaped human antibody Hu2PLAP (against placental alkaline phosphatase; PLAP), labelled with indium-111 and iodine-123, were evaluated for their ability to localise ovarian tumours in sequential studies of our group. Thirty patients with ovarian cancer, aged 40-78 years (median 60 years) were studied with HMFG1/G2: 11, and H317/H17E2: 19 murine mAbs. Six patients with ovarian cancer aged between 36 and 65 years (median 49 years) were studied with the reshaped human Hu2PLAP mAb (5 patients) or the murine H17E2 mAb (2 patients) labelled with 111In via a new macrocyclic chelating agent (DOTA). One of these was imaged twice, with H17E2- and Hu2PLAP-DOTA-111In, respectively. In 20 out of 22 patients with radiologically measurable ovarian cancer, the presence of tumour was confirmed by the murine mAb scan and correlated well with the findings of conventional radiology diagnostic methods. One of these patients with a negative H17E2 scan and a large abdominal mass at laparotomy was found to have a PLAP-negative tumour on immunohistochemistry. Additionally, the antibody scan revealed the presence of active disease, confirmed at laparotomy/laparoscopy, in 6 out of 8 patients considered to be in clinical complete remission. Best images were obtained at 24 and 48 h after the 123I and 111In mAbs, respectively. Successful imaging with the reshaped human antibody, Hu2PLAP, was seen in 2 patients with PLAP-positive tumours. Antibodies to DOTA developed in 2 patients. In conclusion, immunolocalisation of ovarian tumours is feasible with both murine and reshaped human mAbs. The sensitivity and specificity of the method appear very high in this pilot study, and in view of the absence of toxicity, the diagnostic contribution of this approach should be evaluated prospectively. Given the low number of patients without surgically detectable disease in the present study, future investigations should include more patients with no evidence of disease in order to provide more meaningful estimates of specificity.
Subject(s)
Antibodies, Monoclonal , Ovarian Neoplasms/diagnostic imaging , Radioimmunodetection , Aged , Female , Humans , Immunoconjugates/pharmacokinetics , Immunoenzyme Techniques , Middle Aged , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathologyABSTRACT
Ovarian cancer has an overall five-year survival of around 30% in spite of complete remissions being obtained after optimal surgery and platinum-based chemotherapy. Previous studies have indicated a survival advantage for patients treated with radiolabelled monoclonal antibodies (radioimmunotherapy). We report here on the survival of patients who received single-dose intraperitoneal radioimmunotherapy after having achieved complete remission with standard management. Twenty-five patients with epithelial ovarian cancer, stages Ic-IV, received adjuvant intraperitoneal radioimmunotherapy following completion of conventional chemotherapy. On achieving complete remission they receive once 25 mg of HMFG1 labelled with 18 mCi/m2. Controls for cases were sought from the database of the North Thames ovary group (NTOG). Controls were selected on the basis of stage, histological grade and type, and age of patient at diagnosis. Kaplan-Meier survival plots were constructed for cases and controls and subjected to statistical analysis with the log-rank test. Additionally, using a database of 84 NTOG patients known to be disease-free at the end of chemotherapy, estimated survival curves were constructed using Cox's proportional hazards regression model. Close matches were found for 20 of the 25 patients. Median survival has not been reached at a median follow-up of 59 months for cases and 27 months for controls. Survival at five years is 80% for cases and 55% for controls (p=0.0035). The Cox model estimates long-term (10-year) survival of 70% for patients who received radioimmunotherapy, compared to 32% for those that did not (p=0.003). All patients developed serological evidence of human anti-mouse antibody (HAMA). This study shows a likely survival benefit for patients with ovarian cancer who receive intraperitoneal radioimmuno-therapy in the adjuvant setting.
Subject(s)
Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/radiotherapy , Radioimmunotherapy , Adult , Aged , Animals , Antibodies, Monoclonal , Cohort Studies , Combined Modality Therapy , Female , Follow-Up Studies , Humans , Mice , Middle Aged , Neoplasm Staging , Ovarian Neoplasms/mortality , Ovarian Neoplasms/pathology , Patient Selection , Proportional Hazards Models , Regression Analysis , Survival Rate , Time Factors , Yttrium Radioisotopes/therapeutic useABSTRACT
Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.
