Sydney Welt

Phase I/II study of iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancer.

PURPOSE A phase I/II study was designed to determine the maximum-tolerated dose (MTD) of iodine 125-labeled monoclonal antibody A33 (125I-mAb A33), its limiting organ toxicity, and the uptake and retention of radioactivity in tumor lesions. PATIENTS AND METHODS Patients (N = 21) with advanced chemotherapy-resistant colon cancer who had not received prior radiotherapy were treated with a single 125I-mAb A33 dose. 125I doses were escalated from 50 to 350 mCi/m2 in 50-mCi/m2 increments. Radioimmunoscintigrams were performed for up to 6 weeks after 125I-mAb A33 administration. RESULTS All 20 patients with radiologic evidence of disease showed localization of 125I to sites of disease. Twelve of 14 patients, who underwent imaging studies 4 to 6 weeks after antibody administration, had sufficient isotope retention in tumor lesions to make external imaging possible. No major toxicity was observed, except in one patient with prior exposure to mitomycin who developed transient grade 3 thrombocytopenia. Although the isotope showed variable uptake in the normal bowel, gastrointestinal symptoms were mild or absent, and in no case did stools become guaiac-positive. The MTD was not reached at 125I doses up to 350 mCi/m2. However, cytotoxicity assays demonstrated that patients treated with the highest dose had sufficiently high serum levels of 125I-mAb A33 to lyse colon cancer cells in vitro. Among 21 patients, carcinoembryonic antigen (CEA) levels returned to normal in one patient and decreased by 35% and 23%, respectively, in two patients; one additional patient had a mixed response on computed tomography. Additional, significant responses were observed in those patients treated with chemotherapy [carmustine [BCNU], vincristine, flourouracil, and streptozocin [BOF-Strep]) after completion of the 125I-mAb A33 study. CONCLUSION Low-energy emission radioimmunotherapy with doses of up to 350 mCi/m2 of 125I-mAb A33 did not cause bowel or bone marrow toxicity. The modest antitumor activity in these heavily pretreated patients is encouraging because of lack of toxicity at the doses studied. The long radioactivity retention in tumors suggests that isotopes with a long half-life may have a therapeutic advantage, based on calculated dose delivery to tumor versus normal tissue. Due to the low bone marrow dose, further 125I trials with humanized mAb A33 are warranted, and controlled studies must be conducted to evaluate the combination of radioimmunotherapy and chemotherapy.

Antibody localization in human renal cell carcinoma: a phase I study of monoclonal antibody G250.

PURPOSE To define the imaging and biodistribution characteristics of iodine 131-labeled monoclonal antibody (mAb) G250 (131I-mAbG250), which recognizes a cell-surface antigen expressed by human renal cell carcinoma (RCC). PATIENTS AND METHODS G250 is a cell-surface antigen recognized by mAbG250 expressed by RCC but not detected in normal kidney. Clear-cell RCC, the most frequent form of RCC, shows homogeneous expression of G250, whereas non-clear-cell RCC and cancers derived from other organs generally do not express G250. Expression in normal tissues is highly restricted and limited to large bile ducts and gastric epithelium. 131I-mAbG250 was administered intravenously (IV) to 16 patients with RCC 7 to 8 days before surgery at five dose levels, with at least three patients entered at each dose level. RESULTS Clear tumor images were observed in 12 patients with G250-positive tumors and in one of three patients with G250-negative tumors. Imaged lesions in the peritoneal cavity were confirmed at surgery. The smallest lesion visualized was 8 mm in diameter. The specificity of 131I-mAbG250 localization to tumor tissue was established by radioactivity measurements, autoradiography, and immunohistochemistry of biopsied tissues, and technetium 99-human serum albumin blood-flow studies. The fraction of the injected 131I-mAbG250 dose per gram tumor (%ID/g tumor) localized in G250-positive tumors showed a broad range, but reached levels as high as 0.02% to 0.12%. CONCLUSION 131I-mAbG250 localized specifically to G250 antigen-positive RCC and seems to have considerable potential as an imaging agent in RCC patients. 131I-mAbG250 uptake in the tumors, relative as well as absolute, are among the highest reported for tumor biopsies obtained 8 days after IV mAb administration. Based on the specific localization and high accumulation, mAb G250 may have therapeutic potential.

