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Purpose: This study examined a pretarget radioimmunotherapy strategy for treatment of an i.p. tumor model (LS174T). Experimental Design: The strategy used regional administration (i.p.) of a novel targeting molecule composed of four CC49 anti–tumor-associated glycoprotein 72 (TAG-72) single-chain antibodies linked to streptavidin as a fusion protein (CC49 fusion protein); 24 hours later, a synthetic clearing agent was administered i.v. to produce hepatic clearance of unbound CC49 fusion protein/synthetic clearing agent complexes. Four hours later, a low molecular weight radiolabeled reagent composed of biotin conjugated to the chelating agent 7,10-tetra-azacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) complexed with 111In-, 90Y-, or 177Lu-DOTA-biotin was injected. Results: Radiolocalization to tumor sites was superior with i.p. administration of radiolabeled DOTA-biotin as compared with i.v. administration. Imaging and biodistribution studies showed excellent tumor localization of radioactivity with 111In- or 177Lu-DOTA-biotin. Tumor localization of 111In-DOTA-biotin was 43% ID/g and 44% ID/g at 4 and 24 hours with the highest normal tissue localization in the kidney with 6% ID/g at 48 and 72 hours. Therapy studies with 90Y-DOTA-biotin at doses of 400 to 600 μCi or 177Lu-DOTA-biotin at doses of 600 to 800 μCi produced significant prolongation of survival compared with controls (P = 0.03 and P < 0.01). Conclusions: Pretarget radioimmunotherapy using regional administration of CC49 fusion protein and i.p. 90Y- or 177Lu-DOTA-biotin represents a successful therapeutic strategy in the LS174T i.p. tumor model and this strategy may be applicable to human trials in patients with i.p. ovarian cancer.

This trial was designed to evaluate strategies to improve the efficacy of a radiolabeled monoclonal antibody (mCC49) against tumor-associated glycoprotein-72 (TAG-72) in patients with non-small cell lung cancer (NSCLC). The aims of this study were to determine: safety and maximum tolerated dose (MTD) of (90)Y-mCC49 in combination with interferon alpha2beta (IFN); whether calcium disodium versonate (EDTA) or diethylenetriamine penta-acetic acid (DTPA) could reduce myelosuppression; and safety and MTD of paclitaxel (Taxol) in combination with (90)Y-mCC49.Patients with advanced (TAG-72 positive) non-small cell lung cancer were entered in three phases; the first was the dose escalation of a single agent (90)Y-mCC49. In the second phase, the dose escalation of (90)Y-mCC49 was attempted with concurrent EDTA or DTPA chelator therapy. In the third phase, radiosensitization with a continuous infusion of paclitaxel (96-hour) was administered with (90)Y-mCC49. All patients received IFN for TAG-72 up-regulation.Thirty-four patients were evaluable. Reversible Grade 4 neutropenia and thrombocytopenia were the dose-limiting toxicities (DLTs). The MTD of (90)Y-mCC49/IFN was 14 mCi/m(2). EDTA did not alter toxicity, while there was a modest reduction of myelosuppression with DTPA. The MTD of continuous infusion paclitaxel in combination with 14 mCi/m(2) of (90)Y-CC49 was 60 mg/m(2). There were no objective tumor responses.(90)Y-mCC49/IFN was well tolerated at a dose of 14 mCi/m(2). The clinical effect of adjunctive chelating therapy with DTPA was modest. The MTD of coadministered continuous infusion (96-hour) paclitaxel was 60 mg/m(2). Because of the immunogenicity of the murine compound, future studies are planned using a humanized version of CC49.

Pretargeted radioimmunotherapy (RIT) increases the dose of radionuclide delivered to tumor sites while limiting radiation to normal tissues. The three components in Pretarget include a streptavidin-containing targeting molecule, a synthetic clearing agent (sCA), and (90)Y and/or (111)In-DOTA-biotin. This trial determined the feasibility and safety of using a genetically engineered fusion protein directed to TAG-72 as the targeting agent. Nine (9) patients with metastatic colorectal cancer (TAG-72+) received 160 mg/m(2) of CC49Fusion protein intravenously (i.v.), followed by the sCA, 45 mg/m(2) i.v. Twenty-four (24) hours later, patients received radiolabeled DOTA-biotin (either 0.65 or 1.3 mg/m(2)). All patients received 5 mCi of (111)In-DOTA-biotin for imaging and dosimetry purposes and patients 4-9 received 10 mCi/m2 of (90)Y-DOTA-biotin as well. The mean plasma T1/2 of CC49Fusion protein was 23 +/- 6 hours. Greater than 95% of the circulating CC49Fusion protein was eliminated from the circulation within 6 hours of sCA administration. The radiolabeled DOTA-biotin rapidly localized to tumor sites while the unbound fraction was rapidly excreted. The mean tumor-to-marrow radiation dose ratio was 139:1 and mean tumor: whole body was 56:1. No infusion-related, renal, hepatic, or hematologic toxicities were noted. CC49Fusion protein performs well in a pretargeted RIT schema, and further study with escalating doses of (90)Y should be pursued. This strategy has the potential to deliver effective radiation tumor doses to TAG- 72+ tumors.

Radiation dose to the testes from radionuclide therapies is of concern. This study evaluated image-quantification methods for testicular uptake in a phantom and in patients.A 50-mL vial and a large water tank were used to simulate testes and the body, respectively. Activity concentration in the vial and water tank was prepared to generate testes-to-background concentrations of 1.3 and 1.1. Five male lymphoma patients who received a Zevalin (Biogen Idec, Cambridge, MA) regimen and 6 male colorectal cancer patients who received a Pretarget (Neo Rx, Seattle, WA) CC49Fusion protein were evaluated. Testicular activity was quantified using two methods: (1) geometric-mean, background-corrected testicular region of interest (ROI) counts as a fraction of body counts without explicit attenuation correction (Zevalin Kit); (2) background-corrected anterior testicular ROI counts with attenuation correction using known depth in the phantom and CT depth in patients.In the phantom study, Method 1 underestimated 49% and 39%, at image contrast of 1.3 and 1.1, respectively. Quantification was improved using Method 2 (7% for a 1.3 contrast, -17% for a 1.1 contrast). Method 2 was used in patients because background-corrected posterior ROI counts were statistically unreliable due to poor image contrast. In patients receiving Zevalin, the median peak percent injected dose (%ID)/testis was 0.10 (range, 0.08-0.18) with a median biologic half-time (T(bio1/2)) of 156 (range, 91-4200) hours. The median dose was 2.4 (range, 1.5-3.6) Gy/GBq, compared to the originally reported mean dose of 9.1 (range, 5.4-11.4) Gy/GBq (Zevalin package insert). In patients receiving the Pretarget CC49Fusion protein, the median peak %ID/testis was 0.22 (range, 0.05-0.29) with a median T(bio1/2) of 44 (range, 37-64) hours. The median dose was 0.84 (range, 0.3-1.2) Gy/GBq.This study found that testicular doses from Zevalin were much lower than that originally reported in the package insert. The median testicular dose from Pretarget CC49Fusion protein was less than half that of the median testicular dose from Zevalin.

Chlorotoxin, or TM-601, is a peptide derived from the venom of the scorpionLeiurus Quinquestriatus that specifically binds to malignant brain tumors, but not to normal tissues. Targeted radiotherapy using 131I-Chlorotoxin is promising for post-surgery treatment of brain tumors. This study reports dosimetry results of 131I-Chlorotoxin in athymic nude mice with intracranially implanted human glioma xenografts and projected radiation doses in patients receiving 370 MBq of 131I-Chlorotoxin. 125I/131I-Chlorotoxin were injected into the right brain where D54 MG xenografts were implanted. Mice were sacrificed 24-96 h later. The blood, normal organs, and tumors were weighed and counted to determine 131I-Chlorotoxin concentration. The radiation dose from 131I was calculated based on non-penetrating radiation in the mouse model. Assuming similar tissue uptake in mice and patients, radiation doses for patients were extrapolated. Distributions of 125I/131I-Chlorotoxin were only significant in tumor, stomach, kidneys, and brain (injection site), reflecting non-specific uptake of Chlorotoxin in normal tissues. Mean radiation dose (cGy/37 kBq) was 58.2 for tumor, 17.9 for brains, 1.8 for marrow, 27.1 for stomach, 16.0 for kidneys in mice. For intracranial injection of 370 MBq 131I-Chlorotoxin in patients, extrapolated patient dose (cGy) was 70 for brains, 6 for marrow, 35 for stomach, 60 to kidneys, 227 to tumor, suggesting that 3.7 GBq of 131I-Chlorotoxin can be safely administrated to patients. These promising results demonstrated potential in improving patient survival using this novel targeting agent.

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.

