MAb 806 enhances the efficacy of ionizing radiation in glioma xenografts expressing the de2-7 epidermal growth factor receptor.

PURPOSE: Mutations of the epidermal growth factor receptor (EGFR) are common in glioma. The most frequent mutation, de2-7 EGFR/EGFRvIII, occurs in approximately 40% of high-grade gliomas and confers resistance to ionizing radiation (IR). We have previously shown that mAb 806, a novel EGFR-specific antibody, is able to inhibit the growth of U87MG.Δ2-7 glioma xenografts expressing the de2-7 EGFR and may have potential as a therapeutic. METHODS AND MATERIALS: Nude mice bearing U87MG.Δ2-7 xenografts were treated with mAb 806 and/or IR. Comparison of tumor volumes, the effect of treatment on angiogenesis as determined by mean vessel density, and expression changes in prosurvival protein pAkt between treatment groups were undertaken. RESULTS: Treatment of mice bearing U87MG.Δ2-7 xenografts with mAb 806 and IR resulted in schedule-dependent radiosensitization. Maximal benefit was obtained when antibody treatment was given before irradiation, with the greatest inhibition of both tumor angiogenesis and tumor growth. Combination treatment mediated radiosensitization by selectively blocking the phosphorylation of the prosurvival protein Akt at serine 473, a process that is independent of DNA-dependent protein kinase catalytic subunit. CONCLUSIONS: Our results provide a rationale for the use of mAb 806 in combination with IR for the treatment of glioma and potentially other solid tumors bearing the de2-7 EGFR.

Immuno-PET quantitation of de2-7 epidermal growth factor receptor expression in glioma using 124I-IMP-R4-labeled antibody ch806.

UNLABELLED: Overexpression, activation, and mutations of the epidermal growth factor receptor (EGFR) are commonly found in solid tumors. The aim of this study was to develop a PET-based method for detecting the constitutively active mutant de2-7 EGFR, which is associated with disease progression and resistance to chemotherapy and radiotherapy in glioma. METHODS: The chimeric antibody ch806, which selectively binds an epitope of the EGFR that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor, was conjugated to the radiohalogen (124)I via the residualizing ligand IMP-R4, and in vitro properties were characterized. In vivo biodistribution and small-animal PET studies were performed in BALB/c nude mice bearing U87MG.de2-7 glioma xenografts. Imaging results were correlated with measured tumor uptake of the radioconjugate. RESULTS: (124)I-IMP-R4-ch806 had an immunoreactivity of 78.3% and was stable for 7 d when incubated in serum in vitro. The biodistribution analysis of (124)I-IMP-R4-ch806 demonstrated a maximal uptake of 30.95 +/- 6.01 percentage injected dose per gram (%ID/g) in U87MG.de2-7 xenografts at 48 h after injection, with prolonged tumor retention (6.07 +/- 0.80 %ID/g at 216 h after injection). The tumor-to-blood ratio increased from 0.44 at 4 h after injection to a maximum of 4.70 at 168 h after injection. PET of (124)I-IMP-R4-ch806 biodistribution was able to clearly detect the U87MG.de2-7 tumors at 24 h after injection and for at least 168 h after injection. Correlation between tumor PET image quantitation of (124)I-IMP-R4-ch806 and %ID/g determined from resected tissues (r = 0.9350) was excellent. CONCLUSION: These results show that immuno-PET with (124)I-IMP-R4-ch806 is feasible and allows noninvasive quantitation of de2-7 EGFR expression in vivo.

Phase I biodistribution and pharmacokinetic study of Lewis Y-targeting immunoconjugate CMD-193 in patients with advanced epithelial cancers.