Antibody Targeting in Metastatic Colon Cancer: A Phase I Study of Monoclonal Antibody F19 Against a Cell-Surface Protein of Reactive Tumor Stromal Fibroblasts

PURPOSE To define the toxicity, imaging, and biodistribution characteristics of iodine 131-labeled monoclonal antibody F19 (131I-mAbF19). MAbF19 recognizes the fibroblast activation protein (FAP), a cell-surface glycoprotein not present in most normal tissues, but abundantly expressed by reactive stromal fibroblasts of epithelial cancers, including more than 95% of primary and metastatic colorectal carcinomas. PATIENTS AND METHODS 131I-mAbF19 was administered intravenously to 17 patients with hepatic metastases from colorectal carcinoma who were scheduled for resection of localized metastases or insertion of hepatic artery catheter for regional chemotherapy. Seven to 8 days before surgery, patients received 131I-mAbF19 at three dose levels, with at least four patients entered at each level. RESULTS No toxicity associated with intravenous 131I-mAbF19 administration was observed. Tumor images were obtained on planar and single-photon emission tomography (SPECT) scans in 15 of 17 patients with hepatic metastases, tumor-infiltrated portal lymph nodes, and/or recurrent pelvic disease. The smallest lesion visualized was 1 cm in diameter. The optimal time for tumor imaging was 3 to 5 days after 131I-mAbF19 administration. The use of image registration techniques allowed precise anatomic localization of 131I-mAbF19 accumulation. Immunohistochemical analysis of biopsy tissues showed expression of FAP in the tumor stroma (but not in normal liver) in all patients studied and confirmed that the FAP-positive tumor stromal fibroblasts were interposed between the tumor capillaries and the malignant colon epithelial cells. At the time of surgery, tumor-to-liver ratios up to 21:1 and tumor-to-serum ratios up to 9:1 were obtained. The fraction of the injected 131I-mAbF19 dose per gram tumor (%ID/g tumor) localized to hepatic metastases at the time of surgery ranged from 0.001% to 0.016%. CONCLUSION The FAP tumor fibroblast antigen is highly expressed in primary and metastatic colorectal carcinomas and shows limited expression in normal adult tissues. This highly selective expression pattern allows imaging of colorectal carcinoma lesions as small as 1 cm in diameter on 131I-mAbF19 scans. Because of the consistent presence of FAP in the stroma of epithelial cancers and the accessibility of FAP-positive tumor stromal fibroblasts to circulating monoclonal antibodies (mAbs), this study suggests possible diagnostic and therapeutic applications of humanized mAbF19 and mAbF19 constructs with novel immune and nonimmune effector functions.

Serological and immunochemical analysis of Lewis Y (Ley) blood group antigen expression in epithelial ovarian cancer

The expression of Ley blood group antigen in epithelial ovarian cancer tissues and cell lines has been studied using a Ley‐specific monoclonal antibody (MAb 35193). In ovarian cancer specimens, Ley was expressed in 75% of the 140 tumor specimens examined, with strong or moderate expression being observed in 56% of the samples. Seven of the 11 ovarian cancer cell lines studied were Ley‐positive. Using immunochemical approaches, Ley epitopes were found to be expressed on 4 types of carrier molecules: CA125 ovarian cancer antigen, MUC‐I mucins, lower m.w. glycoprotiens and glycolipids. In cell lines, Ley was more commonly expressed on MUC‐I mucin than on CA125, whereas in tumor specimens Ley was commonly found on both CA125 and MUC‐I. The biochemical nature of the smaller Ley glycoproteins was not determined, but it was shown that they were not CEA and LAMP‐I, known Ley carriers in some other tumor types. Glycolipids carrying Ley epitopes were detected in both ovarian cancer cell lines and tumor specimens. The presence of Ley epitopes on a number of different molecular carriers, including 2 major ovarian cancer antigens (CA125 and MUC‐I), explains the high incidence of Ley in ovarian cancer. The high expression of Ley in ovarian cancer and the availability of specific murine and humanized MAbs make Ley an attractive candidate target for clinical studies.

