Adjuvant therapy of ovarian cancer with radioactive monoclonal antibody.

Fifty-two patients with epithelial ovarian cancer were treated with yttrium-90-labelled monoclonal antibody HMFG1 administered intraperitoneally following conventional surgery and chemotherapy as part of an extended phase I-II trial. The treatment was well tolerated and the only significant toxicity observed was reversible myelosuppression as previously described. Following conventional surgery and chemotherapy, 21 out of the 52 patients had no evidence of residual disease and were regarded as receiving treatment in an adjuvant setting. To date, two of these patients have died of their disease (follow-up 3-62 months, median follow-up 35 months). This extended phase I-II study suggests that patients with advanced ovarian cancer who achieve a complete remission following conventional therapy may benefit from further treatment with intraperitoneal radioactive monoclonal antibody.

Coordinate elevation of serum markers in ovarian cancer but not in benign disease.

Effective screening for occult ovarian cancer will require a strategy that is both sensitive and specific. Preliminary data suggest that CA 125 is elevated at diagnosis in a majority of patients with ovarian cancer. Although CA 125 is sufficiently specific to prompt its evaluation as one component of a strategy to detect ovarian cancer in postmenopausal women, a further improvement in specificity would facilitate cost-effective screening. In an attempt to develop a more specific screening strategy, multiple markers were assayed in a panel of sera from 47 patients with ovarian cancer and in a separate panel of sera from 50 individuals with benign disease whose serum CA 125 levels exceeded 35 U/ml. Among the patients with ovarian cancer, elevations of CA 125 (greater than 35 U/ml) were observed in 91%, CA 15-3 (greater than 30 U/ml) in 57%, TAG 72 (greater than 10 U/ml) in 49%, placental alkaline phosphatase (PLAP) in 25%, human milk fat globule protein (HMFG) 1 in 77%, HMFG2 in 62%, and NB/70K in 57%. Among the 50 sera selected from patients with benign disease, CA 125 was more than 35 U/ml in 100% and more than 65 U/ml in 42%. Among those patients with benign disease and elevated CA 125, NB/70K was elevated in 62%, HMFG1 in 26%, and HMFG2 in 12%, whereas TAG 72 and CA 15-3 were elevated in only 6% and 2%, respectively. In addition PLAP appeared promising; elevated enzyme levels were not found in the benign disease group. Among patients with ovarian cancer with CA 125 levels more than 35 U/ml, either TAG 72 or CA 15-3 was elevated in 77%. In the false-positive group, only 6% had elevations of one or the other marker. The CA 125 levels in cancer patients were, however, substantially greater than in patients with benign disease. If sera from patients with ovarian cancer were diluted to a range comparable to that found in benign disease, at least one of the two confirmatory tests was elevated in 63% of the samples from the malignant cases. Consequently, use of CA 15-3 and TAG 72 in combination with CA 125 can increase the apparent specificity of the CA 125 assay for distinguishing malignant from benign disease. Prospective studies will be required to test critically whether the use of additional serum markers in combination with the CA 125 assay would contribute to the specificity of a cost-effective screening strategy for ovarian cancer.

Quantitative measurement of monoclonal antibody distribution and blood flow using positron emission tomography and 124iodine in patients with breast cancer.

The uptake and in vivo quantitation of monoclonal antibodies (MAbs) has been measured non-invasively using positron emission tomography (PET) and 124iodine in 9 patients with breast ductal carcinoma. Blood-flow measurements were also made using 15oxygen-labelled water and PET to evaluate antibody delivery; 7 patients were studied with HMFGI antibody and 2 patients with a non-specific antibody. Tumour uptake ranged from 2-7.7 x 10(-3)% of injected dose per gram of tissue. Values for normal tissues including liver, lung and bone were also obtained. In 2 out of 7 patients studied with the specific antibody, uptake was greater than that seen with the non-specific antibody. There was no correlation between antibody uptake and blood flow. This report exemplifies the potential of PET for the non-invasive and accurate quantitative assessment of targeted antibody which is a prerequisite to therapy.

Intraperitoneal yttrium-90-labeled monoclonal antibody in ovarian cancer.