PURPOSE: This phase I study explored the biodistribution and pharmacokinetics of the immunoconjugate CMD-193 [a humanized anti-Lewis Y (Le(y)) antibody conjugated with calicheamicin in patients with advanced cancers expressing the Le(y) antigen. EXPERIMENTAL DESIGN: The primary objectives were to determine biodistribution and pharmacokinetics of CMD-193. Secondary objectives included response rates and change in tumor metabolism. Patients with progressive, measurable, and Le(y) positive malignancies were eligible for enrollment in one of two dose cohorts, 1.0 and 2.6 mg/m(2). The first cycle was trace labeled with (111)In for biodistribution assessment using gamma camera imaging. Subsequent cycles were administered every 3 weeks up to a maximum of six cycles, depending on toxicity and response. Pharmacokinetic analysis was based on radioassay and ELISA. RESULTS: Nine patients were enrolled in the study. Biodistribution images showed initial blood pool activity, followed by markedly increased hepatic uptake by day 2, and fast blood clearance in all patients. There was low uptake in tumor in all patients. The overall T(1/2)beta of (111)In-CMD-193 was 102.88 +/- 35.67 hours, with no statistically significant difference between the two dose levels. One patient had a partial metabolic response on (18)F-fluorodeoxyglucose-positron emission tomography ((18)F-FDG PET) after four cycles, but no radiological responses were observed. Myelosuppression and effects on liver function were the most significant adverse effects. CONCLUSIONS: CMD-193 shows rapid blood clearance and increased hepatic uptake compared with prior studies of the parental antibody hu3S193. These results highlight the importance of biodistribution and pharmacodynamic assessment in early phase studies of new biologics to assist in clinical development.

Therapeutic efficacy of 177Lu-CHX-A''-DTPA-hu3S193 radioimmunotherapy in prostate cancer is enhanced by EGFR inhibition or docetaxel chemotherapy.

BACKGROUND: This study investigated the biodistribution and therapeutic efficacy of Lutetium-177 (177Lu) radiolabeled anti-Lewis Y monoclonal antibody hu3S193 radioimmunotherapy (RIT) in mice bearing prostate cancer xenografts. The ability of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 and docetaxel chemotherapy to enhance the efficacy of RIT was also assessed in vivo. METHODS: The in vitro cytotoxicity of 177Lu labeled hu3S193 on Le(y) positive DU145 prostate cancer cells was assessed using proliferation assays, with induction of apoptosis measured by ELISA. The in vivo biodistribution and tumor localization of 177Lu-hu3S193 was assessed in mice bearing established DU145 tumor xenografts. The efficacy and maximum tolerated dose of 177Lu-hu3S193 RIT in vivo was determined by a dose escalation study. EGFR inhibitor AG1478 or docetaxel chemotherapy was administered at sub-therapeutic doses in conjunction with RIT in vivo. RESULTS: 177Lu-hu3S193 mediated significant induction of cytotoxicity and apoptosis in vitro. In vivo analysis of 177Lu-hu3S193 biodistribution demonstrated specific targeting of DU145 prostate cancer xenografts, with maximal tumor uptake of 33.2 +/- 3.9%ID/g observed at 120 hr post-injection. In RIT studies, 177Lu-hu3S193 caused specific and dose-dependent inhibition of prostate cancer tumor growth. A maximum tolerated dose of 350 microCi was determined for 177Lu-hu3S193. Combination of 177Lu-hu3S193 RIT with EGFR inhibitor AG1478 or docetaxel chemotherapy both significantly improved efficacy. CONCLUSIONS: 177Lu-hu3S193 RIT is effective as a single agent in the treatment of Le(y) positive prostate cancer models. The enhancement of RIT by AG1478 or docetaxel indicates the promise of combined modality strategies.

Tumor targeting by a multivalent single-chain Fv (scFv) anti-Lewis Y antibody construct.

