Improved tumor detection by anti-CEA chimeric Fab oligomers with disulfide linkages in a pancreatic-carcinoma-xenograft model

We have investigated the effect of Fab oligomerization on imaging efficacy in a pancreatic-carcinoma xenograft model in mice. Recombinant mouse/human chimeric Fab of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A10, which has been shown to react specifically with gastrointestinal cancers, was used in this study. Fab homo-oligomers (dimers and trimers) chemically linked through disulfide bonds (S-S Fab oligomers) were prepared by linkage of chimeric Fab with N-succinimidyl-3-(2-pyridyldithio)-propionate. Oligomers with S-S bonds showed 10-fold higher binding activity against human CEA than Fab, while the binding activity of oligomers was similar to that of F(ab′)₂. In mice bearing pancreatic-carcinoma xenografts, tumor uptake of S-S oligomers was significantly greater than that of monomeric Fab, while there was no difference in tumor uptake between S-S Fab trimers and F(ab′)₂. S-S oligomers showed more rapid clearance rates and uniform percolation in the tumor nodules than F(ab′)₂. At 24 hr after injection, S-S Fab oligomers exhibited higher tumor-to-normal-tissue specificity ratios than did F(ab′)₂. At 18 hr after injection, clear scintigraphic detection of the pancreatic-carcinoma tumors was obtained with ¹²³I-labeled S-S Fab dimers. At 24 hr, improved tumor imaging was shown for ¹²³I-labeled S-S Fab oligomers with slightly visible uptake in normal tissues, similar to that of F(ab′)₂. S-S oligomers of chimeric A10 Fab may be useful as rapid diagnostic tools of pancreatic carcinomas. © 1996 Wiley-Liss, Inc.     

Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: Influence of radiation dose and dose rate on toxicity and anti-tumor efficacy

Whereas bivalent fragments have been widely used for radio-immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of ¹³¹I-labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose-limiting organ toxicity and anti-tumor efficacy. The maximum tolerated doses (MTDs) and dose-limiting organ toxicities of the ¹³¹I-labeled anti-CEA antibody MN-14 [IgG, F(ab′)₂ and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D-lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 ¹³¹I-labeled immunoconjugates [IgG, F(ab′)₂ and Fab], the red marrow was the only dose-limiting organ; MTDs were 260 μCi for IgG, 1,200 μCi for F(ab′)₂ and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab′)₂. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti-tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments. Int. J. Cancer 77:787–795, 1998. © 1998 Wiley-Liss, Inc.     

Differential inhibitory effect of L-lysine on renal accumulation of 67Cu-labelled F(ab′)2 fragments in mice

The basic amino acid L-lysine was administered to mice in an attempt to circumvent unwanted renal accumulation of ⁶⁷Cu-labelled F(ab′)₂ fragments derived from the anti-NCAM IgG₁, SEN7 and anti-CEA IgG₁ monoclonal antibody (MAb)35. In control experiments, significant renal uptake of both ⁶⁷Cu-labelled F(ab′)₂ fragments was observed, radiolabel being primarily localised to proximal tubules in the renal cortex. Following optimised L-lysine dosing protocols, renal uptake of ⁶⁷Cu-MAb35 F(ab′)₂ was inhibited by up to 42%. Surprisingly, little inhibition (<10%) of ⁶⁷Cu-SEN7 F(ab′)₂ uptake was observed. Experiments to investigate this differential inhibition indicated that inhibition of MAb35 F(ab′)₂ uptake was relatively short-lived (approx. 6 hr), whilst no apparent differences were found in blood clearance rates between either ⁶⁷Cu-F(ab′)₂ fragment. L-lysine administration caused a significant diuresis with high levels of intact ⁶⁷Cu-labelled SEN7 and MAb35 F(ab′)₂ appearing in the urine, possibly due to blockade of renal uptake and lysine-induced increases in glomerular membrane permeability. Iso-electric focusing studies failed to identify any charge differences between the ⁶⁷Cu-labelled F(ab′)₂ fragments, although a cathodal migration of all ⁶⁷Cu-labelled samples, presumably due to the net positive charge conferred by addition of ⁶⁷Cu²⁺ ions, was observed. Our results demonstrate that in addition to net charge, other unidentified characteristics may influence renal accumulation of radiometal-labelled F(ab′)₂ fragments and their inhibition by L-lysine. Int. J. Cancer 72:522–529, 1997. © 1997 Wiley-Liss, Inc.     

Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells. In this study the F(ab')2 was the best compromise between the slowly cleared IgG and the poorly localized Fab in tumor imaging.     

Enhanced Tumor Localization of Radiolabeled Fab Fragments of Monoclonal Antibody A7 in Nude Mice Bearing Human Pancreatic Carcinoma Xenografts

Much recent research has been directed toward the use of monoclonal antibodies (MAb) for the inimunodetection of solid tumors. In pancreatic cancer, the results of conventional immunoscintigraphy using intact MAb remain disappointing. Clear immunoseintigrapliy with radiolabeled MAb requires a high tumor tissue/blood ratio of radioactivity and a low normal tissue/blood ratio of radioactivity. In this study, ¹²⁵I-labeled Fab fragments produced by papain digestion of MAb A7 were injected intravenously into nude mice bearing a human pancreatic cancer (HPC-YS) xenograft previously shown to react specifically with MAb A7. The radioactivity of tumors and normal organs was subsequently measured. The tumor tissue/blood ratio of ¹²⁵I-labeled Fab fragments of MAb A7 was 1.00±0.24 and 9.68±2.54 at 2 and 24 h after injection, respectively. The tumor tissue/blood ratio of radioactivity was significantly higher than those of normal organs at 24 h after injection. Moreover, the tumor tissue/blood ratio of ¹²⁵I-labeled Fab fragments of MAb A7 was greater than that of intact MAb A7, although the ¹²⁵I-labeled Fab accumulation level was much less than that of ¹²⁵I-labeled intact MAb A7 in the tumor. When mice bearing tumors which did not react with MAb A7 were studied, ¹²⁵I-labeled Fab fragments did not specifically localize to the tumors. These results suggest that Fab fragments of MAb A7 may be suitable carriers of radionuclides for the immunodetection of human pancreatic cancer.     

Increased tumor localization by monoclonal antibody A7 after F(ab')2 fragmentation in athymic nude mice bearing human pancreatic carcinomas

Much recent research has been directed toward the use of monoclonal antibodies (MoAbs) for the immunodetection of solid tumors. In pancreatic cancer, conventional immunoscintigraphy using intact MoAbs remains disappointing. In this study, ¹²⁵I-labeled F(ab')₂ fragments produced by pepsin digestion of MoAb A7 were injected intravenously into nude mice bearing human pancreatic cancer, HPC-YS, xenografts that have previously been shown to react specifically with MoAb A7. The tumor tissue/blood ratio of ¹²⁵I-labeled F(ab')₂ fragments of MoAb A7 increased with time and was much higher than those for normal tissues. Moreover, the tumor tissue/blood ratio of ¹²⁵I-labeled F(ab')₂ fragments was greater than that of intact MoAb A7, although the F(ab')₂ accumulation was less than that of intact MoAb A7 in the tumor. These results suggest that F(ab')₂ fragments of MoAb A7 may be suitable carriers of radionuclides for immunodetection of human pancreatic cancer. © 1993 Wiley-Liss, Inc.     

Comparative targeting of human colon-carcinoma multicell spheroids using one- and two-step (bispecific antibody) techniques

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. © 1996 Wiley-Liss, Inc.     