The generation of chimeric and complementary-determining region (CDR) grafted monoclonal antibodies (MAb) have reduced the immunogenicity problem in the clinical application of radioimmunotherapy with monoclonal antibodies. However, humanization (Hu) has prolonged the circulation (plasma T1/2) of radiolabeled antibodies, resulting in an increased normal tissue exposure to radioactivity and greater dose-limiting bone marrow suppression. To overcome this problem, a tumor-associated glycoprotein (TAG)-72-specific CDR grafted MAb with C(H)2 domain deletion (DeltaC(H)2) was developed from the MAb CC49. Preclinical studies have demonstrated that HuCC49 DeltaC(H)2 clears more rapidly from the plasma of mice than HuCC49. This preliminary report describes the initial human experience with HuCC49 DeltaC(H)2 radiolabeled with 131I and administered to patients with metastatic colorectal carcinoma. In this pilot study we enrolled four patients who received a single infusion of 20 mg of HuCC49 DeltaC(H)2 (total protein dose) labeled with 10 mCi of 131I. Pharmacokinetics, biodistribution, dosimetry, and immune response were evaluated over 2-6 weeks. No toxicity was observed in this group of patients. A one-compartment bolus model using the non-linear (NLIN) procedures in Statistical Analysis Software (SAS; SAS, Incorporated, Cary, NC) best describes the pharmacokinetics of the 131I-HuCC49 DeltaC(H)2 with a plasma mean T1/2 of 20 +/- 3 hours, a mean residence time (MRT) of 29 +/- 4 hours and a clearance rate (Cl) of 1.5 +/- 0.1 mL/hours/kg. The whole body and marrow radiation dose estimates were 0.55 +/- 0.06 rad/mCi and 1.00 +/- 0.14 rad/mCi, respectively. All patients had positive localization of antibody to metastatic tumor sites. The 131I-HuCC49 DeltaC(H)2 biodistribution was similar to murine CC49. Three patients had no evidence of antibody response to HuCC49 DeltaC(H)2 over 6 weeks of observation, and one patient had a marginal response by week 6. Intravenous administration of HuCC49 DeltaC(H)2 is safe and well tolerated. The deleted C(H)2 construct has a shorter half-life compared with prior studies of murine CC49 but with similar biodistribution and low immunogenicity. These studies support the further clinical investigation of this agent in phase I trials by intravenous and intraperitoneal routes.

CC49 is a second-generation murine antibody with anti-TAG-72 (tumor-associated antigen) reactivity. For cancer therapy, it has the advantage of being expressed on adenocarcinomas but not on most normal tissues. CC49 has been utilized in phase I and II clinical trials at multiple institutions. Therapeutic applications to date have included (131)I-, (90)Y-, and (177)Lu-CC49, with tracer amounts of (111)In-CC49 as a dosimetry surrogate for (90)Y-CC49 therapy. Dosimetry methods and details of their description vary between studies. Biodistribution to normal organs and the effective plasma T(1/2) for various radionuclides were relatively consistent among patients with different diseases and treatment at several institutions. As expected with marrow suppression being the dose-limiting toxicity, higher doses of (177)Lu-CC49 were tolerated via intraperitoneal than IV administration. The biologic response modifier interferon enhanced TAG-72 expression and resulted in a trend of increased uptake of (131)I-CC49 by tumors. Tumor dose estimates were more variable than that of normal organs. Standardization and improved dosimetry may be helpful for comparison among patients in various studies and for establishing dose/toxicity relationships that are useful for predicting safe levels of radioimmunoconjugates.

Simultaneous analysis of common antineoplastic agents potentially hazardous to healthcare workers is of much interest for the evaluation of the overall health risk to these workers. Such analysis could be applied to both air and surface monitoring samples to provide a broader indication of risk to combinations of these agents. It was determined that the ability to simultaneously evaluate five frequently used, potentially hazardous agents was sufficient for general evaluation of exposures to healthcare workers. The approach used to select the five agents was to obtain a list of the agents used most frequently in both a cancer hospital and an outpatient cancer treatment center, then review the list to determine which agents were potentially more hazardous to human health. From these reviews, it was decided to attempt to develop an analytical method able to detect and quantify the presence of 5-fluorouracil, ifosfamide, cyclophosphamide, doxorubicin HCl, and paclitaxel. A reverse-phase high performance liquid chromatograph (HPLC) with a Waters Symmetry C8 column and a UV wavelength of 195 nm was selected for method development. The mobile phase was 22.75 percent acetonitrile in water buffered to a pH of 6.0. The HPLC analytical method developed is able to detect all five agents of interest, and at minimum detectable concentrations of 0.5-microgram/mL for each of the five agents.

Cyclophosphamide is a known human carcinogen. In July 1999, in a report at a conference on cytotoxic drugs in Sweden, it was indicated that cyclophosphamide (CP) was not effectively controlled by high efficiency particulate air (HEPA) filters.((1)) This then raised a concern that the existing air monitoring methods, which utilize polytetrafluoroethylene (a.k.a. PTFE, or Teflon) or glass fiber filters for evaluation of antineoplastics such as CP in air may also be ineffective for collection and quantification of such agents. It was decided that further evaluation of the existing filter method for monitoring antineoplastics in air be conducted. This evaluation determined that the filter method of monitoring was minimally effective for some antineoplastic agents, and that an alternate method of monitoring should be sought. The method subsequently developed utilizes a solid sorbent tube, Anasorb 708, a methacrylic acid polymer. Evaluation of this sorbent tube for adsorption and desorption properties found it had a greater than 90 percent recovery for both CP and ifosfamide. Other agents evaluated included 5-fluorouracil, doxorubicin, and paclitaxel. All three agents were able to be detected and measured by use of Anasorb 708 solid sorbent tube. Validation of the method was then conducted with air pulled through the tubes via attachment to an air manifold system at air flows ranging from 1.5 to approximately 4.0 liters per minute for up to 24 hours. This evaluation did validate the Anasorb 708 tube as an effective media for collection of airborne concentrations of CP from less than 1 microgram up to approximately 2 mg (2000 microgram) per tube. This corresponds to a concentration range of approximately 0.7 microgram/m(3) (0.0007 mg/m(3)) to 0.7 mg/m(3) in a 5.76 m(3) volume of air. This method can provide accurate information on airborne concentrations of CP for purposes of conducting risk assessments or evaluation of risk management methods.

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2‘-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drug-to-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal ef...

A method of evaluating surface contamination caused by selected antineoplastic agents was studied. The antineoplastic agents tested were cyclophosphamide, ifosfamide, doxorubicin hydrochloride, fluorouracil, and paclitaxel. Each agent was reconstituted and prepared as a stock solution. A 0.1-mL portion of each solution was spread evenly over a 600-cm2 area of a stainless steel surface, a resin countertop surface, and a vinyl flooring surface. After drying, the surfaces were wiped with each of two types of commercially available wiping materials (What-man no. 42 filters and Kimberly-Clark Kimwipes). A blend of methanol, acetonitrile, and buffered water was used both as the wetting agent for wiping the surfaces and as a desorbing solution. The desorbate was analyzed for drug concentration by reverse-phase high-performance liquid chromatography (HPLC). Mean +/- S.D. percent total recovery ranged from 72.4% +/- 17.6% to 95.3% +/- 2.9% for the vinyl surface wiped with filters, 91.5% +/- 5.4% to 104.7% +/- 0.8% for the resin surface wiped with filters, 73.9% +/- 2.3% to 95.3% +/- 1.7% for the stainless steel surface wiped with filters, and 18.2% +/- 1.4% to 372.8% +/- 8.0% for the stainless steel surface wiped with Kimwipes. Results were best for ifosfamide and cyclophosphamide. Kimwipes were deemed ineffective for this monitoring method because an ingredient interfered with the quantitative analytical tests. A wipe-sampling, desorption, and HPLC method for monitoring surface contamination by selected antineoplastic agents was sufficiently accurate and sensitive to evaluate surfaces typically found in both the pharmacy and drug administration areas of oncology treatment facilities.

PURPOSE: To evaluate the safety, pharmacokinetics, and efficacy of a chimeric anti–epidermal growth factor receptor monoclonal antibody, cetuximab, in combination with radiation therapy (RT) in patients with advanced squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: We treated 16 patients in five successive treatment schedules. A standard dose escalation procedure was used; three patients entered onto the study at each dose level of cetuximab received conventional RT (70 Gy, 2 Gy/d), and the final three patients received hyperfractionated RT (76.8 Gy, 1.2 Gy bid). Cetuximab was delivered as a loading dose of 100 to 500 mg/m2, followed by weekly infusions of 100 to 250 mg/m2 for 7 to 8 weeks. Circulating levels of cetuximab during therapy were determined using a biomolecular interaction analysis core instrument. Human antichimeric antibody response was evaluated with a double-antigen radiometric assay. The recommended phase II/III dose was defined as the optimal cetuximab dose level based on the pharmacologic parameters and adverse events. RESULTS: The most commonly reported adverse events were fever, asthenia, transaminase elevation, nausea, and skin toxicities (grade 1 to 2 in most patients). Skin toxicity outside of the RT field was not strictly dose-dependent; however, grade 2 or higher events were observed in patients treated with higher dose regimens. There was one grade 4 allergic reaction. Most acute adverse effects were associated with RT (xerostomia, mucositis, and local skin toxicity). No antibodies against cetuximab were detected. All patients achieved an objective response (13 complete and two partial remissions). CONCLUSION: Cetuximab can be safely administered with RT. The recommended dose for phase II/III studies is a loading dose of 400 to 500 mg/m2 and a maintenance weekly dose of 250 mg/m2.