The use of single-chain variable fragment (scFv) constructs has been investigated in cancer radioimmunotherapy (RIT) and radioimmunodetection, as these molecules permit rapid tumor penetration and clearance from the serum relative to whole IgG. Multimerization of scFv constructs has demonstrated improvements in functional affinity (i.e., avidity) and maximal tumor uptake. In this paper, we report the first biodistribution and pharmacokinetics studies of a noncovalent, direct-linked scFv (V(L)-0-V(H)) trimeric/tetrameric "multimer" of the anti-Lewis Y monoclonal antibody, hu3S193. The in vitro binding and in vivo biodistribution of the hu3S193 multimer was characterized alongside the hu3S193 F(ab')(2) following radiolabeling with the Indium-111 ((111)In) radioisotope. Immunoreactivities of the radiolabeled multimer and F(ab')(2) were 73% and 53.2%, and binding affinities (K(a)) were 1.58 x 10(7) M(1) and 4.31 x 10(6) M (1) for the multimer and F(ab')(2), respectively. Maximal tumor uptake in Le(y)-positive MCF-7 breast cancer xenografted BALB/c nude mice was 12.6 +/- 2.5 percent injected dose/per gram (%ID/g) at 6 hours postinjection for the multimer and 15.7 +/- 2.1 %ID/g at 24 hours postinjection for the F(ab')(2). However, limited in vitro stability and high renal localization of radiolabeled constructs were observed, which, despite the observed tumor targeting of the hu3S193 multimer, most likely preclude its use in RIT and imaging modalities.

Radioimmunotherapy with alpha-particle emitting 213Bi-C-functionalized trans-cyclohexyl-diethylenetriaminepentaacetic acid-humanized 3S193 is enhanced by combination with paclitaxel chemotherapy.

PURPOSE: Previous experience in solid tumor radioimmunotherapy studies has indicated that greatest therapeutic efficacy is achieved in the treatment of small-volume disease. alpha-Particle-emitting radioisotopes possess several physical characteristics ideally suited to the treatment of minimal residual disease. Therefore, we have investigated the efficacy of the alpha-particle-emitting bismuth-213 (213Bi) radioimmunotherapy using the humanized anti-Lewis Y (Ley) monoclonal antibody humanized 3S193 (hu3S193). EXPERIMENTAL DESIGN: The intracellular localization of hu3S193 in Ley-positive MCF-7 breast carcinoma cells was assessed by confocal microscopy. Cytotoxicity of 213Bi-hu3S193 and apoptosis was assessed using [3H]thymidine incorporation assay and ELISA, respectively. Immunoblotting for gamma-H2AX assessed DNA strand breaks. In vivo efficacy of 213Bi-hu3S193 was assessed using a minimal residual disease model in BALB/c nude mice, with radioconjugate [15, 30, and 60 microCi (9.2 microg)] injected 2 days after s.c. implantation of MCF-7 cells. Radioimmunotherapy was also combined with a single injection of 300 microg paclitaxel to explore improved efficacy. Further, mice with established tumors received 30, 60, or 120 microCi (14.5 microg) of 213Bi-hu3S193 to assess the effect of tumor volume on treatment efficacy. RESULTS: hu3S193 is internalized via an endosomal and lysosomal trafficking pathway. Treatment with 213Bi-hu3S193 results in >90% cytotoxicity in vitro and induces apoptosis and increased gamma-H2AX expression. 213Bi-hu3S193 causes specific and significant retardation of tumor growth even in established tumors, and efficacy was enhanced by paclitaxel to produce defined complete responses. CONCLUSIONS: These studies show the potency of alpha-particle radioimmunotherapy and warrant its further exploration in the treatment of micrometastatic disease in Ley-positive malignancies.

A pilot study of monoclonal antibody cG250 and low dose subcutaneous IL-2 in patients with advanced renal cell carcinoma.