Phase-I/II radio-immunotherapy study with iodine-131-labeled anti-CEA monoclonal antibody F6 F(ab′)2 in patients with non-resectable liver metastases from colorectal cancer

Experimental studies in nude mice with human colon-carcinoma grafts demonstrated the therapeutic efficiency of F(ab′)₂ fragments to carcinoembryonic antigen (CEA) labeled with a high dose of ¹³¹Iodine. A phase I/II study was designed to determine the maximum tolerated dose of ¹³¹I-labeled F(ab′)₂ fragments (¹³¹I-F(ab′)₂) from anti-CEA monoclonal antibody F6, its limiting organ toxicity and tumor uptake. Ten patients with non-resectable liver metastases from colorectal cancer (9 detected by CT scan and 1 by laparotomy) were treated with ¹³¹I-F(ab′)₂, doses ranging from 87 mCi to 300 mCi for the first 5 patients, with a constant 300-mCi dose for the last 5 patients. For all the patients, autologous bone marrow was harvested and stored before treatment. Circulating CEA ranged from 2 to 126 ng/ml. No severe adverse events were observed during or immediately following infusion of therapeutic doses. The 9 patients with radiologic evidence of liver metastases showed uptake of ¹³¹I-F(ab′)₂ in the metastases, as observed by single-photon-emission tomography. The only toxicity was hematologic, and no severe aplasia was observed when up to 250 mCi was infused. At the 300-mCi dose, 5 out of 6 patients presented grade-3 or -4 hematologic toxicity, with a nadir for neutrophiles and thrombocytes ranging from 25 to 35 days after infusion. In these 5 cases, bone marrow was re-infused. No clinical complications were observed during aplasia. The tumor response could be evaluated in 9 out of 10 patients. One patient showed a partial response of one small liver metastasis (2 cm in diameter) and a stable evolution of the other metastases, 2 patients had stable disease, and 6 showed tumor progression at the time of evaluation (2 or 3 months after injection) by CT scan. This phase-I/II study demonstrated that a dose of 300 mCi of ¹³¹I-F(ab′)₂ from the anti-CEA Mab F6 is well tolerated with bone-marrow rescue, whereas a dose of 200 mCi can be infused without severe bone-marrow toxicity. Int. J. Cancer 75:615–619, 1998. © 1998 Wiley-Liss, Inc.     

Radiolabeled F(ab′)<Subscript>2</Subscript>-cetuximab for theranostic purposes in colorectal and skin tumor-bearing mice models

Purpose

This study aimed to investigate theranostic strategies in colorectal and skin cancer based on fragments of cetuximab, an anti-EGFR mAb, labeled with radionuclide with imaging and therapeutic properties, ¹¹¹In and ¹⁷⁷Lu, respectively.

Methods

We designed F(ab′)₂-fragments of cetuximab radiolabeled with ¹¹¹In and ¹⁷⁷Lu. ¹¹¹In-F(ab′)₂-cetuximab tumor targeting and biodistribution were evaluated by SPECT in BalbC nude mice bearing primary colorectal tumors. The efficacy of ¹¹¹In-F(ab′)₂-cetuximab to assess therapy efficacy was performed on BalbC nude mice bearing colorectal tumors receiving 17-DMAG, an HSP90 inhibitor. Therapeutic efficacy of the radioimmunotherapy based on ¹⁷⁷Lu-F(ab′)₂-cetuximab was evaluated in SWISS nude mice bearing A431 tumors.

Results

Radiolabeling procedure did not change F(ab′)₂-cetuximab and cetuximab immunoreactivity nor affinity for HER1 in vitro. ¹¹¹In-DOTAGA-F(ab′)₂-cetuximab exhibited a peak tumor uptake at 24 h post-injection and showed a high tumor specificity determined by a significant decrease in tumor uptake after the addition of an excess of unlabeled-DOTAGA-F(ab′)₂-cetuximab. SPECT imaging of ¹¹¹In-DOTAGA-F(ab′)₂-cetuximab allowed an accurate evaluation of tumor growth and successfully predicted the decrease in tumor growth induced by 17-DMAG. Finally, ¹⁷⁷Lu-DOTAGA-F(ab′)₂-cetuximab radioimmunotherapy showed a significant reduction of tumor growth at 4 and 8 MBq doses.

