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The toxicity and therapeutic efficacy of escalating doses of anti-carcinoembryonic antigen x anti-N alpha-(diethylenetriamine-N,N,N',N''-tetraacetic acid)-In bispecific monoclonal antibody (F6-734) and iodine 131-labeled bivalent hapten were determined in a Phase I/II trial. A total of 26 patients with recurrences of medullary thyroid cancer documented by imaging and a rise in serum thyrocalcitonin were enrolled. Twenty to 50 mg of F6-734 and 40-100 mCi of 131I-hapten were injected 4 days apart. Quantitative scintigraphy was performed after the second injection for dosimetry estimations in eight cases. Clinical, biological, and morphological follow-up was carried out for 1 year after treatment. The mean percentage of injected activity per gram of tumor at the time of maximum uptake was 0.08% (range, 0.003-0.26%). The tumor biological half-life ranged from 3 to 95 days, and tumor doses ranged from 2.91 to 184 cGy/mCi. The estimated tumor-to-nontumor dose ratios were 43.8 x 53.4, 29.6 x 35.3, 10.9 x 13.6, and 8.4 x 10.0 for total body, red marrow, liver, and kidney, respectively. Grade III/IV hematological toxicity was observed in seven patients, most of them with bone metastases. Among the 17 evaluable patients, 4 pain reliefs, 5 minor tumor responses, and 4 biological responses with decrease of thyrocalcitonin were observed. Nine patients developed human anti-mouse antibody. Dose-limiting toxicity was hematological, and maximum tolerated activity was 48 mCi/m2 in this group of patients, most of whom had suspected bone marrow involvement. The therapeutic responses observed in patients mainly with a small tumor burden are encouraging for the performance of a Phase II trial with minimal residual disease.

As small cell lung carcinoma (SCLC) is frequently a widespread disease at diagnosis, highly radiosensitive and often only partially responsive to chemotherapy, radioimmunotherapy (RIT) would appear to be a promising technique for treatment. We report the preliminary results of a Phase I/II trial of RIT in SCLC using a two-step method and a myeloablative protocol with circulating stem cells transplantation. Fourteen patients with proved SCLC relapse after chemotherapy were treated with RIT. They were first injected i.v. with a bispecific (anti-carcinoembryonic antigen/anti-diethylenetriaminepentaacetic acid) monoclonal antibody (20-80 mg in 100 ml of saline solution) and then 4 days later with di-(In-diethylenetriaminepentaacetic acid)-tyrosyl-lysine hapten labeled with 1.48-6.66 GBq (40-180 mCi) of I-131 and diluted in 100 ml of saline solution. In patients receiving 150 mCi or more, circulating stem cells were harvested before treatment and reinfused 10-15 days later. Treatment response was evaluated by CT and biochemical data during the month before and 1, 3, 6, and 12 months after treatment. All patients received the scheduled dose without immediate adverse reactions to bispecific antibody or 1-131 hapten. Toxicity was mainly hematological, with two cases of grade 2 leukopenia and three cases of grade 3 or 4 thrombopenia. Body scanning 8 days after injection of the radiolabeled hapten generally showed good uptake at the tumor sites. Estimated tumor dose was 2.6-32.2 cGy/mCi. Among the 12 patients evaluated to date, we have observed 9 progressions, 2 partial responses (one almost complete for 3 months), and 1 stabilization of more than 24 months. Efficiency and toxicity were dose-related. The maximal tolerable dose without hematological rescue was 150 mCi. These preliminary results are encouraging, and dose escalation is currently continuing to reach 300 mCi. RIT should prove to be an interesting therapeutic method for SCLC, although repeated injections and hematological rescue will probably be required, as well as combination with other treatment modalities.