ANTIBODY-BASED IMMUNOLOGICAL THERAPIES

Clinical research in the area of antibody-based tumor-targeted therapy has been driven for many years by the prospect of identifying cell surface antigens with sufficient restrictive tissue expression patterns to allow for the selective and specific accumulation of antibody in tumor tissue. Few, if any, such antibody-antigen systems have been identified which can effectively deliver a large fraction of an administered therapeutic agent to metastatic cancer. Despite this limitation, however, a greater understanding of the biological and physiological principles of tumor-targeted therapy has resulted in successful antibody-based therapy of lymphoma, colon cancer and breast cancer in recent clinical trials.

A Phase I Trial of Humanized Monoclonal Antibody A33 in Patients with Colorectal Carcinoma: Biodistribution, Pharmacokinetics, and Quantitative Tumor Uptake

Purpose: To determine the in vivo characteristics of huA33, a CDR-grafted humanized antibody against the A33 antigen, we have conducted an open-label, dose escalation, biopsy-based phase I trial of huA33 in patients with colorectal carcinoma. Experimental Design: Patients with colorectal carcinoma were infused with [131I]huA33 (400 MBq: 10 mCi) and [125I]huA33 (40 MBq: 1 mCi) 1 week before surgery. There were four huA33 dose levels (0.25, 1.0, 5.0, and 10 mg/m2). Adverse events, pharmacokinetics, biodistribution, tumor biopsies, and immune responses to huA33 were evaluated. Results: There were 12 patients entered into the trial (6 males and 6 females; age range, 39-66 years). No dose-limiting toxicity was observed. The biodistribution of huA33 showed excellent uptake of [131I]huA33 in metastatic colorectal carcinoma. Pharmacokinetic analysis showed no significant difference in terminal half-life (T1/2β) between dose levels (mean ± SD, 86.92 ± 22.12 hours). Modeling of colon uptake of huA33 showed a T1/2 of elimination of 32.4 ± 8.1 hours. Quantitative tumor uptake ranged from 2.1 × 10−3 to 11.1 × 10−3 %ID/g, and tumor/normal tissue and tumor/serum ratios reached as high as 16.3:1 and 4.5:1, respectively. Biosensor analysis detected low-level human anti-human antibody responses in four patients following huA33 infusion. Conclusions: huA33 shows selective and rapid localization to colorectal carcinoma in vivo and penetrates to the center of large necrotic tumors, and colon elimination half-life of huA33 is equivalent to basal colonocyte turnover. The excellent targeting characteristics of this humanized antibody indicate potential for the targeted therapy of metastatic colorectal cancer in future trials.

Anti-renal-cell carcinoma chimeric antibody G250 facilitates antibody-dependent cellular cytotoxicity with in vitro and in vivo interleukin-2-activated effectors

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy, whereas treatment with biologics has achieved limited success. Although monoclonal antibodies able to recognize human RCC have been identified, most induce little complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC), and thus are of limited potential as therapeutic modalities in their natural conformation. We evaluated a human/ mouse chimeric derivative of the previously described G250 murine monoclonal antibody (mAb), reactive with RCC, to identify a reagent for potential immunotherapy. This chimeric antibody (ch-G250) is composed of the murine variable region from the G250 mAb, which recognizes a tumor-associated antigen expressed on 95% of primary and 86% of metastatic renal cell carcinomas. The constant region of the ch-G250 is comprised of the human IgG1 isotype domains. This chimeric antibody does not bind to normal renal tissue or other normal human tissues, with the exception of gastric mucosal cells and large bile-duct epithelium. Clinical radiolocalization studies have demonstrated the relative tumor-targeting potential of this radiolabeled antibody. This ch-G250 antibody facilitated potent ADCC against several RCC lines when using in vitro and in vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells obtained from healthy control donors and patients with cancer, respectively. This lymphocyte-mediated ADCC was specific for RCC cells recognized by the ch-G250 antibody. Using flow cytometry, we found that the level of ADCC was directly related to the degree of binding of ch-G250 to the renal cell target. These in vitro data suggest that this antibody may improve efficacy of IL-2 therapy by targeting cytokine-activated effector cells directly to the tumor and facilitating in vivo ADCC. Clinical studies combining this chimeric antibody with IL-2 treatment will be needed to test the antitumor effects of this ADCC effect in vivo.