Conclusions

¹¹¹In-DOTAGA-F(ab′)₂-cetuximab is a reliable and stable tool for specific in vivo tumor targeting and is suitable for therapy efficacy assessment. ¹⁷⁷Lu-DOTAGA-F(ab′)₂-cetuximab is an interesting theranostic tool allowing therapy and imaging.

     

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacokinetics,biodistribution and biological effects of intravenously administered bispecific monoclonal antibody OC/TR F(ab′)2 in ovarian carcinoma patients

The bispecific monoclonal antibody (biMAb) OC/TR combines the anti-ovarian-cancer reactivity of the MOv18 monoclonal antibody (MAb) with the reactivity of an anti-CD3 MAb. Pre-clinical studies have indicated that this biMAb is able to redirect the cytolytic activity of T cells towards tumour cells, resulting in efficient tumour-cell lysis. To assess the clinical potential of systemic biMAb-based cancer therapy we initiated a study in ovarian-cancer patients. Five patients suspected of ovarian cancer received ¹²³I-OC/TR F(ab′)₂ i.v. Unexpectedly, the first patient developed side effects (grade III–IV toxicity) starting 30 min after infusion (p.i.) of 1 mg of OC/TR F(ab′)₂. After approval of the Ethical Committee, the study was continued at lower dose levels (0.1 mg; 0.2 mg). However, at the 0.2-mg dose level similar side effects were observed. FACS analysis indicated that all peripheral T cells were coated with biMAb immediately following the infusion. The cytokines tumour necrosis factor-α, interferon-γ and interleukin-2 showed maximum serum concentrations 2 h p.i. Tumour uptake ranged from 0.8 to 1.9% ID/kg, resulting in tumour/background ratios of 3 to 8. Our results suggest that at higher antibody dose levels OC/TR F(ab′)₂ causes T-cell activation with acute release of cytokines. Only low doses of biMAb can be administered safely. Despite the interaction with T cells, OC/TR F(ab′)₂ preferentially localizes in tumours following i.v. administration, thus offering therapeutic perspectives. © 1996 Wiley-Liss, Inc.     

Selective tumor localization of radiolabeled anti-human melanoma monoclonal antibody fragment demonstrated in the nude mouse model

Monoclonal antibodies (Mab) directed against distinct epitopes of the human 240 kD melanoma-associated antigen have been evaluated for their capacity to localize in human melanoma grafted into nude mice. A favorable tumor to normal tissue ratio of 13 was obtained with intact 131I-labeled MAb Me1-14. This ratio was further increased to 43 and 23 by the use of F(ab')2 and Fab fragments, respectively. The specificity of tumor localization was demonstrated by the simultaneous injection of F(ab')2 fragments from MAb Me1-14 and anti-CEA MAb 35, each labeled with a different iodine isotope, into nude mice grafted with a melanoma and colon carcinoma. The fragments from both MAb localized with perfect selectivity in their relevant tumor as shown by differential whole body scanning and by direct measurement of the two isotopes in tumors and normal tissues. These in vivo experimental results suggest that the F(ab')2 fragment from MAb Me1-14 is suitable for melanoma detection by immunoscintigraphy in patients.     

Evaluation of L6, an anti-carcinoma murine monoclonal antibody, in tumor-bearing nude mice

L6 is a murine monoclonal antibody (MAb) binding to cells of most human carcinomas, mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and inhibiting tumor growth in nude mice [10]. Fab and F(ab')2 fragments of L6, as well as intact MAb, have been evaluated for immunospecific localization in nude mice xenografted with a human lung carcinoma. They were compared with preparations of an isotype-matched control immunoglobulin, P1.17, after labelling with 125I or 131I. L6 Fab fragments prepared from MAb L6 and labelled with 67Ga via desferrioxamine were also tested. The data suggest that MAb L6 may be useful for in vivo detection of human carcinomas.     