The chimeric monoclonal antibody cG250 recognizes the CAIX/MN antigen. cG250 induces antibody-dependent cellular cytotoxicity (ADCC) responses in vitro that can be enhanced by IL-2. We studied the effects of adding daily low-dose subcutaneous IL-2 to cG250 for treatment of clear cell renal cell carcinoma (RCC). The primary endpoints of the trial were toxicity and immunological effects (human anti-chimeric antibodies [HACA], ADCC, natural killer [NK] and lymphokine-activated killer cell [LAK] activity); secondary endpoints were cG250 biodistribution and pharmacokinetics (PK) and tumour response rates. Eligible patients had unresectable metastatic or locally advanced clear cell RCC with measurable or evaluable disease. Nine patients were treated with six doses of cG250 (10 mg/m(2)/week, first and fifth doses trace-labelled with (131)I), and 1.25 x 10(6) IU/m(2)/day IL-2 for six weeks. Treatment was generally well tolerated with no adverse events attributable to cG250. Two patients required a 50% dose reduction of IL-2 due to toxicity. No HACA was detected. (131)I-labeled cG250 showed excellent targeting of tumour deposits. (131)I cG250 PK: T(1/2)alpha 20.16 +/- 6.59 h, T(1/2)beta 126.21 +/- 34.04 h, CL 39.67 +/- 23.06 mL/h, Cmax 5.12 +/- 0.86 microg/mL, V(1) 3.88 +/- 1.05 L. IL-2 did not affect cG250 PK. A trend for increased percentage of circulating CD3-/CD16+CD56+ NK cells was observed. Some patients showed enhanced ADCC or LAK activity. No antitumour responses were observed. In conclusion, weekly cG250 with daily low-dose subcutaneous IL-2 is well tolerated. IL-2 does not influence cG250 biodistribution or increase HACA.

A phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody Hu3s193 in patients with advanced epithelial cancers that express the Lewis-Y antigen.

PURPOSE: We report a first-in-man trial of a humanized antibody (hu3S193) against the Le(y) antigen. EXPERIMENTAL DESIGN: Patients with advanced Le(y)-positive cancers received four infusions of hu3S193 at weekly intervals, with four dose levels (5, 10, 20, and 40 mg/m(2)). The first infusion of hu3S193 was trace labeled with Indium-111, and biodistribution, pharmacokinetics, tumor uptake, and immune response were evaluated in all patients. RESULTS: A total of 15 patients (7 male/8 female; age range, 42-76 years; 6 breast, 8 colorectal cancer, and 1 non-small-cell lung cancer) were entered into the study. Transient grade 1 to 2 nausea and vomiting was observed following infusion of hu3S193 at the 40 mg/m(2) dose level only. There was one episode of dose-limiting toxicity with self-limiting Common Toxicity Criteria grade 3 elevated alkaline phosphatase observed in one patient with extensive liver metastases. The biodistribution of (111)In-hu3S193 showed no evidence of any consistent normal tissue uptake, and (111)In-hu3S193 uptake was observed in cutaneous, lymph node, and hepatic metastases. Hu3S193 displayed a long serum half-life (T(1/2)beta = 189.63 +/- 62.17 h). Clinical responses consisted of 4 patients with stable disease and 11 patients with progressive disease, although one patient experienced a 89% decrease in a lymph node mass, and one patient experienced inflammatory symptoms in cutaneous metastases, suggestive of a biological effect of hu3S193. No immune responses (human anti-human antibody) to hu3S193 were observed. CONCLUSION: Hu3S193 is well tolerated and selectively targets tumors, and the long half-life and biological function in vivo of this antibody makes it an attractive potential therapy for patients with Le(y)-expressing cancers.

A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.

An array of cell-surface antigens expressed by human cancers have been identified as targets for antibody-based therapies. The great majority of these antibodies do not have specificity for cancer but recognize antigens expressed on a range of normal cell types (differentiation antigens). Over the past two decades, our group has analyzed thousands of mouse monoclonal antibodies for cancer specificity and identified a battery of antibodies with limited representation on normal human cells. The most tumor-specific of these antibodies is 806, an antibody that detects a unique epitope on the epidermal growth factor receptor (EGFR) that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor in cancer. In vitro immunohistochemical specificity analysis shows little or no detectable 806 reactivity with normal tissues, even those with high levels of wild-type (wt)EGFR expression. Preclinical studies have demonstrated that 806 specifically targets a subset of EGFR expressed on tumor cells, and has significant anti-tumor effects on human tumor xenografts, primarily through abrogation of signaling pathways. The present clinical study was designed to examine the in vivo specificity of a chimeric form of mAb 806 (ch806) in a tumor targeting/biodistribution/pharmacokinetic analysis in patients with diverse tumor types. ch806 showed excellent targeting of tumor sites in all patients, no evidence of normal tissue uptake, and no significant toxicity. These in vitro and in vivo characteristics of ch806 distinguish it from all other antibodies targeting EGFR.