The pharmacologic efficacy of the promising antitumor agent paclitaxel (Taxol) may be potentially enhanced through derivatization of the drug to a water-soluble tumor-recognizing conjugate. This work reports the design and synthesis of the first tumor-directed derivative of paclitaxel. A 7-amino acid synthetic peptide, BBN[7-13], which binds to the cell surface bombesin/gastrin-releasing peptide (BBN/GRP) receptor, was conjugated to the paclitaxel-2'-hydroxy function by a heterobifunctional poly(ethylene glycol) linker. The resulting conjugate, designated PTXPEGBBN[7-13], was soluble to the upper limit of tested concentrations (250 mg/mL). The conjugate completely retained the receptor binding properties of the attached peptide as compared with those of the unconjugated BBN[7-13]. In experiments with NCI-H1299 human nonsmall cell lung cancer cells, the cytotoxicity of the PTXPEGBBN[7-13] conjugate at a 15 nM dose was enhanced by a factor of 17.3 for 24 h and 10 for 96 h exposure times, relative to paclitaxel. The IC(50) of the conjugate, tested against the same cell line, was lower than the free drug by a factor of 2.5 for both 24 h and 96 h exposures. These results describe, for the first time, the design and synthesis of a soluble tumor-directed paclitaxel prodrug which may establish a new mode for the utilization of this drug in cancer therapy.

A procedure utilizing an activated ester approach for conjugation of unprotected hydroxamic acids to antibodies and peptides was recently reported. Here, an alternative method with advantages over the activated ester strategy is described. This protocol utilizes the hydrazone formation between a hydrazide derivative of the trihydroxamate ligand trisuccin and either a ketone derivative of antibody or the aldehyde groups, generated by oxidation of the carbohydrate residues. Thus, the trisuccin carboxylic acid (1) was derivatized with tert-butyl carbazate to the hydrazide 2, and the protecting groups were removed by catalytic hydrogenation and acidolysis with TFA to afford the hydroxamate hydrazide 4. Conjugation of 4 to monoclonal antibody CC49 was effected by two approaches: attachment through the amine (e.g., lysine) residues of the antibody or oxidation of the carbohydrate residues. The extent of conjugations were monitored by MALDI, through evaluation of the increases in molecular weights of the conjugates compared to the unconjugated antibody. The first approach utilizing a ketone linker (6-oxoheptanoic acid, OHA) which served as a hydrazide anchor, is being introduced in this report as a new technique for conjugation of hydrazide derivatives to proteins. The OHA approach proved to be a superior strategy over the aldehyde approach in the ease of the procedure and yield of protein recovery. It also had the advantage of yielding more control in adjusting the ligand-to-protein ratio and was therefore selected for protocol optimization. All conjugates resulting from both approaches were radiolabeled with 125I and screened for their immunoreactivity. Furthermore, the conjugates prepared through the optimized OHA protocol were radiolabeled with both 99mTc and 125I for which the radiolabeling yields and immunoreactivities are reported.

Twenty-seven ovarian cancer patients who failed chemotherapy entered a phase I/II trial of intraperitoneal 177Lu-CC49 antibody.Patients had disease confined to the abdominal cavity +/- retroperitoneal lymph nodes, adequate organ function, and no previous radiation.The most common side effects were delayed, transient arthralgia (10/27) and marrow suppression with 1.665 GBq/m2 (45 mCi/m2), which was considered the maximum tolerated dose. One of thirteen patients with gross disease had50% tumor reduction after therapy, whereas most others with gross disease progressed (one went off study with stable disease at 11 weeks). Seven of nine patients with1-cm nodules progressed inor =21 months, and two of nine remain without evidence of disease at 4 to 5 months. Of patients with microscopic or occult disease, one relapsed at 10 months and four of five remain without evidence of disease at6 to 35 months.Marrow suppression was the dose-limiting toxic effect of intraperitoneal immunotherapy with 177Lu-CC49. Antitumor effects were noted against chemotherapy-resistant ovarian cancer, even at lower dose levels, and resulted in prolonged disease-free survival of most patients with microscopic disease. This form of treatment deserves further study.

We performed a phase Ia/Ib trial of chimeric anti-GD2 monoclonal antibody 14.18 (ch14.18) in combination with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to determine the maximum tolerated dose as well as immunologic and biologic responses to the regimen. Sixteen patients with metastatic malignant melanoma received escalating doses of ch14.18 (15-60 mg/m2) administered intravenously for 4 h on day 1. Twenty-four hours later, subcutaneous injections of rhGM-CSF were administered daily for a total of 14 days. Significant side effects were related to ch14.18 infusion and consisted of moderate to severe abdominal and/or extremity pain, blood pressure changes, headache, nausea, diarrhea, peripheral nerve dysesthesias, myalgias, and weakness. Dose-limiting toxicity was observed at 60 mg/m2 and consisted of severe hypertension, hypotension, and atrial fibrillation in one patient each, respectively. Significant increases in white blood cell count, granulocyte count, eosinophil count, and monocyte count occurred after rhGM-CSF treatment. Significant enhancement of in vitro and in vivo monocyte and neutrophil tumoricidal activity and antibody-dependent cellular cytotoxicity along with significant elevations in C-reactive protein and neopterin were observed. Despite these immunological and biological changes, no antitumor activity was seen. In short, the combination of ch14.18 and rhGM-CSF resulted in toxicity similar to that observed with ch14.18 alone without improvement in tumor response.

In a phase I trial, eight patients with non-Hodgkin's B-cell lymphoma received mouse IgG1k monoclonal antibody HD37 specific for CD19 conjugated to deglycosylated ricin A chain (dgA) administered in four doses at 4-h intervals with total doses ranging from 4-12 mg/m2. This schedule generated serum levels of immunotoxin which were sustained over 36 h. The plasma half-life of HD37-dgA was 17 +/- 4 (SD) h. The HD37-dgA conjugate was stable in vivo as demonstrated by serum levels of HD37-dgA conjugate comparable to those of total HD37 antibody. Peak serum levels attained after the fourth dose ranged from 0.36 to 5.63 micrograms/ml. Two of seven evaluable patients developed modest human anti-immunotoxin antibody responses. Toxicity in patients 1-7 consisted of dose-dependent capillary leak syndrome with hypoalbuminemia, orthostatic hypotension, and weight gain. Patient 8 died on day 8 with severe capillary leak, bronchopneumonia, and rhabdomyolysis. All patients had progressive disease at 4 weeks except patient 8, who exhibited a near-complete remission before his death. This intensive schedule appears to produce inordinate toxicity with a maximal tolerated total dose of 8 mg/m2.

Background. Although treatment with radiolabeled monoclonal antibodies (MoAb) against tumor-associated antigens offers the potential for targeted therapy, the efficacy of this approach is limited by the low dose-rate delivered. This could be overcome by increasing tumor sensitivity through the use of radiation sensitizers. Methods and Results. In vitro studies using LS174T human colon cancer cells showed that exposure to 1 μM bromodeoxyuridine (BrdUrd), a thymidine-analog radiation sensitizer, at a plasma concentration easily achievable through systemic administration in both animals and patients, increased the cytotoxicity of continuous low dose-rate irradiation delivered by a cesium-137 irradiator (at 12 cGy/h which resembles the dose-rate delivered by radiolabeled MoAb therapy)

Monoclonal antibodies either of mouse, mouse/human chimeric, or human origin have been used safely in human trials for a decade. Considerable effort has been committed in investigations of manipulating endogenous immunological activity against tumors and targeting various cytotoxic agents to cancers. These studies have identified several problems that need to be resolved before any such reagents can be used routinely in patients. One of these problems has been the immunogenicity of these monoclonal antibodies. This review article discusses what is known regarding human immune response to monoclonal antibodies and their clinical consequences.