We have developed a pretargeting strategy, called the Affinity Enhancement System (AES), which uses bispecific antibodies (BsF(ab')2) to target radiolabeled bivalent haptens to tumor cells. We performed several radioimmunotherapy (RIT) experiments in nude mice grafted with LS174T colon carcinoma or TT medullary thyroid cancer. Mice were treated with 131I-labeled di-DTPA-indium-tyrosyl-lysine bivalent hapten (75-112 MBq) administered 15-48 h after anti-CEA x anti-DTPA-indium BsF(ab')2. Immunohistological studies were performed on tumors at their minimal relative volume (TT), on stabilized tumor nodules (LS174T), and on regrowing tumors (TT and LS174T). Untreated tumors were used as controls. On microscopic examination, regrowing tumors (2 months posttherapy) were similar to untreated tumors with cells showing their respective typical morphology (large cells with a high nucleocytoplasmic ratio for TT, small and very undifferentiated cells for LS174T). However, regrowing tumors showed larger necrotic areas and a higher mitotic index correlated with Ki-67 antigen staining. Immunostaining for CEA was as strong as for controls. By contrast, the immunohistology of TT tumors at their minimal relative volume (1 month posttherapy) or of LS174T residual nodules (8 months posttherapy) showed decreased mitotic indices correlated with poor Ki-67 antigen staining. Some clusters of LS174T presented with features of glandular lumen, which suggested a more differentiated and less aggressive status. In TT tumors, CEA expression remained unchanged (80-100% membrane and cytoplasmic staining), whereas only 70% of the LS174T tumors were stained, with 58% loss of the membrane expression. Repeated treatment early after the tumor has reached its minimal relative volume should thus be efficient and improve the overall efficacy of AES RIT.

Unlabelled: The "affinity enhancement system," a two-step targeting technique using bispecific antibody and radiolabeled bivalent hapten, has been reported to be useful for carcinoembryonic antigen-expressing tumors. The purpose of this study was to evaluate the efficacy of this method for targeting human small cell lung cancer using an antineural cell adhesion molecule antibody., Methods: Antineural cell adhesion molecule/antihistamine bispecific antibody NK1NBL1-679 was prepared by coupling an equimolecular quantity of a Fab' fragment of NK1NBL1 to a Fab fragment of antihistamine 679. Athymic mice inoculated with NCI-H69 small cell lung cancer cells expressing neural cell adhesion molecule were administered bispecific antibody and then 48 h later 125I-labeled bivalent histamine hapten. 125I-labeled intact NK1NBL1 was injected into other groups of mice. Biodistributions were examined as a function of time., Results: In mice of the two-step targeting, tumor uptake was 2.5 +/- 0.2, 3.2 +/- 0.4, 6.4 +/- 2.0, 7.2 +/- 2.7, 6.1 +/- 2.1 and 2.2 +/- 0.4 %ID/g at 5, 30 min, 5, 24, 48 and 96 h, and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios were 1.4 +/- 1.1, 10.8 +/- 13.2 and 4.6 +/- 4.7, respectively, at 5 h, whereas 125I-labeled NK1NBL1 showed a tumor uptake of 5.7 +/- 0.4 %ID/g and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios of 0.3 +/- 0.1, 1.1 +/- 0.2 and 0.9 +/- 0.1, respectively, at 5 h. These results were confirmed by autoradiographic studies, which demonstrated clear tumor-to-normal tissue contrast. Dosimetry showed that the affinity enhancement system could enhance the therapeutic potential of the antineural cell adhesion molecule antibody NK1NBL1., Conclusion: This two-step targeting method seems promising for the diagnosis and therapy of small cell lung cancer.