From March 1987 to March 1988, a phase I to II study was carried out in 25 patients with ovarian cancer. They received escalating doses of intraperitoneally (IP) administered yttrium-90 (Y-90)-labeled monoclonal antibody, HMFG1, against a tumor cell-surface antigen. Myelosuppression prevented an escalation of the administered Y-90 activity above 25 mCi. Y-90-labeled antibody was absorbed from the peritoneal cavity into the circulation. Maximum blood Y-90 activity was observed 40 hours after the IP injection with a mean of 21% of the injected activity (range, 14.2% to 26.4%) in the circulation. The radiation dose the bone marrow received from circulating Y-90-labeled antibody (the blood radiation dose) was calculated by applying the Medical Internal Radiation Dose (MIRD) formulation to the measured Y-90 activity in patients blood. Myelosuppression occurred following calculated blood radiation doses to bone marrow of only 10 to 30 cGy. The excessive myelosuppression following such modest radiation doses from circulating Y-90-labeled antibody could be explained by the uptake of Y-90 by bone. In an attempt to reduce bone absorption of Y-90, seven patients received an intravenous (IV) infusion of EDTA (Sinclair Pharmaceuticals Ltd, Godalming, United Kingdom). This increased the urinary excretion of Y-90 from a mean of 11.1% to 32.3% of the injected activity (P = .0001). Fourteen patients had assessable tumor at laparoscopy. Tumor regression was observed in one patient, and palliation of ascites in a further patient.

Immunolocalisation of testicular tumours using radiolabelled monoclonal antibody to placental alkaline phosphatase.

Tumour associated monoclonal antibody against placental alkaline phosphatase (H17E2) was radiolabelled with Indium-111 and Iodine-123 and administered intravenously in 33 patients with primary and/or metastatic testicular tumour, as well as in 8 patients who were in complete remission after surgical excision of the tumour. The presence of a tumour was confirmed and correlated well with conventional diagnostic techniques and, in addition, the antibody scan revealed the presence of active disease in 2 patients with negative conventional imaging and with elevated serum markers. In addition, in one patient the CT produced a false positive result where the antibody scan was negative. Finally, the absence of tumour was confirmed in all 8 cases of patients in complete remission. All patients studied with Indium-labelled antibody had observable concentrations of the radiolabel in the liver (estimated to be approximately 30% of the administered dose), as well as in the kidneys and spleen. The patients studied with the Iodine-123 labelled antibody had observable concentrations in the thyroid gland and the stomach. The best images were seen at 48 and 24 hrs after the Indium and Iodine radiolabelled antibody respectively. No human anti-mouse antibody was detected in any of our patients, even in those who received 2 and 3 administrations, with the highest amount of administered protein being 800 micrograms. No toxicity was encountered in any of our patients in 4 months of follow-up. This method may be of clinical value in patients with testicular neoplasma and represents a new addition to current imaging techniques. A positive scan indicates the definite presence of a tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraperitoneally administered 90Y-labelled monoclonal antibodies as a third line of treatment in ovarian cancer. A phase 1-2 trial: problems encountered and possible solutions.

A phase 1-2 trial of 90Y-labelled monoclonal antibody, HMFG1 administered intraperitoneally to 30 patients with ovarian carcinoma is presented. The problems encountered with myelotoxicity are described, and the steps which have so far been taken to overcome this and to increase the dose of 90Y to an estimated tumouricidal level. Bone deposition of free 90Y limits the dose which can be administered without severe bone marrow toxicity. The intravenous use of a chelating agent, Ledclair (EDTA) has allowed the dose to be increased from 18 to 30 mCi without causing severe myelotoxicity. 90Y-DTPA MAb is an unstable immunoconjugate in vivo and in vitro. The use of a more stable linkage such as a macrocycle should enable the administered dose to be increased without increasing the amount of free 90Y which becomes available to be deposited in bone. This would be expected to reduce toxicity and increase therapeutic efficacy.

Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase.

Twenty-seven patients with brain glioma were scanned using 123I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment.

Localization of monoclonal antibody AUA1 and its F(ab')2 fragments in human tumour xenografts: an autoradiographic and immunohistochemical study.