Clinical trials with genetically engineered chimeric mouse/human monoclonal antibodies have demonstrated that mouse variable regions differ dramatically in their degree of immunogenicity. These observations led us to search for an animal model that could predict mouse variable region (V region) immunogenicity prior to human clinical trials. We selected monoclonal antibodies 17-1A and B72.3 for study because human trials have demonstrated the very low immunogenicity of the mouse 17-1A V region and the high immunogenicity of the mouse B72.3 V region. Random-bred New Zealand white rabbits were injected intravenously with mouse 17-1A, B72.3, or both using a dose and schedule comparable to human trials. After initial injection only two of ten rabbits developed an antibody response to mouse 17-1A, while all five animals receiving a second injection developed antibody that was entirely mouse constant region-specific. On the other hand, nine of ten rabbits demonstrated an antibody response to initial infusion of mouse B72.3 that was greater than 90% specific for the complementarity-determining region components of the V regions. Competitive inhibition with isolated heavy and light chains demonstrated specificity for heavy or light chains (approximately 60%) or a requirement for both (40%). Thus, as in humans, the V region of 17-1A elicited little or no immune response in rabbits, while the V region of B72.3 was highly immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

In a phase 1 trial, patients with metastatic melanoma received the anti-GD2 murine mAb 14G2a. All patients developed human anti-14G2a antibodies including anti-Id antibodies. Peripheral blood MNCs from one such patient were fused with the murine myeloma cell line Ag8. Four human anti-14G2a secreting hybridomas were generated and the mAb product of one of the hybridomas was characterized. The human mAb 4B5 (hu-IgG, lambda) binds to the variable region of murine 14G2a (anti-Id). The 4B5 binds to the antigen-combining site of 14G2a and inhibits its binding to GD2 expressing Mel-21 cells. Rabbits were immunized with the human anti-Id 4B5. Sera from the immunized rabbits demonstrated anti-4B5 antibodies and anti-Mel-21 and anti-GD2 reactivity. Furthermore, rabbit sera competitively inhibited binding of 14G2a to Mel-21 cells. Rabbits immunized with 4B5 developed a DTH response when challenged with 4B5 antibody and Mel-21 cells. These studies demonstrate that the human anti-Id 4B5 mimics the GD2 antigen and is capable of eliciting both a humoral and cellular anti-GD2 immune response. This antibody could be potentially used as a human anti-Id vaccine in patients with malignant melanoma.

In an effort to generate bifunctional chelating agents (BCAs) with improved labeling, conjugation, and biodistribution properties, the synthesis of trisuccin is reported. This new hydroxamate BCA, after synthesis and characterization, was used for conjugation and radiolabeling of monoclonal antibodies with 99mTc. This new class of synthetic BCAs may be useful in the radioimmunodiagnosis and radioimmunotherapy of cancer.

Tumor imaging, using monoclonal antibodies (MAb) carring radioisotopes, is a promising approach toward improving early diagnosis of cancer. The PR81 is a new murine anti-MUC1 MAb that was generated by imunizing BALB/c mice with homogenized human breast cancerous tissues. This study was performed to develop an indirect labeling of this antibody via the HYNIC with 99mTc in order to use it in radioimmunoscintigraphy (RIS) of breast tumor in mouse model. A 20 molar excess of dissolved HYNIC was added to a solution of the MAb. The solution was stirred for 5 hr at room temperature followed by dialysis against citrate buffer overnight. The resulting conjugate was labeled with 99mTc using tricine as a co-ligand. The labeling efficiency determined by ITLC was 89.2%±4.7. Radiocolloides measured by cellulose nitrate electrophoresis were 3.4%±0.9. In vitro stability of labeled product determined in human serum by gel filtration chromatography (FPLC) was 78.6%±5.7 over 24 hr. The integrity of labeled MAb was checked by means of SDS-PAGE and no significant fragmentation was seen. The results of cell-binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF-7 cells. Biodistribution studies and tumor imaging performed in female BALB/c mice with breast tumor xenografts at 4, 16 and 24 hr after the 99mTc-HYNIC-PR81 injection were demonstrated definite localization of the compound at the site of tumors with high sensitivity. These findings show that the new radiopharmaceutical can be considered as a promising candidate for imaging of human breast cancer in nuclear medicine.

Purpose TM-601 binds to malignant brain tumor cells with high affinity and does not seem to bind to normal brain tissue. Preclinical studies suggest that iodine-131 (131I) –TM-601 may be an effective targeted therapy for the treatment of glioma. We evaluated the safety, biodistribution, and dosimetry of intracavitary-administered 131I-TM-601 in patients with recurrent glioma. Patients and Methods Eighteen adult patients (17 with glioblastoma multiforme and one with anaplastic astrocytoma) with histologically documented recurrent glioma and a Karnofsky performance status of ≥ 60% who were eligible for cytoreductive craniotomy were enrolled. An intracavitary catheter with subcutaneous reservoir was placed in the tumor cavity during surgery. Two weeks after surgery, patients received a single dose of 131I-TM-601 from one of three dosing panels (0.25, 0.50, or 1.0 mg of TM-601), each labeled with 10 mCi of 131I. Results Intracavitary administration was well tolerated, with no dose-limiting toxicities observed. 131I-TM-601 bound to the tumor periphery and demonstrated long-term retention at the tumor with minimal uptake in any other organ system. Nonbound peptide was eliminated from the body within 24 to 48 hours. Only minor adverse events were reported during the 22 days after administration. At day 180, four patients had radiographic stable disease, and one had a partial response. Two of these patients further improved and were without evidence of disease for more than 30 months. Conclusion A single dose of 10 mCi 131I-TM-601 was well tolerated for 0.25 to 1.0 mg TM-601 and may have an antitumoral effect. Dosimetry and biodistribution from this first trial suggest that phase II studies of 131I-TM-601 are indicated.

The application of intraperitoneal (i.p.) radioimmunotherapy to treat i.p. tumor loci has been limited by bone marrow toxicity secondary to circulating radiolabeled antibodies. The generation of novel genetically engineered monoclonal antibodies, which can achieve high tumor uptake and rapid blood clearance, should enhance the therapeutic index of i.p. radioimmunotherapy. In this regard, a novel humanized anti-TAG-72 monoclonal antibody with a deleted CH2 region (HuCC49DeltaCH2) has been described, which localized well to subcutaneous xenograft tumors and had a rapid plasma clearance. The aim of this study was to examine the characteristics of this radiolabeled reagent when administered through the i.p. route in mice bearing i.p. tumor (LS174T). The DeltaCH2 molecule and intact humanized CC49 (HuCC49) monoclonal antibody were conjugated to PA-DOTA and radiolabeled with (177)Lu. Both molecules retained high-affinity binding to TAG-72 positive LS174T tumor cells in vitro. The radiolabeled DeltaCH2 molecule had a modest decrease in tumor localization, as compared to the intact molecule when administered i.p. to tumor-bearing mice and a dramatically shorter plasma disappearance T(1/2) at 2.7 hours compared to 61.2 hours for the intact antibody. The radiolabeled DeltaCH2 molecule thus had very high tumor:blood ratios. Using an (131)I-labeled system, the maximum tolerated dose of DeltaCH2 was3x that of intact HuCC49. Autoradiography of tumors showed low radiation dose rates at tumor centers early (1 and 4 hours), as compared to higher dose rates at tumor periphery but a more uniform distribution by 24 hours. Dose-rate distributions were similar for both reagents. Animals bearing LS174T i.p. tumors were treated with 300 microCi of (177)Lu-labeled DeltaCH2 or intact HuCC49 by i.p. route daily x 3. The (177)Lu-DeltaCH(2) molecule mediated an increase in median survival compared to controls (67.5 +/- 7.5 days versus controls of 32 +/- 3.3) while the same dose of (177)Lu-HuCC49 produced early toxic deaths. These studies suggest that i.p. radioimmunotherapy using radiolabeled HuCC49DeltaCH2 should allow higher radiation doses to be administered with less marrow toxicity and potentially improved efficacy.

Pretargeted radioimmunotherapy (RIT) using CC49 fusion protein, comprised of CC49-(scFv)4 and streptavidin, in conjunction with 90Y/111In-DOTA-biotin (DOTA = dodecanetetraacetic acid) provides a new opportunity to improve efficacy by increasing the tumor-to-normal tissue dose ratio. To our knowledge, the patient-specific dosimetry of pretargeted 90Y/111In-DOTA-biotin after CC49 fusion protein in patients has not been reported previously.Nine patients received 3-step pretargeted RIT: (a) 160 mg/m2 of CC49 fusion protein, (b) synthetic clearing agent (sCA) at 48 or 72 h later, and (c) 90Y/111In-DOTA-biotin 24 h after the sCA administration. Sequential whole-body 111In images were acquired immediately and at 2-144 h after injection of 90Y/111In-DOTA-biotin. Geometric-mean quantification with background and attenuation correction was used for liver and lung dosimetry. Effective point source quantification was used for spleen, kidneys, and tumors. Organ and tumor 90Y doses were calculated based on 111In imaging data and the MIRD formalism using patient-specific organ masses determined from CT images. Patient-specific marrow doses were determined based on radioactivity concentration in the blood.The 90Y/111In-DOTA-biotin had a rapid plasma clearance, which was biphasic with10% residual at 8 h. Organ masses ranged from 1,263 to 3,855 g for liver, 95 to 1,009 g for spleen, and 309 to 578 g for kidneys. The patient-specific mean 90Y dose (cGy/37 MBq, or rad/mCi) was 0.53 (0.32-0.78) to whole body, 3.75 (0.63-6.89) to liver, 2.32 (0.58-4.46) to spleen, 7.02 (3.36-11.2) to kidneys, 0.30 (0.09-0.44) to lungs, 0.22 (0.12-0.34) to marrow, and 28.9 (4.18-121.6) to tumors.Radiation dose to normal organs from circulating radionuclide is substantially reduced using pretargeted RIT. Tumor-to-normal organ dose ratios were increased about 8- to 11-fold compared with reported patient-specific mean dose to liver, spleen, marrow, and tumors from 90Y-CC49.