Unlabelled: The purpose of this study was to compare the toxicity and efficacy of two-step radioimmunotherapy using a bispecific anticarcinoembryonic antigen (CEA)/anti-diethylenetriamine pentaacetic acid (DTPA) antibody (F6-734 bispecific monoclonal antibodies (BsMAbs) and an 131I-di-DTPA-TL bivalent hapten with F(ab')2 fragments of the same directly labeled anti-CEA 131-F6., Methods: Eight groups of nude mice subcutaneously grafted with the human TT medullary thyroid cancer cell line were injected once tumor volume reached about 200 mm3. Two groups received 37 or 92.5 MBq (1 or 2 nmol) 131I-di-DTPA-TL 48 h after injection of 2 or 4 nmol F6-734 BsMAb and two groups received 37 or 92.5 MBq (250 microg) 131I-F6. Four control groups were treated respectively with (a) 92.5 MBq nonspecific 131I-734 fragments, (b) 92.5 MBq 131I-di-DTPA-TL 48 h after injection of a mixture of irrelevant F6-679 (anti-CEA/anti-histamine) and G7A5-734 (anti-melanoma/anti-DTPA) BsMAb, (c) 250 microg nonradiolabeled F6, and 250 microg F6-734 BsMAb and then 48 h later 1.25 nmol of nonradiolabeled hapten. A control group received no injections. Toxicity was evaluated by determining animal weight and the number of leukocytes and platelets, and efficacy by variation in tumor volume and thyrocalcitonin during a 90-d period. Histological analysis of tumors and statistical studies were performed., Results: The time required for the tumor to double in size was respectively 57 and 86 d with 37 and 92.5 MBq F6-734/131I-di-DTPA-TL and 44 and 65 d with 37 and 92.5 MBq 131I-F6. Changes in thyrocalcitonin levels were parallel to those in tumor volume. Weight loss was 5%, leukocyte nadirs respectively 1640+/-838 and 1560+/-1160/mm3 and platelet nadirs 1.46+/-0.52 10(6)/mm3 and 0.73+/-0.38 10(6)/mm3 after injections of 37 and 92.5 MBq F6-734/1311-di-DTPA-TL. Weight loss was respectively 8% and 16%, leukocyte nadirs 50+/-100/mm3 and 175+/-50/mm3 and platelet nadirs 0.71+/-0.18 10(6)/mm3 and 0.48+/-0.11 10(6)/mm3 after injections of 37 and 92.5 MBq 131I-F6., Conclusion: Two-step radioimmunotherapy was as efficient as the one-step system and markedly less toxic.

Unlabelled: The purpose of this study was to evaluate biodistributions and absorbed doses of anti-carcinoembryonic antigen (CEA)/anti-diethylenetriamine pentaacetic acid (DTPA)-indium (anti-DTPA-In) bispecific monoclonal antibody (BsMAb) F6-734 and 125I-labeled DTPA-indium dimer hapten (125I-di-DTPA-In hapten) in athymic mice xenografted with human medullary thyroid cancer., Methods: Bispecific monoclonal antibodies F6-679 (anti-CEA/antihistamine) and G7A5-734 (antimelanoma/anti-di-DTPA-In) were used as irrelevant BsMAbs. Athymic mice inoculated with TT medullary thyroid cancer cells expressing CEA were administered BsMAbs F6-734, F6-679 or G7A5-734 and then, 48 hr later, 125I-di-DTPA-In hapten. Iodine-125-labeled F6 F(ab')2 fragment was injected into other groups of mice. Biodistributions were examined at 30 min and 5, 24, 48 and 96 hr after injection of 125I-di-DTPA-In hapten or 125I-labeled F6 F(ab')2., Results: In mice injected with BsMAb F6-734 and 125I-di-DTPA-In hapten, tumor uptake was 9.1%+/-2.1%, 8.7%+/-3.5%, 8.0%+/-2.3%, 5.1%+/-0.9% and 3.5%+/-1.5% of the injected dose/g at 30 min and 5, 24, 48 and 96 hr, and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios were 37.0+/-12.5, 32.3+/-10.9 and 10.4+/-2.7 at 24 hr. Iodine-125-F6 F(ab')2 fragment showed a tumor uptake of 7.39% injected dose/g and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios of 1.8+/-0.6, 7.3+/-2.9 and 3.6+/-1.6 at 24 hr. In mice injected with F6-679 or G7A5-734, tumor uptake and tumor-to-normal tissue ratios were much lower than in the mice injected with F6-734. These results were confirmed by autoradiographic studies that demonstrated clear tumor-to-normal tissue contrast., Conclusion: This two-step targeting method seems very potent for the diagnosis and therapy of human medullary thyroid cancer and other CEA-producing tumors because it combines high tumor uptake and low normal tissue background.