The mouse monoclonal antibody (MAb) AUA1, when applied on LoVo tumour sections, reacts by staining all tumour cells, on their cell surfaces. To investigate the accessibility of these sites to antibody when the tumour is present as a solid mass in vivo, subcutaneous xenografts of LoVo were first prepared in nude mice. The mice were then injected intravenously with either 125I-labelled AUA1, 125I AUA1 F(ab')2 or with 125I-labelled HMFG2 (negative control antibody). Animals were killed at various time intervals. Gross and micro-autoradiography as well as immunohistochemistry were performed on tissue samples of tumour and control organs. The in vivo injected antibody, in contrast to that studied in vitro, was localized only, as detected by autoradiography, on a thin layer of tumour cells adjacent to the vascularized stroma. On microscopically small tumour islands the antibody penetration was complete. Most of the radioactivity was on the cell surfaces, as seen on in vitro immunostaining. With intact antibody, similar autoradiographic results were obtained at days 1, 3 and 6. With F(ab')2 fragments there was deeper penetration into the tumour at days 1 and 3, though less radioactivity was found; by day 6 the activity had greatly decreased. Radioactivity in the control organs was limited to the blood pool. Negative control antibody HMFG2 showed no localization on the tumour cells. These results were not due to differences in antigenic expression of the tumour cells but reflect the problem of accessibility of antigenic sites in vivo.

Antibody-guided irradiation of hepatic metastases using intrahepatically administered radiolabelled anti-CEA antibodies with simultaneous and reversible hepatic blood flow stasis using biodegradable starch microspheres.

Two monoclonal antibodies to carcinoembryonic antigen (CEA) were radiolabelled with 131I and used for the treatment of hepatic metastases in a patient who had a primary colonic carcinoma. Approximately 100 mCi of 131I-labelled antibody were administered via the hepatic artery on two occasions. On the second occasion, radiolabelled antibody was given concurrently with biodegradable starch microspheres in an attempt to enhance tumour uptake of antibody by achieving temporary stasis or delay of hepatic blood flow. The procedure was carried out uneventfully. There was clinical improvement and a fall in circulating CEA levels after each course of treatment. Furthermore, after the second course of therapy the clinical improvement was sustained for a longer period (more than 3 months) and there was evidence of diminution in the size of some of the liver metastases. Regional administration of 131I-labelled anti-CEA antibody concurrently with biodegradable starch microspheres appears to be a promising new method for the treatment of hepatic metastases from colonic carcinoma.

A low pH enzyme linked immunoassay using two monoclonal antibodies for the serological detection and monitoring of breast cancer.

A new, simple and sensitive low pH ELISA method has been developed to measure serum levels of tumour associated antigens detectable by monoclonal antibodies HMFG1 and HMFG2. We examined sera from healthy controls, patients with neoplastic and non-neoplastic conditions of breast, liver and gastrointestinal tract. The majority of patients with metastatic breast cancer had elevated serum antigens (69% HMFG1, 72% HMFG2) compared to healthy controls (6.3% HMFG1, 3.0% HMFG2) or patients with benign breast disease (17% HMFG1, 4% HMFG2). There was no discrimination using these assays between patients with neoplastic and non-neoplastic conditions of liver and gastrointestinal tract. This new method promises to be of value in the assessment and management of patients with breast cancer.

A new immunoassay using monoclonal antibodies HMFG1 and HMFG2 together with an existing marker CA125 for the serological detection and management of epithelial ovarian cancer.

A new method with a low pH step to dissociate serum complexes has been developed to measure serum levels of antigens associated with ovarian cancer. The antigens are detected by monoclonal antibodies HMFG1 and HMFG2 and have been compared to an existing ovarian cancer associated antigen detected by the antibody CA125. Elevated HMFG1 was found in 56%, and elevated HMFG2 in 65% of 924 sera from 85 patients with ovarian cancer. CA125 was elevated in 85% of these sera. When the three markers were used in conjunction, 95% of sera from patients with ovarian cancer were positive--compared with 7% in sera from healthy control subjects. Therefore, the combination of HMFG1, HMFG2 and CA125 increases the diagnostic accuracy. If all three markers are normal in a patient previously treated for ovarian cancer then no further positive information regarding disease status can be obtained by ultrasound and CT scanning.

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 irradiation of malignant pleural and pericardial effusions.