CC49 is an antitumor monoclonal antibody that is promising for use in radioimmunoguided surgery (RIGS). However, the murine antibody has been limited by human antimouse antibody (HAMA) response and slow clearance. This study examined the pharmacokinetics and tissue localization of a humanized domain-deleted CC49 antibody (HuCC49DeltaC(H)2 MAb) in humans.Twenty-one patients with colorectal carcinoma were given 1 mg intravenous (I.V.) bolus of HuCC49DeltaC(H)2 MAb radiolabeled with 2 mCi (125)I after thyroid blockade. The level of circulating HuCC49DeltaC(H)2 MAb was measured daily as precordial counts using a handheld gamma-detecting probe. Each patient underwent an exploratory laparotomy on postinjection days 3-20. Gamma counts were measured at normal organs, aortic bifurcation (AB), and both clinically evident and occult tumors.Precordial and AB gamma counts showed an excellent linear correlation. HuCC49DeltaC(H)2 MAb followed a two-compartment pharmacokinetic model. Normal organs and AB showed similar exposures to HuCC49DeltaC(H)2 MAb, while HuCC49DeltaC(H)2 MAb favorably distributed into tumors from day 3. Intestinal and metastatic liver lesions showed the highest partition coefficients. All patients showed no HAMA response.C(H)2 region deletion of HuCC49DeltaC(H)2 MAb did not alter the pharmacokinetics compared to murine CC49. The favorable partition coefficient K of HuCC49DeltaC(H)2 MAb into tumors supports its use in RIGS.

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.

Background Fanolesomab (NeutroSpec) is a murine monoclonal 9 9 m Tc labelled anti-CD15 IgM antibody that localizes collections of human polymorphonuclear neutrophils (PMNs) at sites of infection. Objectives The objectivesof this study were to evaluate the safety of repeated injections of fanolesomab and the extent of induction of human anti-mouse antibody (HAMA) response. Methods Thirty healthy adults (15 men and 15 women) were enrolled in the study. Subjects were injected on two separate occasions, separated by 21 days, with 125 μg of fanolesomab that had been labelled with decayed 9 9 m Tc. HAMA assays were performed on blood samples drawn prior to each injection, and at 7 and 28 days following the second injection. Safety was determined by monitoring for adverse events, and for changes in vital signs, physical examination and clinical laboratory measurements. Results Five subjects exhibited induction of HAMA (16.7%; 95% Cl, 6.3-34.2%). Two were considered marginal responses (increase from 5 to 31, and 5 to 20 and 24 ng ml - 1 ), and three were considered moderate (7 to 228, 7 to 140 and 270, and 7 to 35 and 450 ng.ml - 1 ). There were no strong responses (greater than 1000 ng.ml - 1 ). Seven subjects experienced adverse events, most of which were coincidental to administration of fanolesomab. There were no serious or severe adverse events. Conclusions Repeated fanolesomab injections at clinically useful doses does not appear to induce a strong HAMA response nor does it present a risk for serious adverse events.

Background: CC49 is a monoclonal antibody directed against a pancarcinoma antigen (TAG-72) expressed by colorectal cancers. The use of murine CC49 in radioimmunoguided surgery (RIGS) was problematic because of the human anti-mouse antibodies (HAMA) generated. This study was designed to assess the clearance, safety, and effectiveness of localization of a complimentarity determining region (CDR)-grafted humanized domain-deleted antitumor CC49 antibody (HuCC49°CH2). Methods: After thyroid blockade, 1 mg of HuCC49°CH2 radiolabeled with 2 mCi of iodine-125 was administered. All patients subsequently underwent traditional exploration followed by a survey with the gamma-detecting probe. In five patients, exploration was performed 10 to 24 days after injection, when precordial counts were sufficiently low (

Ovarian carcinoma has a high mortality rate, because most ovarian carcinomas are detected at a late stage. Traditional therapies, such as surgical debulking and chemotherapy, have not been successful in improving the long-term survival of these patients. Alternative therapies targeting various biomarkers, such as carcinoembryonic antigen (CEA), Tag-72, and Lewis-Y antigen, have been developed to treat patients with advanced ovarian cancers. To ensure that therapies targeting these biomarkers are effective, it is imperative to determine whether there is any differential expression of these targeted biomarkers between primary and metastatic ovarian carcinomas. In the present study, primary and metastatic lesions from 68 and 58 patients, respectively, including primary and matched metastatic lesions from 31 patients, were evaluated for cytoplasmic and membranous expression of CEA (clone Col-1), Tag-72 (clone CC-49), and Lewis-Y antigen (clone BR-96) by immunohistochemistry. No significant differences were observed with cytoplasmic and membranous expression of Tag-72 (CC-49) and Lewis-Y antigen (BR-96) in the primary and metastatic, matched and unmatched lesions (Wilcoxon signed-rank test). Although there was no statistically significant difference in the scores of CEA (Col-1) between primary and metastatic lesions, 5 of 11 (45%) cases with positive staining with CEA (Col-1) demonstrated discordant results between primary and metastatic lesions. There was a moderate positive correlation of the cytoplasmic and membranous expression of Tag-72 (CC-49), as well as cytoplasmic expression of BR-96 between primary and metastatic ovarian carcinomas. There was a weak negative correlation between the membranous expression of CEA (Col-1) and that of Lewis-Y antigen (BR-96); however, the difference was not statistically significant. No correlation was observed with other combinations of biomarkers. Our findings suggest that samples from either primary or metastatic ovarian carcinomas can be used for the evaluation of the expression of Tag-72 (CC-49) and Lewis-Y antigen (BR-96) to identify targets for novel therapies in patients with disseminated ovarian carcinomas. CEA (Col-1), due to its low expression and variation in phenotypic expression between primary and metastatic lesions, should be evaluated carefully in metastatic lesions before targeting the CEA antigen with CEA (Col-1)-like antibodies.

Although marrow suppression is usually the dose-limiting toxicity in non-marrow ablative radionuclide therapy, calculated marrow dose has rarely been used for prescribing the radioactivity to be administered. This study assesses the correlation of myelotoxicity with mCi/m(2), patient-specific lean body dose, marrow dose from blood and body of reference man, or from blood and body using the patient-specific mass. Fourteen prostate cancer patients were treated with (131)I-CC49. Radioactivity in blood and body was determined and used to calculate their contributions to the marrow dose. Platelet nadir expressed as percentage (%) of the initial baseline was used as an indicator for myelotoxicity. Correlation between platelet nadir (%) and myelotoxicity predictors was evaluated. Platelet nadirs (%) varied substantially (5-33%) for a small range of injected radioactivity/m(2) (68-78 mCi/m(2), 2.5-2.9 GBq/m(2)). Patient-specific total body dose based on lean body mass exhibited a weak correlation (r = 0.48) with platelet nadir. Marrow dose from blood and body of reference man had a better correlation (r = 0.73). Patient-specific marrow dose from blood and body (or lean body) had a similar correlation (r = 0.74 or 0.73). Radioactivity in the remainder of the body contributed only 28% of the total dose, and thus changes to this dose component had small impact on total marrow dose. Marrow dose was a better predictor for myelotoxicity than mCi/m(2) or lean total body dose in this non-marrow targeting (131)I-antibody therapy with high blood contributions to total dose.

Angiogenesis plays an important role in colorectal cancer progression. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in endothelial mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain-containing receptor (KDR) appear to be principally up-regulated during tumorigenesis. A chimeric anti-KDR antibody, IMC-1C11, blocks VEGFR-KDR interaction and inhibits VEGFR-induced endothelial cell proliferation. This trial seeks to assess the safety, tolerability and feasibility of targeting an important pathway in tumorigenesis.In a dose-escalation, single-agent study of IMC-1C11, we enrolled 14 patients with colorectal carcinoma and hepatic metastases. Safety-, pharmacokinetic-, immunogenicity-, and magnetic resonance imaging-assessed alteration of vascular effects of IMC-1C11 were evaluated in this trial. IMC-1C11 was infused weekly at 0.2 mg/kg (n = 3), 0.6 mg/kg (n = 4), 2.0 mg/kg (n = 3), and 4.0 mg/kg (n = 4) for 4 weeks, which constituted a cycle.No grade-3 or -4 IMC-1C11-related toxicities were observed. Minor grade-1 bleeding events were observed in four patients [0.2 mg/kg (n = 1) and 0.6 mg/kg (n = 3)]. Each resolved quickly and required no intervention. The starting dose of IMC-1C11 was selected to achieve a C(max) of approximately 5 micro g/ml. This concentration prevented KDR phosphorylation in vitro. Pharmacokinetic analysis demonstrated that the plasma t(1/2) and C(max) were dose dependent with a plasma t(1/2) of 67 +/- 3 h at the 4-mg/kg dose level. Human antichimeric antibodies were detected in 7 of 14 patients. The antibodies to IMC-1C11 inhibited the circulation of the agent in two patients. One patient had prolonged stable disease for seven cycles (28 weeks). The mean changes in tumor-influx volume-transfer constant k(in) (min(-1)) and enhancement factor after 4 weeks of therapy were significantly decreased compared with pretreatment values in 11 patients.IMC-1C11 was both safe and well tolerated. Drug levels of IMC-1C11 were reliably predicted. Further clinical investigation of anti-VEGFR/KDR agents is warranted.