Unlabelled: Pretargeting labeled bivalent hapten with bispecific antibodies has proven feasible in the clinic, and our earlier results have suggested the technique may be very sensitive for detecting small recurrences and metastases. Medullary thyroid carcinoma (MTC) is an example where this technique may be the most useful since local recurrences and isolated metastases are removed surgically when detected, and thyrocalcitonin provides a specific and sensitive tumor marker. In our current study, we evaluated pretargeted immunoscintigraphy in a larger number of MTC patients., Methods: Anti-carcinoembryonic antigen (CEA) x anti-diethylenetriaminepentaacetic acid (DTPA) indium bispecific antibody and 111In-labeled bivalent DTPA hapten were administered sequentially (4-5 days apart) to 44 patients with elevated circulating calcitonin after resection of primary MTC. Immunoscintigraphy was performed 2, 5 and 24 hr after hapten injection and, when necessary, at longer time intervals. When available, a handheld gamma probe was used during surgery., Results: Fifteen patients had known tumor sites before immunoscintigraphy. Tumors were imaged in 12 (80%) of these patients, including 3 with liver metastases. Five unknown tumor sites were detected. For the 29 patients with occult disease, immunoscintigraphy detected high-activity uptake sites in 21 patients (72%), including 5 in the liver. Twelve were confirmed by surgery, 1 by guided morphologic imaging and 1 by venous catheterization. There were 2 false-positive patients. The other 5 patients have not yet been confirmed. All detected liver metastases were high-activity uptake areas. Radioimmunoguided surgery was used in 14 patients. It was considered helpful by the surgeon in 12 patients, including 4 patients where it determined the resection of small, not palpable nor visible, tumor-involved lymph nodes. Surgical resection resulted in a significant decrease (8 patients) or normalization (1 patient) of circulating calcitonin and CEA., Conclusion: This technique affords high sensitivity and specificity for detecting small tumor lesions including liver metastases. Its use for immunoscintigraphy and guided surgery should improve the therapeutic management of recurrent MTC.

Unlabelled: The purpose of this study was to estimate the dose delivered to tumor targets and normal tissues after two-step injection of an anti-CEA/anti-DTPA-In (F6-734) bispecific antibody and a 131I-labeled di-DTPA in-TL bivalent hapten in patients with medullary thyroid carcinoma (MTC) and small-cell lung cancer (SCLC)., Methods: Five patients with persistent disease or recurrences of MTC and five patients with primary SCLC or relapse were studied. In a first step, 0.1 to 0.3 mg/kg of F6-734 bispecific antibody was injected intravenously. Four days later, 6 nmole (5.8 to 9.8 mCi) of 131I-labeled di-DTPA in-TL bivalent hapten were injected. Quantitative imaging was performed during one week after the second injection., Results: All 5 patients with MTC showed positive immunoscintigraphy (IS). In the smallest visualized and resected tumor (0.8 g), the fraction of injected activity per gram (% ID/g) was 0.1% at Day 3. IS was positive in 4 of the 5 patients with SCLC. The volume of the smallest visualized SCLC tumor was estimated at 11 +/- 2 ml, and tumor uptake was about 0.009% ID/g. Tumor dose estimates ranged from 4.2 to 174 cGy/mCi in patients with MTC and from 1.7 to 8 cGy/mCi in patients with SCLC., Conclusion: High absorbed dose values were calculated for small MTC recurrences. For SCLC recurrences the values were smaller but in the same range as those obtained by other investigators with the one-step technique in lymphoma.

In the perspective of radioimmunotherapy (RIT) of micrometastases, we compared, in multicell spheroids (MS), the uptake and retention kinetics of 125I-F(ab)'2 F6 anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), and the affinity enhancement system (AES) using an anti-CEA/anti-DTPA-indium bispecific antibody (BsMAb) and a 125I-labeled di-DTPA-In-tyrosine-lysine bivalent hapten. We used MS of colorectal tumor cell lines expressing CEA strongly (LS 174T), weakly (HT-29) or not at all (HRT-18). Uptake and retention kinetics of 125I-F(ab)'2 F6 and 125I-BsMAb used alone gave similar results. The highest uptake values, obtained with LS 174T MS, were slightly lower with AES than with 125I-F(ab)'2 F6. However, effective retention half-lives were longer for AES than for 125I-F(ab)'2 F6 or for 111In-labeled monovalent hapten after pre-incubation of spheroids with BsMAb. Autoradiography showed the same slow and heterogeneous distribution of 125I-F(ab)'2 F6 and 125I-BsMAb. These results indicate that the 2-step technique is more favorable for RIT: uptake values were approximately the same but uptake kinetics were more rapid, and retention half-life was longer than with the one-step technique.