Intratumoral administration of neocarzinostatin conjugated to monoclonal antibody A7 in a model of pancreatic cancer

We investigated the following in athymic nude mice with xenografts of a human pancreatic carcinoma: 1) clearance of the murine monoclonal antibody A7 from the carcinoma; and 2) the antitumor effect of neocarzinostatin conjugated to MAb A7 (A7-NCS) on the carcinoma following intratumoral injection. Compared with ¹²⁵I-labeled normal mouse IgG, a significantly larger amount of ¹²⁵I-labeled A7 remained in the tumor after intratumoral injection. Neocarzinostatin conjugated to MAb A7 showed a greater antitumor activity against human pancreatic cancer than neocarzinostatin alone after intratumoral administration. The conjugate completely suppressed tumor growth macroscopically during the experiment. Tumor tissue in mice became necrotic 32 days after injection with A7-NCS. These observations suggest that the intratumoral injection of A7-NCS offers promise in treating pancreatic carcinoma. © 1993 Wiley-Liss, Inc.     

Radiolocalization of xenografted human lung cancer with monoclonal antibody 8 in nude mice

A monoclonal antibody (MAb) 8 [immunoglobulin G3 (IgG3)], directed against a Mr 48,000 human lung cancer-associated antigen, was radiolabeled with either 125I or 131I, and its biodistribution was studied in nude mice bearing human lung cancer (TKB-2) over a 7-day period. 125I-labeled MAb 8 increased its binding to the tumor during the period, while the binding of 125I-labeled control IgG3 declined after initial uptake. At Day 7, percentages of injected dose of 125I-labeled MAb 8 bound to the tumor rose to 7.4%, which was a 4.4-fold increase from Day 1 and 16-fold binding of 125I-labeled control IgG3 at the same day. Tumor:blood ratios became 2.7:1 at Day 7, and tumor:liver, tumor:spleen, and tumor:kidney ratios were more than 9:1. Normal organs showed no significant uptake of 125I-labeled MAb 8, compared with those of 125I-labeled control IgG3. A clear image of the xenografted tumor was obtained at Day 5, and it further improved at Day 7, when 60% of whole body radioactivity was localized to the tumor. Autoradiography of the mouse with tumor confirmed the excellent localization of 125I-labeled MAb 8 to the tumor, although the radioactivity of the tumor was not uniformly distributed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that most of the radioactivity was present at the tumor in the form of degraded immunoglobulin. MAb 8 has a potential usefulness in the diagnosis and treatment of lung cancer.     

Selection of radioimmunoconjugates for the therapy of well-established or micrometastatic colon carcinoma

In order to optimize radioimmunotherapy (RAIT) as a cancer-treatment modality, it is necessary to select the appropriate radionuclide and antibody carrier. We evaluated the therapeutic potential of a single cycle of Mu-9 anti-CSAp monoclonal antibody (MAb) labeled with 3 different radionuclides, ¹³¹I, ⁹⁰Y and ¹⁸⁸Re. Intact antibodies and bivalent fragments with different blood clearance kinetics, normal organ distribution and varying tumor accretion and retention are also evaluated. Efficacy of treatment for large and small tumor burden was assessed in nude mice bearing s.c. GW-39 human colonic-carcinoma xenografts or intrapulmonary micrometastatic GW-39 colonies at the maximal tolerated dose of each agent. The magnitude and duration of myelosuppression associated with each radioantibody was considered by monitoring peripheral blood counts, marrow colony-forming unit activity and hematopoietic tissue weight. Radiation-dose estimates were calculated based on the kinetics of antibody accretion and elimination from tumor and normal tissues, and the results were correlated with tumoricidal activity and dose-limiting toxicity results. These studies, therefore, represent a detailed analysis, in a well-defined experimental tumor system, of several parameters (antibody form, radioisotope, tumor size) influencing the overall outcome of RAIT using equitoxic doses. It was found that myelosuppression is the primary dose-limiting toxicity for all radioantibodies except ⁹⁰Y-F(ab′)₂, even though the different agents showed varied organ distribution. In a single-cycle treatment schedule of Mu-9 MAb, the ¹³¹I-labeled IgG is the radioimmunoconjugate of choice for the treatment of s.c. and intrapulmonary growth of the GW-39 human colonic-carcinoma xenograft in nude mice. Int. J. Cancer 72:477–485, 1997. © 1997 Wiley-Liss, Inc.     