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2'-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drug-to-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal effect of paclitaxel as compared to those of the free drug, the intact antibody, and a physical mixture of the two (the controls). In A431 cells, the conjugate showed 25.2% +/- 2.2% of apoptosis induction as compared to little or no apoptosis caused by the controls. Biodistribution analysis of the PTXC225 in tumor-implanted nude mice and a tyrosine-kinase assay showed that conjugation of the drug did not interfere with the immunoreactivity of the antibody. The 24-h tumor uptake of C225 and PTXC225 were 11.7% +/- 6.0% and 7.1% +/- 3.6% of the injected dose per gram of tissue (%ID/g), respectively, which were not significantly different. Also, in A431-implanted nude mice, the conjugate and C225 showed tumor growth inhibition effects of 57.2% and 41.2%, respectively, against a saline-treated control, which were not significantly different from each other. This lack of difference in the in vivo antitumor activity of the MAb-delivered drug and free PTX may be due to either a relatively low dose of the antibody-delivered drug (346 microg/kg), or an untimely release of it, or both. The tumor growth inhibition pattern of the conjugate, however, was identical to that of C225, indicating that the attachment of PTX did not affect the antigen-binding and growth inhibitory features of the MAb. These preliminary results demonstrate the potential of tumor-targeted delivery of taxol as a promising strategy in cancer treatment and warrant further work to develop more suitable drug-MAb linkers as well as improved dosage and treatment protocols.

The purpose of this study was to determine the feasibility and maximum tolerated dose of (90)Yttrium-CC49 ((90)Y-CC49) as the radioimmunotherapy (RIT) component of an i.p. combined modality treatment for recurrent ovarian cancer.A Phase I trial of (90)Y-CC49 RIT was conducted in ovarian cancer patients who had persistent or recurrent intra-abdominal disease, had failed one or two prior chemotherapy regimens, and demonstrated TAG-72 expression. Patients were treated with a previously established combined modality treatment protocol of s.c. IFN alpha2b, i.p. paclitaxel, and increasing dosages of i.p. (90)Y-CC49. Patients were monitored for toxicity, generation of human antimouse antibody response, and clinical efficacy.Twenty eligible patients were treated per study specifications. All patients had been treated with debulking and paclitaxel/carboplatin-based chemotherapy at initial diagnosis. The patients included 11 patients with persistent disease at the time of second look laparotomy and 9 patients with delayed recurrence. Patients were treated with i.p. (90)Y-CC49 given in combination with s.c. IFN alpha2b (dose of 3 x 10(6) units for a total of four doses) and i.p. paclitaxel (dose of 100 mg/m(2)). RIT treatment was associated with primarily hematological toxicity. The maximum tolerated dose of i.p. (90)Y-CC49 was established at 24.2 mCi/m(2) in this combined regimen. Of nine patients with measurable disease, two had partial responses lasting 2 and 4 months. Of 11 patients with nonmeasurable disease, median time to progression was 6 months in 7 patients who recurred; 4 of these patients remain no evidence of disease at 9+, 18+, 19+, and 23+ months.(90)Yttrium-CC49-based RIT in combination with IFN alpha2b and i.p. paclitaxel is feasible and well tolerated at a dose ofor =24.2 mCi/m(2).

For calculation of radiation dose to the marrow, standard dosimetry for radiopharmaceuticals that do not bind to the marrow includes dose contributions from radioactivity in blood and the remainder of the body. For a pure beta -emitter such as (90)Y, marrow dose is usually determined by the blood contribution. However, myelotoxicity from (90)Y-antibody therapy often correlates poorly with marrow dose estimated using the blood method. This study proposes a method to address 2 possible factors affecting marrow dose estimates. These include (a) recycled (90)Y in bone/marrow space after (90)Y-antibody has been processed in the liver and (b) use of the marrow mass of Reference Man for individual patients.Thirty-three patients with advanced non-small cell lung cancer were treated with (90)Y-anti-TAG-72 murine antibody (CC49). TAG-72 is often expressed in epithelial-derived tumors but not in normal marrow. (111)In-CC49 was used as a tracer. The marrow doses from blood were calculated on the basis of radioactivity concentrations in blood. Marrow dose in the lumbar vertebrae was estimated from images for (111)In-CC49 uptake in L2-L4. In 20 patients who had CT images, trabecular bone volumes of L2-L4 were measured from CT images to estimate patient-specific marrow mass in L2-L4. The fraction of baseline platelet counts at nadir was used as an indicator of myelotoxicity.Marrow dose per unit injected radioactivity estimated from blood was lower than that from L2-L4 uptake values. Prediction of myelotoxicity using marrow dose estimated from blood was poorer than that using injected dose per body surface area (GBq/m(2)) (r = 0.31 vs. 0.51). Prediction was improved using marrow dose estimated from L2-L4 uptake, assuming the marrow mass of Reference Man (r = 0.67 for n = 33; r = 0.70 for n = 20). Prediction was worse if reference marrow mass was adjusted by body weight (r = 0.56 for n = 33; r = 0.63 for n = 20). Prediction was not improved if adjusted by body surface area or lean body mass but was improved if adjusted by height (r = 0.72 for n = 33; r = 0.78 for n = 20). The best prediction was obtained (r = 0.85 for n = 20) using patient-specific L2-L4 marrow mass estimated from CT.Marrow dose estimated from the blood radioactivity method was not a good predictor of myelotoxicity for non-marrow-targeting (90)Y-antibody therapy. Thrombocytopenia in this group of patients correlated much better with dose estimated from lumbar vertebrae imaging and patient-specific marrow mass than with that estimated from GBq/m(2) or standard marrow dose based on blood.

A phase I trial was designed to examine the feasibility of combining interferon and Taxol with intraperitoneal radioimmunotherapy (177Lu-CC49). Patients with recurrent or persistent ovarian cancer confined to the abdominal cavity after first line therapy, Karnofsky performance status60, adequate liver, renal and hematologic function, and tumor that reacted with CC49 antibody were enrolled. Human recombinant alpha interferon (IFN) was administered as 4 subcutaneous injections of 3 x 10(6) U on alternate days beginning 5 days before RIT to increase the expression of the tumor-associated antigen, TAG-72. The addition of IFN increased hematologic toxicity such that the maximum tolerated dose (MTD) of the combination was 40 mCi/m2 compared to 177Lu-CC49 alone (45 mCi/m2). Taxol, which has radiosensitizing effects as well as antitumor activity against ovarian cancer, was given intraperitoneally (i.p.) 48 hrs before RIT. It was initiated at 25 mg/m2 and escalated at 25 mg/m2 increments to 100 mg/m2. Subsequent groups of patients were treated with IFN + 100 mg/m2 Taxol + escalating doses of 177Lu-CC49. Three or more patients were treated in each dose group and 34 patients were treated with the 3-agent combination. Therapy was well tolerated with the expected reversible hematologic toxicity. The MTD for 177Lu-CC49 was 40 mCi/m2 when given with IFN + 100 mg/m2 Taxol. Interferon increased the effective whole body half-time of radioactivity and the whole body radiation dose. Taxol did not have a significant effect on pharmacokinetic or dosimetry parameters. Four of 17 patients with CT measurable disease had a partial response (PR) and 4 of 27 patients with non-measurable disease have progression-free intervals of 18+, 21+, 21+, and 37+ months. The combination of intraperitoneal Taxol chemotherapy (100 mg/m2) with RIT using 177Lu-CC49 and interferon was well tolerated, with bone marrow suppression as the dose-limiting toxicity.

We investigated the expression of human endogenous retroviral (HERV) sequences in breast cancer.Reverse transcription-PCR (RT-PCR) was used to examine expression of the envelope (env) region of ERV3, HERV-E4-1, and HERV-K in breast cancer cell lines, human breast tumor samples, adjacent uninvolved breast tissues, nonmalignant breast tissues, and placenta. Expression of HERV transcripts was confirmed by Northern blot analysis and in situ hybridization (ISH). To evaluate coding potential, amplified HERV sequences were cloned into vectors for expression and sequence analysis.No expression of ERV3 or HERV-E4-1 RNA was detected in the analyzed breast samples. In contrast, HERV-K transcripts were detected in most breast cancer cell lines and many breast tumor tissues. Expression was detected in a small percentage of matched, uninvolved breast tissues and in placentas but not nonmalignant breast tissues. In HERV-K-positive breast cancer tissues, Northern blot analysis demonstrated full-length proviral and spliced env transcripts. ISH demonstrated expression of HERV-K transcripts in breast tumor cells but not in normal or uninvolved breast epithelial cells. Independently isolated clones of HERV-K env cDNA generated recombinant proteins of the expected size. Sequence analysis of env cDNA clones derived from four breast tumor samples revealed97% identity with the type I HERV-K102, with no premature termination codons. Independent isolates from the same breast tumor sample showed nucleotide sequence differences, suggesting that multiple loci may be transcribed.These data indicate that HERV-K transcripts with coding potential for the envelope region are expressed frequently in human breast cancer.