Tumour-associated monoclonal antibodies (HMFG1, HMFG2 and AUA1) radiolabelled with iodine-131 were given intracavitary (intrapleurally and intrapericardially) to patients with malignant effusions. Ten out of 13 effusions (3 pericardial and 7 pleural) responded completely with no fluid reaccumulation between 3 and 18 months. No clinical or other toxicity was observed. This new method of treatment for recurrent malignant effusions is non-toxic and effective resulting in improved quality of life, and, in some cases, prolongation of survival.

Serum levels of c-myc and c-ras oncogene products in normal subjects and in patients with neoplastic and non neoplastic conditions.

A new, simple and sensitive low pH ELISA system has been developed and used to measure serum levels of c-myc and c-ras oncogene products in healthy blood donors and patients with neoplastic and non neoplastic conditions. Blood donors had significantly lower serum levels of oncogene products than patients with cancer or other pathologies (p-value less than 0.01). There was, however, no difference between patients with neoplastic and non-neoplastic conditions. Although c-myc and c-ras oncogene products in the serum appear to discriminate between healthy state and pathological conditions they do not discriminate between neoplastic and non-neoplastic conditions.

Selective induction of stalk-cell-specific proteins in Dictyostelium.

We compared the proteins synthesized and accumulated by Dictyostelium discoideum amoebae in response to the morphogenetic factor termed differentiation-inducing factor (DIF) to assess the proposed ability of DIF to regulate the choice of differentiation pathway. When amoebae of a mutant strain with low endogenous DIF levels were given DIF, they dramatically increased the expression of 21 of 23 proteins preferentially found in stalk cells, but drastically repressed 4 major spore-specific proteins. Most of the induced proteins were also expressed in amoebae of a developmentally competent strain developing at low cell densities and exposed to DIF, low extracellular pH, or the proton pump inhibitor diethylstilbestrol; this suggests that an intracellular acidification may be a key part of the mechanism of DIF action. We conclude from the similar morphology and extensive homology of proteins of DIF-induced and stalk cells that most stalk-pathway functions are DIF dependent.

Purification of stalk-cell-inducing morphogens from Dictyostelium discoideum.

We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low-Mr factors, which can induce isolated cells to differentiate into stalk cells in the presence of cyclic AMP [Town, C. D., Gross, J. D. and Kay, R. R. (1976) Nature (Lond.) 262, 717-719; Town, C. D. and Stanford, E. (1979) Proc. Natl Acad. Sci. USA, 76, 308-312]. These differentiation-inducing factors (DIF) have now been purified by a procedure involving binding to and elution from XAD-2 resin, extraction into hexane and two steps of reverse-phase high-pressure liquid chromatography (HPLC). Our results show the following. HPLC resolves a major stalk-cell-inducing activity (DIF-1) and at least four minor and more polar activities (DIFs 2-5). DIF-1 has been purified at least 3000-fold over the starting dialysed medium with a recovery of about 2%. This low recovery of DIF-1 can be explained in part by the loss of non-specific stimulatory ('helper') factors during the purification. A few micrograms purified DIF-1 were obtained from 10(12) cells. This material could induce stalk cell differentiation in the standard assay at less than 0.2 nM. The biological activity of DIFs 1, 2 and 3 was sensitive to borohydride reduction, suggesting the presence of an essential carbonyl group. DIF-5 was partially sensitive and DIF-4 resistant. Other properties of DIF-1 suggest that it is a non-polar molecule of Mr less than 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties. The purification of DIF should make possible its eventual identification by sensitive physical techniques, such as mass spectroscopy, and will allow further investigation of its biological effects.

Dictyostelium mutants lacking DIF, a putative morphogen.

DIF is an endogenous extracellular signal that may control differentiation of D. discoideum cells. It is a dialyzable, lipid-like factor that induces stalk cell formation among isolated amebae incubated in vitro with cAMP. To examine the consequences of DIF deprivation, we have isolated several mutant strains that are impaired in DIF accumulation, and whose inability to make stalk cells in vitro and during normal development on agar can be corrected by the addition of exogenous DIF. Little DIF is made by the mutants, and morphological development on agar stops after the cells have aggregated, but before a slug forms. In these DIF-deprived conditions, prespore cells can differentiate, but prestalk cells cannot.