Phase I studies of treatment of malignant gliomas and neoplastic meningitis with131I-radiolabeled monoclonal antibodies anti-tenascin 81C6 and anti-chondroitin proteoglycan sulfate Me1-14 F (ab′)2-a preliminary report

Summary The advent of monoclonal antibody (MAb) technology has made Ehrlich's postulate of the magic bullet an attainable goal. Although specific localization of polyvalent antibodies to human gliomas was demonstrated in the 1960s, the lack of specific, high affinity antibody populations and of defined target antigens of sufficient density precluded therapeutic applications. Not until the identification of operationally specific tumor-associated antigens (present in tumor tissue but not normal central nervous system tissue); production of homogeneous, high affinity MAbs to such antigens; and the use of compartmental administration (intrathecal or intracystic), has the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms been recognized. We report here preliminary data from Phase I studies of the compartmental administration of the anti-tenascin MAb 81C6 and F(ab2)₂ fragments of MAb Mel-14, which recognizes the proteoglycan chondroitin sulfate-associated protein of gliomas and melanomas, to patients with primary central nervous system tumors or tumors metastatic to the central nervous system. Phase I dose escalation studies of intracystically administered¹³¹I-labeled anti-tenascin MAb 81C6 to either spontaneous cysts of recurrent gliomas or surgically created cystic resection cavities have resulted in striking responses. Of five patients with recurrent cystic gliomas treated, four had partial responses, clinically or radiographically. Similarly, in patients with surgically created resection cavities, a partial response at the treatment site and extended stable disease status has been obtained following intracystic administration of¹³¹I-labeled 81C6. No evidence of hematologie or neurologic toxicity has been observed in either patient population, with the exception of transient exacerbation of a pre-existing seizure disorder in a single patient. Dosimetry calculations indicated high intracystic retention for four to six weeks with little or no systemic dissemination; estimated total doses intracystically ranged from 12,700–70,290 rad.Intrathecal administration of labeled MAbs to patients with neoplastic meningitis is more difficult to assess in terms of clinical responsiveness. Of patients so treated with either¹³¹I-labeled 81C6 or¹³¹I-labeled Mel-14 F(ab)₂, cerebrospinal fluid and radiographie responses have been achieved, and survival prolongation through maintenance of stable disease has been observed in several cases.Initial results from Phase I dose escalation trials are encouraging in terms of the proportion of cases of disease stabilization and partial and complete responses obtained. Importantly, neurotoxicity has been virtually nonexistent, and hematologie toxicity rare and rapidly responsive to treatment. In the intracompartmental setting, then, the promise of chimerized MAb molecules or of dimeric or monomeric single-fragment chains, either radiolabeled or drug- or toxin-conjugated, is great. The possibilities of MAb-mediated, targeted therapy for tumors of the central nervous system are many and promising. Future work will be with newly defined antigens of exquisite tumor specificity, such as the variant epidermal growth factor receptor III molecule. New labeling technology will allow halogens such as¹³¹I and²¹¹At to be used for internalized or membrane-localized antigens. Internalized MAbs will be able to be used as immunotoxins or labeled with chemotherapeutic agents.     