Enhanced efficacy of 90Y-radiolabeled anti-Lewis Y humanized monoclonal antibody hu3S193 and paclitaxel combined-modality radioimmunotherapy in a breast cancer model.

UNLABELLED: Radioimmunotherapy (RIT) of solid tumor is often limited in efficacy because of restrictions in achieved tumor dose. In an effort to overcome this, the combination of RIT with other therapeutic modalities was investigated in an animal model of breast carcinoma. The rationale for this combined-modality RIT (CMRIT) was to increase the therapeutic efficacy of RIT through the use of paclitaxel to arrest cells in the radiosensitive G(2)/M phase of the cell cycle. METHODS: In this study, the biodistribution and therapeutic efficacy of (90)Y-radiolabeled humanized anti-Lewis Y hu3S193 monoclonal antibody ((90)Y-hu3S193) RIT in combination with paclitaxel chemotherapy was explored in a Lewis Y-expressing MCF-7 tumor xenografted BALB/c nude mouse model of breast cancer. RESULTS: Biodistribution studies demonstrated excellent tumor targeting and limited normal tissue uptake by (90)Y-hu3S193. A therapeutic study with established tumors assessed (90)Y-hu3S193 as a single agent and demonstrated significant antitumor effects in all animals receiving a single intravenous 1.85 or 3.70 MBq dose of this treatment compared with phosphate-buffered saline placebo controls (P = 0.0008 vs. P < 0.0001). Complete responses were observed in all animals in the 3.70 MBq study arm for the duration of the study. Single-dose (90)Y-hu3S193 plus paclitaxel (600 microg) CMRIT displayed improved efficacy over single-modality therapies, with a significant difference (P < 0.0001) between the mean percentage change in tumor volume in mice receiving 0.46 MBq (90)Y-hu3S193 alone and when combined with 600 mug paclitaxel. CONCLUSION: The significant efficacy of (90)Y-hu3S193 and paclitaxel CMRIT at low radiation doses in this model of breast carcinoma indicates its therapeutic potential and warrants further investigation into this promising therapeutic approach.

Adoptive transfer of T cells modified with a humanized chimeric receptor gene inhibits growth of Lewis-Y-expressing tumors in mice.

In this study, human T cells were provided with a reactivity against the Lewis-Y (Le(Y)) carbohydrate antigen, which is overexpressed on 70% of epithelial-derived tumors, but not normally recognized by T cells. Antitumor reactivity was achieved by transduction of T cells with a gene encoding a cell-surface chimeric receptor composed of single-chain anti-Le(Y) antibody linked to an enhanced cytoplasmic signaling domain made up of CD28 and CD3-zeta. Importantly, the single-chain antibody was humanized to try to reduce potential problems of human anti-mouse antibody responses in patients receiving chimeric receptor-modified T cells in future clinical trials. T cells expressing the chimeric receptor were demonstrated to secrete cytokines and proliferate in response to receptor ligation and lysed Le(Y+) tumors in vitro. Another aspect of this study was the finding that no activity was observed against normal tissue, as represented by autologous neutrophils that express low levels of Le(Y). Significantly, systemic delivery of anti-Le(Y) T cells dramatically inhibited established s.c. human ovarian OVCAR-3 tumors (a recognized difficult model to treat) in mice. Finally, we demonstrated that anti-Le(Y) T cells preferentially expanded or accumulated in the tumor compared with control empty vector T cells, thereby providing mechanistic insight into the specific antitumor response. This study supports the use of humanized gene-modified T cells as a potential therapy for Le(Y+) malignancies.