Subject(s)
Endoribonucleases/chemistry , Immunoglobulin Fragments/chemistry , Immunotoxins/chemistry , Lymphokines/chemistry , Recombinant Fusion Proteins/chemistry , Sialoglycoproteins/chemistry , Alkaline Phosphatase/immunology , Dimerization , Endoribonucleases/genetics , Humans , Immunoglobulin Fragments/genetics , Immunotoxins/genetics , Immunotoxins/isolation & purification , Immunotoxins/metabolism , Immunotoxins/therapeutic use , Lymphokines/genetics , Placenta/enzymology , Protein Structure, Tertiary , Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/therapeutic use , Sialoglycoproteins/genetics , Tumor Cells, CulturedABSTRACT
BACKGROUND: Preliminary experiments in an animal model have shown the favorable tumor targeting potential in vivo of radiolabeled BC-1, an immunoglobulin (Ig)G1 monoclonal antibody (MoAb) that recognizes the human fibronectin isoform (B+) containing the ED-B oncofetal domain. This antigen has extremely restricted distribution in normal adult tissues. Instead, it is highly expressed in fetal and tumor tissues, especially in high grade astrocytomas and malignant gliomas of the brain, in which the process of neoangiogenesis linked to tumor growth is particularly important. METHODS: This study was carried out with five patients who had malignant brain tumors (four gliomas and one malignant angioblastic meningioma). The BC-1 MoAb was labeled with technetium-99m (99mTc) by MDP transchelation. Planar and single photon emission computed tomography (SPECT) imaging was acquired at 4-6 and 20 hours after intravenous injection of about 450 MBq/0.2 mg 99mTc-BC-1 and was compared with the nonspecific indicator of blood-brain barrier disruption, 99mTc-diethylenetriamine pentaacetic acid (DTPA). Plasma pharmacokinetic analysis was based on serial blood sampling. All patients underwent potentially curative surgery at the end of the study. RESULTS: The plasma clearance curves were biexponential, with average T(1/2) values of 2-4 hours and 28-33 hours, respectively. 99mTc-BC-1 showed very low nonspecific uptake in the bone marrow, liver, and spleen. Planar and SPECT imaging with 99mTc-BC-1 visualized brain tumors in all patients, with a pattern of intratumor distribution that specifically identified areas of peripheral tumor growth more accurately than the nonspecific indicator, 99mTc-DTPA. Tumor uptake of 99mTc-BC-1 was correlated with the expression of the specific oncofetal fibronectin, as shown by immunohistochemistry on surgical samples. CONCLUSIONS: These results indicate the diagnostic potential of MoAb 99mTc-BC-1 for immunoscintigraphy in cancer patients, at least when neoangiogenesis induced by cancer is particularly important.
Subject(s)
Antibodies, Monoclonal , Antigens, Neoplasm/analysis , Brain Neoplasms/diagnostic imaging , Fibronectins/analysis , Technetium , Adult , Antibodies, Monoclonal/pharmacokinetics , Antigens, Neoplasm/immunology , Female , Fibronectins/immunology , Humans , Immunohistochemistry , Male , Middle Aged , Pilot Projects , Radionuclide ImagingABSTRACT
Although much progress has been made in the production of recombinant antibodies and their fusions, there are still problems with solubility and folding. Useful antibodies produced from cloned hybridomas do not always result in scFvs behaving favourably. We report here further work on an scFv (H17E2) against the oncofetal antigen human placental alkaline phosphatase. The overall expression was greatly improved and the H17E2 scFv was redesigned by manipulation of the interdomain linker, resulting in much higher expression levels of the soluble scFv in its active conformation at 0.2-0.5 mg/l of bacterial culture. We show that the new soluble version of this scFv has similar characteristics to the refolded version in terms of antigen and tumour cell binding, stability and in vivo pharmacokinetics. The final tumour uptake behaviour of these scFvs is superior to that of the parental whole antibody with respect to tumour:organ ratios, but still requires further development before considering it as a suitable molecule for clinical use in ovarian or testicular cancer.
Subject(s)
Alkaline Phosphatase/immunology , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/chemistry , Antibodies, Neoplasm/genetics , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Immunoglobulin G/genetics , Placenta/enzymology , Animals , Antibodies, Monoclonal/blood , Antigens, Neoplasm/immunology , Biomarkers, Tumor , Cloning, Molecular/methods , Gene Expression/genetics , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , KB Cells , Mice , Protein Binding/genetics , Protein Binding/immunology , Tissue Distribution/physiologyABSTRACT
Four murine IgG1 monoclonal antibodies, each with specificity for a human tumour-associated antigen, have been tested for their in vivo immunogenicity using a rabbit model. Surprisingly, one of these antibodies, MR6, was significantly more immunogenic than the remaining three reagents. This enhanced MR6 immunogenicity was not restricted to the immunoglobulin molecule itself, but also applied to a hapten (fluorescein isothiocyanate, FITC) when conjugated to the monoclonal antibody. In addition, the secondary immune response to an independent antigen, human haemoglobin, was higher when the antigen was administered simultaneously with MR6 than when co-injected with an isotype-matched control monoclonal antibody. The presence of the target antigen, gp200-MR6, on both rabbit and human leucocytes and epithelium, and its known association with human IL-4 function, raises the possibility that antibody MR6 may not only target immunogens to antigen-presenting cells, but may also enhance the ability of these cells to present antigen to the immune system. Antibodies to the gp200-MR6 may therefore find important clinical application as in vivo adjuvants.