Development and Characterization of Chimeric Anti-carcinoembryonic Antigen Monoclonal Antibodies and Their Fab Fragments

In an attempt to reduce the immunogenicity of two different murine anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs), KM10 and A10, we produced recombinant mouse/human chimeric MAbs and the respective Fab fragments carrying the variable regions of the murine MAbs. Chimeric A10 Fab fragment was expressed in Escherichia coli , and produced in large quantities in a mini-jar fermentation system. In competitive binding assays, chimeric MAbs and their Fab fragments showed identical specificity to human CEA epitopes, as compared to the parental MAbs or Fab fragments. Both chimeric Fab fragments exhibited strong immunohistochemical reactivity with various gastrointestinal carcinomas and no reactivity with CEA-related antigens, such as NCA (nonspecific cross-reacting antigen) or BGPI (biliary glycoprotein I). Furthermore, chimeric KM10 MAb elicited substantially higher antibody-dependent cellular cytotoxicity than the murine MAb. Complement-dependent cytotoxicity in vitro was much weaker with chimeric KM10 MAb. These results indicate that chimeric MAbs or Fab fragments could potentially replace the parental murine antibodies or their Fab fragments in therapy or diagnosis of human gastrointestinal carcinomas.     

Engineering high affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab′)2 for efficient lysis of p185HER2 overexpressing tumor cells

We previously constructed a humanized anti-p185^HER2/anti-CD3 bispecific antibody variant, BsF(ab′)₂ v1 which retargets the cytotoxic activity of human T cells in vitro against human breast tumor cells which overexpress the p185^HER2 product of the HER2/neu (c-erbB-2) protooncogene. Subsequently we identified an improved anti-CD3 variant, v9, which binds to T cells with approx. 100-fold higher affinity than the original variant, v1. Here we demonstrate that BsF(ab′)₂ v9 is more potent than BsF(ab′)₂ v1 in stimulating the proliferation of both resting peripheral blood lymphocytes (PBL) and IL-2-activated, long-term cultured T lymphocytes (ATL). In addition, at low concentrations (0.01-1 ng/ml) BsF(ab′)₂ v9 is much more efficient than BsF(ab)₂ v1 in directing lysis of p185^HER2-overexpressing tumor cells by IL-2 activated PBL. In contrast, at higher concentration BsF(ab′)₂ v9 and BsF(ab′)₂ v1 have similar potency in retargeted cytotoxicity. At BsF(ab′)₂ v9 concentrations of ? 1 ng/ml the susceptibility of p185^HER2-expressing tumor cells to lysis is apparently independent of the level of p185^HER2 expression. At lower concentrations of BsF(ab′)₂ v9 and/or lower ratios of effector to target cells the extent of lysis is reduced, in some cases improving the selectivity of lysis of high p185^HER2 expressors over low expressors. Thus selection of a high affinity anti-CD3 arm is likely important in the design of BsF(ab′)₂ for retargeting the cytotoxicity of T cells to tumors. The dose of BsF(ab′)₂ v9 in any future clinical evaluation will require optimization to maximize anti-tumor efficacy whilst minimizing potential toxicity against normal tissue expressing p185^HER2. © 1995 Wiley-Liss Inc.     

Effects of neocarzinostatin-chimeric Fab conjugates on the growth of human pancreatic carcinoma xenografts.

Neocarzinostatin (NCS) was bound covalently to human/mouse chimeric Fab fragments of MAb A7 (chA7Fab) directed against human pancreatic carcinoma. The anti-tumour effect of chA7Fab-NCS was tested in a nude mouse model on pancreatic carcinoma and compared with A7-NCS or NCS alone. The anti-tumour effect of chA7Fab-NCS increased in a dose-dependent manner and was significantly greater than either A7-NCS or NCS. Tumour growth was completely suppressed after the administration of chA7Fab-NCS. An enzyme-linked immunosorbent assay with rabbit anti-mouse immunoglobulin was performed to examine the antigenicity of chA7Fab. ChA7Fab had less reactivity with rabbit anti-mouse immunoglobulin than either whole antibody A7 or murine Fab fragments of A7. Thus, chA7Fab-NCS can inhibit human pancreatic cancer growth in an animal and may be useful for targeting chemotherapy to pancreatic cancer in humans.