The angiogenic response of a progressing malignancy is characterized by a shift in the balance of stimulatory and inhibiting factors of angiogenesis. Recognition of the regulated steps in tumor angiogenesis provides unique targets for developing anti-tumor therapy. Vitaxin is a humanized monoclonal antibody, which has specificity for the integrin alpha v beta 3 (vitronectin receptor). This antibody can impair the vascular response of endothelial cell growth factors in vitro and inhibit tumor cell mediated angiogenesis in pre-clinical animal models. Patients with metastatic cancer who failed standard therapy received intravenous doses of 10, 50 or 200 mg in cohorts of three patients. The unlabeled dose of Vitaxin was infused on days 0 and 21 of a treatment cycle. All patients received a pre-therapy imaging dose of 1 mg of Tc-99m Vitaxin with gamma camera imaging studies. There was no significant toxicity noted in these three dose levels. There were no objective anti-tumor responses. Three patients received two cycles of therapy and had stable disease at day 85 when taken off study. Radioimaging of tumor vasculature was unsuccessful although one patient with alpha v beta 3 positive melanoma had imaging of tumor sites. There was no immune response to Vitaxin in any patient. Patients receiving 10 mg doses of Vitaxin had poor plasma recovery of injected doses and brief circulation in plasma. Doses of 50 and 200 mg had plasma recovery that better approximated the predicted levels in plasma and circulation half-lives of approximately 7 days. This data suggests that an every three-week schedule of Vitaxin at doses of 200 mg (2.5-3.5 mg/kg) can maintain circulating levels of antibody with little or no toxicity. Future studies will be challenged to define anti-tumor activity in malignancy or appropriate surrogates of anti-tumor effect and explore escalating doses and alternate schedules of administration.

Our previous results indicated that the trihydroxamate ligand, trisuccin, was a promising bifunctional chelating agent (BCA) for radiometal labeling of monoclonal antibodies with rhenium and technetium. An interest was developed to evaluate structural modifications of this compound from both synthetic and biological points of view. In this report we describe the synthesis of an esterified trisuccin (referred to as trisester), and conjugation of this new derivative to MAb CC49, radiolabeling of this conjugate with rhenium-188 (188Re), and biodistribution of the labeled conjugate in athymic nude mice. Thus, trisuccin (1) was esterified with benzyl 4-hydroxybenzoate in a DCC/DMAP reaction followed by removal of all benzyl protecting groups with catalytic hydrogenation. The resulting product was conjugated to CC49 by the active ester technique, through formation of the 2-nitrophenyl ester 6, and the conjugate was radiolabeled with generator-produced 188Re. The lead molecule trisuccin 1 was also conjugated to CC49 through the active ester 5 and the conjugate was radiolabeled by the same procedure to serve as the control conjugate. Biodistributions of the labeled conjugates were studied in athymic nude mice, transplanted s.c. with LS174T human colon cancer xenografts. Although an increase in the radiolabeling yield was observed for the esterified ligand-CC49 conjugate, as well as some increase in its immunoreactivity, as compared to those for the parent trisuccin molecule, there were no significant differences in their biodistribution. This new compound therefore may be useful in improving the conjugation and radiolabeling chemistries of this trihydroxamate ligand system.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

A concise synthesis of the bifunctional chelating agent 1,4,7,10-tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N′,N″,N‴-tris(acetic acid)cyclododecane (PA-DOTA) is reported. Difficulties involving the production of partially alkylated products and their removal have been addressed and obviated. After the pure nitro form of PA-DOTA was obtained, conversion to the isothiocyanato form PA-DOTA ( 1 Download : Download high-res image (142KB) Download : Download full-size image Figure 3 . Synthetic scheme of PA-DOTA. , conjugation to HuCC49 and HuCC49ΔCH2 monoclonal antibodies was achieved. Subsequent radiolabeling with 177Lu was performed, demonstrating a useful bifunctional chelating agent suitable for clinical radioimmunotherapy applications.

To determine the safety and immunogenicity of an idiotypic anti-dsDNA vaccine in patients with nonactive systemic lupus erythematosus (SLE) and stable lupus nephritis.Patients with SLE with a history of nephritis were randomized for vaccination with the murine anti-dsDNA monoclonal antibody (Mab) 3E10 in a dose ranging, double blind, placebo controlled study (phase I).Of the 9 patients injected with Mab 3E10, 5 showed a human anti-mouse antibody (HAMA) response, in large part antiidiotypic, which developed within the first 3 months in 3 strong HAMA responders, and more than one year after immunization in an initially weak HAMA responder. All but one nonresponder were receiving low dose prednisone. No adverse events, in particular no evidence of lupus flares, and no untoward laboratory findings were reported over a followup of 2 years.In patients with stable lupus nephritis, immunization with Mab 3E10 appears safe and can generate a significant antiidiotypic response. Idiotypic vaccination may be an approach to specific immunotherapy of autoimmune lupus nephritis.

One of the problems in achieving cures with radioimmunotherapy is that hematological toxicity limits the quantity of radiolabeled monoclonal antibody (MAb) that can be administered. The MAb CC49 binds with high affinity to the TAG-72 antigen expressed in many human adenocarcinomas. We investigated tumor growth inhibition, survival, and tumor and bone marrow dosimetry after multiple bolus injections or continuous infusion of 131I-labeled CC49 MAb in a human colon cancer xenograft model to determine which method of administration results in the highest therapeutic ratio. Groups of athymic nude mice bearing established s.c. LS174T human colon cancer xenografts received three i.p. bolus injections (3X) of 131I-labeled CC49 (3X, days 0, 3, and 7) or were implanted i.p. with mini-osmotic pumps delivering 131I-labeled CC49 over 7 days. The total radionuclide doses administered were broken down into low-dose (or = 450 microCi), medium-dose (450-800 microCi), and high-dose (800 microCi) groups. At the medium-dose level, the bolus-therapy animals did not have a significantly longer survival time but did have a significantly longer time-to-tumor doubling than the pump-therapy animals. The median survival for medium-dose bolus and pump therapy was 157 and 105 days, respectively, and the median time-to-tumor doubling was at least 114 and 77 days, respectively. At the low-dose level, the bolus-therapy animals had a significantly longer survival time but not a significantly longer time-to-tumor doubling than the pump-therapy animals. The median survival for low-dose bolus and pump therapy was 95.5 and 59 days, respectively, and the median time-to-tumor doubling was 73 and 38 days, respectively. The high-bolus dose was toxic. A comparison of the overall survival rate of pump therapy versus bolus therapy, excluding high-dose, resulted in the bolus-therapy animals having a longer survival time and a longer time-to-tumor doubling than the pump-therapy animals. Serial section autoradiography was used to reconstruct tumor activity density distributions over time. Average dose values calculated from total uptake data for 900 microCi administered activity yielded 158 Gy (3X) and 141 Gy (pump). Average three-dimensional doses using the radial histograms to calculate the absorbed fractions were 139 Gy and 123 Gy, respectively. This calculation includes energy loss external to the tumor. With cell proliferation parameters set to single fraction 60Co recurrence results, the effective dose (D(eff)) for local control was 11 Gy and 9 Gy, respectively. Three bolus injections resulted in a more uniform dose rate over a longer period, resulting in a calculated 19% improvement in D(eff) compared with pump administration. Dose to bone marrow was calculated assuming an activity concentration in bone marrow of 0.24 times the concentration in blood and an absorbed fraction of 0.63. For the 900-microCi 131I-labeled CC49 injected activity, pump administration resulted in an 80% higher calculated D(eff) to bone marrow compared with 3X bolus injection. These results demonstrate that 3X bolus injections were clearly superior to pump administration in terms of survival, tumor growth inhibition, tumor absorbed dose, and bone marrow dose.