Anti-CEA F(ab')2 monoclonal antibody fragments [F6 MAb F(ab')2] were conjugated to two bifunctional semi-rigid chelating agents derived from trans-1,2-diaminocyclohexane tetraacetic acid (CDTA), the monolithium salt of N-[methyl(2-isothiocyanatoethyl)carbamide] trans-1,2-diaminocyclohexane-N,N',N'-triacetic acid (SCN), and 4 isothiocyanato-trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (4-ICE) and labelled with 111In to obtain IIIIn-labelled-F6 MAb F(ab')2 conjugates (111In-F6-SCN and 111In-F6-4-ICE respectively). Biodistribution in mice and clinical studies were undertaken to assess the potential of these two ligands in the detection of colorectal adenocarcinoma recurrences and metastases in humans. Toxicity studies were carried out on guinea pigs and Swiss mice injected with a dose proportionally 100 times greater than that used in human studies. Clinical studies were performed in patients with clinically and/or biologically suspected adenocarcinoma recurrences. No immunoconjugate-induced toxicity was found. The biodistribution studies in mice gave better visualization of tumour sites with 111In-F6-SCN and 111In-F6-4-ICE than with 111In-F6-DTPA. Ten patients were included in the clinical protocol. 111In-F6-SCN and 111In-F6-4-ICE effectively visualized adenocarcinoma recurrences. However, in this small series, 111In-F6-4-ICE performed somewhat better than 111In-F6-SCN. The present study has demonstrated the potential of new bifunctional semi-rigid chelating agents coupled to antibody and labelled with 111In to localize recurrences (especially in liver) in humans using a one-step targeting method.

In two-step targeting technique using bifunctional antibodies, a nonradiolabeled immunoconjugate with slow uptake kinetics (several days) is initially injected, followed by a small radiolabeled hapten with fast kinetics (several hours) that binds to the bispecific immunoconjugate already taken up by the tumor target. In patients with colorectal or medullary thyroid cancer, clinical studies performed with an anti-CEA/anti-DTPA-indium bifunctional antibody and an indium-111-labeled di-DTPA-TL bivalent hapten showed that tumor uptake was not modified compared to results for F(ab')2 fragments of the same anti-CEA antibody directly labeled with indium-111, whereas the radioactivity of normal tissues was significantly reduced (3- to 6-fold). The fast tumor uptake kinetics (several hours) and high or very high tumor-to-normal tissue ratios obtained with the bifunctional antibody technique are favorable parameters for efficient radioimmunotherapy.

Eleven patients with primary colorectal carcinoma tumors (4 +/- 2 cm) were given intravenous injections of 1-10 mg of an anti-CEA, anti-In-DTPA bispecific Fab'-Fab monoclonal antibody, and 2-8 days later, were injected with 1.2-4.2 nmol of an 111In-labeled DTPA dimer (6 mCi). The bispecific antibody exhibited good stability and F(ab)'2-like pharmacokinetics. After injection, the 111In-DTPA dimer distributed in a large volume (88 ml/kg-180 ml/kg) and cleared through the kidneys (mean residence time in the whole body: 9 hr-16 hr). Uptake of 111In by the tumor using this two-step technique (1.8%-17.5% injected dose ID/kg, measured from surgical samples 48 hr after hapten injection) was not found significantly lower than that achieved with our reference 111In-labeled anti-CEA F(ab)'2 1 to 4 days after injection in six patients with similar clinical status (5.5%-30.2% ID/kg). In addition, tumor-to-blood and tumor-to-liver uptake ratios were significantly improved (blood 7.8 versus 4.2, liver 2.8 versus 0.8). As a result, low background images allowed detection of 12 of 13 lesions, 4 hr and 24 hr after hapten injection. However, 7 of 11 patients developed HAMA.