Enhanced efficacy of radioimmunotherapy with 90Y-CHX-A''-DTPA-hu3S193 by inhibition of epidermal growth factor receptor (EGFR) signaling with EGFR tyrosine kinase inhibitor AG1478.

PURPOSE: Monoclonal antibodies and tyrosine kinase inhibitors specific for the epidermal growth factor receptor (EGFR) have been shown to enhance the effect of external beam radiation on EGFR-positive tumors. The effect of EGFR signaling abrogation by EGFR tyrosine kinase inhibitor on the efficacy of radioimmunotherapy has not been reported previously. This study investigated the effect of EGFR tyrosine kinase inhibition on the efficacy of radioimmunotherapy in a human cancer xenograft model. EXPERIMENTAL DESIGN: The humanized anti-Lewis Y antibody hu3S193 and the EGFR tyrosine kinase inhibitor AG1478 were studied. BALB/c nude mice were engrafted with A431 squamous carcinoma cells. Initial biodistribution properties of the 90Y-CHX-A''-DTPA-hu3S193 were evaluated in this model. In therapy experiments, cohorts of four to five xenografted mice were treated with saline as placebo, 0.4 mg AG1478 i.p. (six doses over 2 weeks), single i.v. injections of unlabeled hu3S193, or 90Y-CHX-A''-DTPA-hu3S193 (12.5, 25, 50, or 100 microCi). The combination of 0.4 mg AG1478 i.p. and 25 microCi 90Y-CHX-A''-DTPA-hu3S193 i.v. was subsequently evaluated in the A431 model. RESULTS: 90Y-CHX-A''-DTPA-hu3S193 retained excellent immunoreactivity after radiolabeling. The biodistribution study showed excellent uptake in tumor (90.33 +/- 38.84%ID/g) peaking at 24 to 72 hours after injection and with prolonged retention. 90Y-CHX-A''-DTPA-hu3S193 significantly inhibited A431 xenograft growth at 25, 50, and 100 microCi doses. The combination of 0.4 mg AG1478 with a single dose of 25 microCi 90Y-CHX-A''-DTPA-hu3S193 resulted in a significant enhancement of efficacy compared with either agent alone (P = 0.013). CONCLUSIONS: The efficacy of radioimmunotherapy with 90Y-CHX-A''-DTPA-hu3S193 is significantly enhanced by EGFR tyrosine kinase inhibitor AG1478. Further investigations of dosing regimens using EGFR tyrosine kinase inhibitors and radioimmunotherapy in the treatment of EGFR expressing tumors are warranted.

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 (T1/2beta) 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 x 10(-3) to 11.1 x 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.

Phase I trial of 131I-huA33 in patients with advanced colorectal carcinoma.

PURPOSE: Humanized monoclonal antibody A33 (huA33) targets the A33 antigen which is expressed on 95% of colorectal cancers. A previous study has shown excellent tumor-targeting of iodine-131 labeled huA33 (131I-huA33). Therefore, we did a phase I dose escalation trial of 131I-huA33 radioimmunotherapy. EXPERIMENTAL DESIGNS: Fifteen patients with pretreated metastatic colorectal carcinoma each received two i.v. doses of 131I-huA33. The first was an outpatient trace-labeled "scout" dose for biodistribution assessment, followed by a second "therapy" dose. Three patients were treated at 20, 30, and 40 mCi/m2 dose levels, and six patients at 50 mCi/m2 to define the maximum tolerated dose. RESULTS: Hematologic toxicity was 131I dose-dependent, with one episode of grade 4 neutropenia and two episodes of grade 3 thrombocytopenia observed at 50 mCi/m2. The maximum tolerated dose was determined to be 40 mCi/m2. There were no acute infusion-related adverse events, and gastrointestinal toxicity was not observed despite uptake of 131I-huA33 in bowel. Seven patients developed pruritus or rash, which was not related to 131I dose. There was excellent tumor-targeting of 131I-huA33 shown in all patients. The serum T1/2beta of 131I-huA33 was (mean +/- SD) 135.2 +/- 46.9 hours. The mean absorbed tumor dose was 6.49 +/- 2.47 Gy/GBq. Four patients developed human anti-human antibodies. At restaging, 4 patients had stable disease, whereas 11 patients had progressive disease. CONCLUSION: Radioimmunotherapy using 131I-huA33 shows promise in targeting colorectal tumors, and is deliverable at a maximum tolerated dose of 40 mCi/m2. Further studies of 131I-huA33 in combination with chemotherapy are planned.