Novel strategies to increase the therapeutic ratio in clinical radioimmunotherapy studies are needed. Limitations to radioimmunotherapy include bone marrow suppression due to the long circulating half-life of radiolabeled monoclonal antibodies (mAbs) and heterogeneous tumor penetration of the high-molecular-weight mAb. An approach to overcome these problems is the use of genetically engineered mAbs. The engineered mAb discussed in this paper contains a deletion in the constant region of the mAb that increases its tumor penetration and blood clearance compared with the intact mAb. Radiolabeling of this mAb should lead to a similar radiation-absorbed dose to tumor compared with the intact mAb, but reduce the radiation absorbed dose to bone marrow. In addition, low or variable expression of tumor-associated target antigens or receptors may lead to low or heterogeneous tumor uptake of radiolabeled mAbs. This report also discusses a novel approach toward systemic radiotherapy that combines gene transfer techniques (to increase tumor receptor expression) with radiolabeled peptides that target the induced receptor. The radiolabeled peptides achieve good tumor uptake, rapid tumor penetration, and rapid blood clearance. A humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 mAb, as well as a construct with the CH2 region deleted (HuCC49deltaCH2), were labeled with 131I and 177Lu. Biodistribution of the radiolabeled constructs was evaluated 24 h after regional i.p. injection in athymic nude mice bearing i.p. LS174T human colon cancer xenografts. The 131I-HuCC49deltaCH2 showed a median tumor uptake of 5.5% ID/g which was similar to that of 131I-HuCC49 at 5.2% ID/g. However, the median blood concentration of 131I-HuCC49deltaCH2 was 0.2% ID/g which was significantly lower than 0.8% ID/g for 1311-HuCC49. The uptake of the constructs in other normal tissues were similar. The 177Lu-HuCC49deltaCH2 showed a median tumor uptake of 9.4% ID/g, which was slightly higher than that of 177Lu-HuCC49 at 7.9% ID/g. The median blood concentration of 177Lu-HuCC49deltaCH2 was 0.2% ID/g, which was significantly lower than 0.4% ID/g for 177Lu-HuCC49. The uptake of the antibody constructs in other normal tissues were similar except for the kidney. The tumor:blood ratios of 177Lu-HuCC49 and 177Lu-HuCC49deltaCH2 were 19.4 and 60.2, respectively, at 24 h after injection. The purpose of the second aspect of the study was to determine the biodistribution of 64Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)-octreotide in a human ovarian cancer model induced to express human somatostatin receptor subtype 2 (SSTr2) using gene transfer techniques as a prelude to future therapy studies. Mice bearing i.p. SKOV3.ip1 tumors transduced with an adenoviral vector encoding the cDNA for SSTr2 (AdSSTr2) and injected i.p. with 64Cu-TETA-octreotide showed a median uptake of 24.3% ID/g in tumor at 4 h postinjection compared with 4.9% ID/g at 18 h after injection. Also, tumor uptake of 64Cu-TETA-octreotide at 4 h was not significantly different when administered either 2 or 4 days after injection of AdSSTr2 (P = 0.076). 64Cu-TETA-octreotide should be useful for targeted radiotherapy against tumors that are genetically induced to express high levels of SSTr. These two novel targeting strategies show promise for improved cancer radioimmunotherapy.

The trihydroxamate bifunctional chelating agent (BCA), trisuccin, has been shown to be a potential ligand for radiolabeling of monoclonal antibodies (MAbs) with rhenium radioisotopes, through an indirect postconjugation approach. The use of this trihydroxamate BCA made it possible to prepare stable BCA-MAb conjugates in pure form that could be radiolabeled with carrier-free 188Re. The anti-TAG-72 murine MAb, CC49, and its humanized derivatives are promising agents in the treatment of a number of malignancies with the CH2 domain-deleted MAb (HuCC49deltaCH2), which is of particular interest due to its rapid blood clearance. The biodistribution of 188Re-labeled conjugates of trisuccin with both humanized CC49 (HuCC49) and HuCC49deltaCH2 in athymic nude mice implanted i.p. with LS174T human colon carcinoma was studied. Trisuccin-MAb conjugates were synthesized at different BCA:MAb ratios by the 6-oxoheptanoic acid method using trisuccin hydrazide. The conjugates were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy for the number of incorporated trisuccin molecules. The conjugates were radiolabeled with carrier-free, generator-produced 188Re and purified by gel filtration on Sephadex G-25. Labeling yields and homogeneity of the labeled conjugates were analyzed by high-pressure liquid chromatography and instant TLC. Athymic nude mice were injected i.p. with LS174T human colon carcinoma cells, 7 days prior to injection of the labeled antibodies. 188Re-labeled MAbs were injected i.p., and the mice were sacrificed 24 h postinjection. Matrix-assisted laser desorption/ionization time-of-flight analyses showed stable incorporation of trisuccin into each MAb, with the measured ligand:MAb values positively correlating with the theoretical ratios. Labeling of the conjugates with 188Re proceeded with high yields, producing homogeneous 188Re-MAbs with good stabilities as shown by instant TLC and biodistribution analyses. Biodistribution of the radiolabeled MAbs at 24 h after injection showed median tumor uptake values of 23.5%ID/g and 17.6%ID/g for the 188Re-HuCC49deltaCH2 and 188Re-HuCC49, respectively. The blood clearance of the domain-deleted MAb was faster than that of the intact antibody. The blood values at 24 h after injection were 0.7%ID/g for 188Re-HuCC49deltaCH2 and 3.2%ID/g for 188Re-HuCC49. The results indicate that trisuccin is a promising agent for postconjugation labeling of antibodies with 188Re. Additionally, these results illustrate the potential of 188Re-HuCC49deltaCH2 in radioimmunodiagnosis and radioimmunotherapy of cancer.

Adjuvant Interferon (IFN) was given to increase tumor antigen expression and enhance localization with 131I-labeled CC49 radioimmunotherapy in a Phase II trial for hormone resistant metastatic prostate cancer. Patients received four doses of alpha-IFN (3 x 10(6) IU) s.c. on alternate days, from day -5 to day +1 of 75 mCi/m2 131I-CC49 treatment. Toxicity was well tolerated, with the majority of patients experiencing transient grade 3 or 4 neutropenia and/or thrombocytopenia (maximal at 4-6 weeks). The absorbed dose was25 Gy in four of eight tumors visualized, which represents an increase of20 fold over whole body radiation dose. Two patients had radiographic minor responses by 6 weeks post-therapy, whereas five of six patients experiencing pain had symptom relief without radiographic changes. The protocol provided modest antitumor effects (pain relief in five of six patients and two minor radiographic responses). This study suggests that the addition of IFN enhanced tumor uptake and antitumor effects as compared to a prior Phase II trial of 131I-CC49 alone.

The principal objectives of this trial were twofold: (a) to examine the safety and relative efficacy of intradermal needle injection versus s.c. jet administration of a carcinoembryonic antigen (CEA)-encoding recombinant vaccinia virus (rV-CEA) over a limited dose range and (b) to evaluate CEA-specific immune responses or antitumor effects induced by rV-CEA vaccination. Patients were randomly assigned to one of two groups, depending upon the technique of vaccine administration. All 20 patients received two doses of 10(7) or 10(8) pfu of rV-CEA at a 4-week interval. Toxicity was limited to modest local inflammation at the inoculation site as well as low-grade fever and increased fatigue, each affecting fewer than 20% of the patients. No evidence of CEA-specific lymphoproliferation, interleukin 2 release, delayed-type hypersensitivity, or antibody response was observed. Thus, the efficacy comparison between the two administration techniques was based upon the induction of immune responses to the vaccinia virus vector. Both techniques induced vaccinia-specific lymphoproliferation, interleukin 2 release, and antibody responses of comparable magnitude and frequency as well as protected 80% of patients against pustule formation following vaccinia scarification. Thus, there is no compelling reason to recommend one administration technique over the other based upon toxicity or efficacy. We have selected s.c. jet injection for subsequent trials of rV-CEA based on the ability to accommodate larger injection volumes, enhanced standardization between clinicians, and avoidance of needles that could transmit the vaccine or blood-borne pathogens to health care workers. We recommend use of 10(8) pfu doses for subsequent trials of recombinant vaccinia virus vaccines based upon the favorable toxicity profile and more consistent local pustule formation indicative of an adequate inoculation of live virus. No objective clinical responses to the rV-CEA vaccine were observed among this population of patients with widely metastatic adenocarcinoma.

In preparation for a Phase I trial of DNA immunization against carcinoembryonic antigen (CEA) in patients with colorectal carcinoma, we have produced a single plasmid DNA encoding CEA and hepatitis B surface antigen (HBsAg) under transcriptional regulatory control of two separate cytomegalovirus promoters within separate eukaryotic expression cassettes, designated pCEA/HBsAg. Hepatitis B surface antigen was included to provide an internal positive control for the efficacy of this immunization strategy without regard to the issue of breaking tolerance to a self-antigen. In the present work, we sought to examine the immunogenicity of this plasmid in a nonhuman primate model with close phylogenetic relationship to humans. Groups of pig-tailed macaques were immunized with pCEA/ HBsAg by i.m. injection or particle bombardment of the skin according to a dose and schedule thought to be optimal for the respective technique of DNA immunization. Both administration techniques produced humoral and lympho-proliferative responses of comparable magnitude. However, delayed type hypersensitivity to CEA and CEA-specific interleukin-2 release were observed only in the i.m. group, suggesting a qualitative difference in the character of the immune response elicited by the two techniques of DNA immunization. The antibody responses to CEA and HBsAg were surprisingly persistent in that all immunized animals maintained moderate antibody titers against both antigens for more than 15 months after the last boost. No toxicity was observed during 2 years of follow-up, including no measurable levels of anti-DNA antibody. This antitumor immunization strategy is presently being examined in patients with metastatic colorectal carcinoma using pCEA/HBsAg administered by i.m. injection.