Purification and Characterization of a Novel Restricted Antigen Expressed by Normal and Transformed Human Colonic Epithelium

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab′)2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

Immune and nonimmune effector functions of IgG3 mouse monoclonal antibody R24 detecting the disialoganglioside GD3 on the surface of melanoma cells

R24, an IgG3 mouse monoclonal antibody reactive with the disialoganglioside GD3, was found to be a potent mediator of human complement cytotoxicity and human effector cell cytotoxicity. Cytotoxicity correlated with the degree of antibody binding (GD3 cell surface expression) for each of the melanoma cell lines and melanocyte cell cultures tested. Melanoma cell lines binding low amounts of R24 (low GD3 cell surface expressors) were not lysed in R24-directed immune reactions, suggesting that a threshold number of R24 molecules bound per cell is necessary to initiate these cytotoxic mechanisms. Since both complement- and cell-mediated reactions lysed the same subpopulations of cells in each cell line, both mechanisms appeared to depend on similar threshold quantities of bound R24 molecules. However, due to the heterogeneity of R24 binding in each cell line, the numerical value for this threshold could not be determined. Only in cell lines binding greater than 10(7) R24 molecules per cell were greater than 90% of the cells lysed. Normal melanocytes in culture were not lysed by R24-directed immune mechanisms, due to their low GD3 expression, indicating that monoclonal antibodies such as R24 may show tumor specificity with regard to effector functions even though normal cells express the relevant antigen. In contrast to the potent in vitro activity of R24, treatment of nu/nu mice bearing human melanoma grafts resulted in tumor inhibition only when started within 3 days of tumor cell inoculation. No effect was seen on established tumors. Thus, this in vivo mouse model failed to predict the clinical and pathological findings observed in treatment trials of R24 in human melanoma patients--urticaria involving skin metastases, cellular infiltration of tumor tissue, and tumor regression. In addition to activating immunologic effector functions, R24 had direct effects on melanoma cells, blocking their ability to attach to surfaces and causing tumor cell aggregation. These effects were again related to the number of R24 molecules bound to the cell surface; no aggregation was seen with cell lines binding less than 4 X 10(5) molecules per cell. Both immune and nonimmune effector functions may be involved in the tumor inhibitory activity of R24 in humans.

Pharmacokinetics and microdistribution of polyethylene glycol-modified humanized A33 antibody targeting colon cancer xenografts

Therapeutic proteins have been conjugated with polyethylene glycol (PEGylation) to reduce immunogenicity and enhance circulating dose. Here we have investigated the effect of PEGylation on immunogenicity, pharmacokinetics, and histologic microdistribution of tumor‐targeting antibodies with humanized A33 antibody (huA33) as a model system. Conjugation of huA33 with methoxy‐PEG of Mr 5,000 (32%–34% of primary amines modified) or Mr 20,000 (16%–18% modification) preserved >50% of native huA33 binding to SW1222 colon cancer cells. In mice, both PEGylated forms cleared from serum moderately slower than native huA33. After repeated immunization with PEG‐huA33, antiantibody titers in immunocompetent mice were <5% of those in huA33‐treated controls. Both PEG‐huA33 forms reached approx. 75% of the maximum tumor dose of huA33 in SW1222‐xenografted mice, but their tumor:blood ratios were considerably reduced. To demonstrate immunologic specificity of PEG‐huA33 targeting in SW1222 tumor‐bearing mice, antigenic sites were presaturated by injecting excess native huA33. This reduced subsequent uptake of PEG‐huA33 by up to 80%, whereas presaturation with hu3S193 control antibody had no significant effect. To assess the microdistribution of antibody uptake in the same xenograft model, tumor tissue resected at different time points after antibody administration was examined for human IgG by immunohistochemistry. Both PEG preparations achieved the same peak staining intensity and homogeneity as native huA33 with a delay of several hours. Given the measured reduction in immunoreactivity in vitro, these results demonstrate that the tumor targeting potential of huA33 in vivo is preserved at PEGylation levels sufficient to suppress immunogenicity. Int. J. Cancer 87:382–390, 2000.