Immunological effects of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma.

We conducted an open label dose-escalation phase I trial of chimeric anti-GD3 mAb KM871 in patients with metastatic melanoma. Patients were entered into one of five dose levels (1, 5, 10, 20, and 40 mg/m2) and received three infusions of KM871 at 2-wk intervals. A metastatic melanoma site was biopsied at day 7-10. Pharmacokinetics, immune function, and mechanism of action of KM871 were analysed. A total of 17 patients were entered into the trial; 15 were evaluable. KM871 had a serum half-life (T1/2-beta) based on ELISA of 10.39 +/- 1.12 d (mean +/- SD). Trough levels >1.0 microg/mL KM871 at 2 wk postinfusion were seen with the 10 mg/m2 and higher dose levels. There were no significant changes in white blood cell subsets or serum complement levels during KM871 treatment. KM871 was stable in vivo and maintained binding affinity and complement-dependent cytotoxicity (CDC) function up to 2 wk postinfusion. No significant trends in CDC or antibody-dependent cellular-cytotoxicity (ADCC) activity in patients were observed during treatment. Analysis of tumour biopsies demonstrated a significant increase in CD4+ T cell infiltrates compared to control patient tumours (P = 0.010), and in patients with either stable disease (2 patients) or a clinical partial response (1 patient) at restaging, a significant increase in CD3 and CD4 infiltrates in tumour over nonresponding patients was observed. The favourable immune properties of KM871, combined with this preliminary clinical data, indicate that KM871 has potential for the treatment of metastatic melanoma.

Generation of anti-idiotype antibodies for application in clinical immunotherapy laboratory analyses.

The chimeric monoclonal antibody ch806 specifically targets the tumor-associated mutant epidermal growth factor receptor (de 2-7EGFR or EGFRVIII) and is currently under investigation for its potential use in cancer therapy. The humanised monoclonal antibody hu3S193 specifically targets the Lewis Y epithelial antigen and is currently in Phase I clinical trials in patients with advanced breast, colon, and ovarian carcinomas. To assist the clinical evaluation of ch806 and hu3S193, laboratory assays are required to monitor their serum pharmacokinetics and quantitate any immune responses to the antibodies. Mice immunized with ch806 or hu3S193 were used to generate hybridomas producing antibodies with specific binding to ch806 or hu3S193 and competitive for antigen binding. These anti-idiotype antibodies (designated Ludwig Melbourne Hybridomas, LMH) were investigated as reagents suitable for use as positive controls for HAHA or HACA analyses and for measuring hu3S193 or ch806 in human serum. Anti-idiotypes with the ability to concurrently bind two target antibody molecules were identified, which enabled the development of highly reproducible, sensitive, specific ELISA assays for determining serum concentrations of hu3S193 and ch806 with a 3 ng/mL limit of quantitation using LMH-3 and LMH-12, respectively. BIAcore analyses determined high apparent binding affinity for both idiotypes: LMH-3 binding immobilized hu3S193, Ka = 4.76 x 10(8) M(-1); LMH-12 binding immobilised ch806, Ka = 1.74 x 10(9) M(-1). Establishment of HAHA or HACA analysis of sera samples using BIAcore was possible using LMH-3 and LMH-12 as positive controls for quantitation of immune responses to hu3S193 or ch806 in patient sera. These anti-idiotypes could also be used to study the penetrance and binding of ch806 or hu3S193 to tumor cells through immunohistochemical analysis of tumor biopsies. The generation of anti-idiotype antibodies capable of concurrently binding a target antibody on each variable domain provides reagents with high sensitivity for the assessment of safety and pharmacokinetic profiles of target antibodies administered clinically.

Expression and targeting of human fibroblast activation protein in a human skin/severe combined immunodeficient mouse breast cancer xenograft model.

Antigens and receptors that are highly expressed on tumor stromal cells, such as fibroblast activation protein (FAP), are attractive targets for antibody-based therapies because the supporting stroma and vessel network is essential for a solid neoplasm to grow beyond a size of 1-2 mm. The in vivo characterization of antibodies targeting human stromal or vessel antigens is hindered by the lack of an appropriate mouse model system because xenografts in standard mouse models express stromal and vessels elements of murine origin. This limitation may be overcome by the development of a human skin/mouse chimeric model, which is established by transplanting human foreskin on to the lateral flank of severe combined immunodeficient mice. The subsequent inoculation of breast carcinoma MCF-7 cells within the dermis of the transplanted human skin resulted in the production of xenografts expressing stromal and vessel elements of human origin. Widespread expression of human FAP-positive reactive stromal fibroblasts within xenografts was seen up to 2 months posttransplantation and postinjection of cells. Human blood vessel antigen expression also persisted at 2 months posttransplantation and postinjection of cells with murine vessels coexisting with the human vascular supply. The model was subsequently used to evaluate the biodistribution properties of an iodine-131-labeled humanized anti-FAP monoclonal antibody (BIBH-7). The results showed high specific targeting of the stromal compartment of the xenograft, indicating that the model provides a useful and novel approach for the in vivo assessment of the immunotherapeutic potential of molecules targeting human stroma and angiogenic systems.

Targeting primary human Ph(+) B-cell precursor leukemia-engrafted SCID mice using radiolabeled anti-CD19 monoclonal antibodies.

UNLABELLED: The Philadelphia chromosome translocation (Ph(+)) confers a poor prognosis in patients with acute lymphocytic leukemia (ALL). CD19 is highly expressed (CD19(+)) on ALL cells and is an attractive target for antibody-based therapies. CLB-CD19 is an IgG1kappa murine monoclonal antibody (mAb) directed against an epitope on the CD19 antigen. METHODS: Radiolabeled CLB-CD19 antibody was evaluated for targeting ALL in a severe combined immunodeficient (SCID) mouse model engrafted with primary human leukemia cells. Lodgment of CD19(+) ALL cells in spleen and liver was confirmed using immunohistochemistry analyses. Circulating CD19(+) ALL cells in blood were also detected by flow cytometry. RESULTS: Antibody was labeled directly with the radiohalogen (125)I and radiometal (111)In via the bifunctional metal ion chelate CHX-A"-diethylenetriaminepentaacetic acid (DTPA) with retention of immunoreactivities. After intravenous injection of radioconjugates, biodistribution studies showed rapid localization of the (111)In-conjugate to leukemia-infiltrated spleen, reaching a maximum (mean +/- SD) of 72.78 +/- 13.67 % injected dose per gram of tissue (%ID/g) by 24 h after injection. In contrast, peak localization of coinjected (125)I-CLB-CD19 occurred by 4 h and was significantly lower (11.41 +/- 12.79 %ID/g) (P < 0.001). Uptake of (111)In-conjugate in the liver containing tumor was also evident but not in other normal tissues. Uptake of radiolabeled CLB-CD19 in tumor-bearing organs was specific, as uptake of radiolabeled isotype-matched antibody control was low. Gamma-camera imaging detected the uptake of (111)In-CHX-A"-DTPA CLB-CD19 in enlarged tumor-bearing spleen of engrafted mice. A single injection of 32 micro g CLB-CD19 mAb had a delayed suppressive effect on the level of circulatory leukemia cells in surviving mice and extended the median survival from 48.5 to 58 d (n = 8; P = 0.03). CONCLUSION: The radiolabeled anti-CD19 antibody showed specific targeting and rapid internalization in ALL cell-engrafted SCID mice and may also be used for selective intracellular delivery of cytotoxic radionuclides with beta-, Auger, or alpha-emissions.