First-in-human phase 0 study of 111In-CHX-A"-DTPA trastuzumab for HER2 tumor imaging.

INTRODUCTION: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis. Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit. We report on a Phase 0 study of the imaging agent, 111In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status. METHODS: 111In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. RESULTS: No safety issues were identified. Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient. The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t1/2=46.9h (R2=0.97; 95%CI 41.8 to 53h). This was followed by high gastrointestinal (GI) tract activity peaking by 52h. Linear regression predicted GI clearance by 201.2h (R2 =0.96; 95%CI 188.5 to 216.9h). Blood pool had lower activity with its maximum on the initial images. Non-linear regression fit projected a t1/2=34.2h (R2 =0.96; 95%CI 25.3 to 46.3h). Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R2=0.96; 95%CI 186.1 to 489.2h). CONCLUSION: 111In-CHX-A"-DTPA trastuzumab can be safely imaged in humans. The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients. Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.

Association of obesity, serum glucose and lipids with the risk of advanced colorectal adenoma and cancer: a case-control study in Korea.

BACKGROUND: Previous studies on colorectal cancer risk suggest that obesity, serum lipids and glucose might be related to colorectal carcinogenesis. This case-control study was conducted to investigate the association between obesity, serum lipids and glucose, and the risk of advanced colorectal adenoma and cancer. METHODS: Patients with histologically confirmed colorectal cancers (n=105), same number of patients with advanced colorectal adenomas matched by age and sex, and the same number of controls matched by age and sex were selected in Hanyang University Guri Hospital between January 2002 and June 2004. RESULTS: Adenoma and cancer group showed significantly higher levels of mean body mass index and serum glucose. Cancer group also showed significantly lower mean serum lipids levels than controls. We used an unordered polytomous logistic model to calculate multivariate odds ratios for advanced adenoma and cancer relative to controls. Higher serum glucose level was more strongly associated with increased risk of cancer relative to controls (odds ratio, 3.0; 95% confidence interval, 0.9-9.8) than with increased risk of advanced adenoma (odds ratio, 2.1; 95% confidence interval, 0.9-5.4). Higher body mass index was strongly associated with increased risk of advanced adenoma (odds ratio, 10.8; 95% confidence interval, 4.6-25.3), but associated with attenuated risk of cancer (odds ratio, 2.3; 95% confidence interval, 0.9-5.8). Serum triglycerides and cholesterol levels were strongly associated with reduced risk of cancer (odds ratio, 0.3; 95% confidence interval, 0.1-0.8 and odds ratio, 0.2; 95% confidence interval, 0.1-0.6, respectively). CONCLUSIONS: Obesity and hyperglycaemia are positively related to advanced colorectal adenoma formation. Furthermore, hyperglycaemia plays an important role in progression to cancer. Findings on an inverse relationship between serum triglyceride and cholesterol levels and the risk of colorectal cancer may be the secondary results from metabolic or nutritional changes in advanced colorectal cancer patients and should be clarified in further studies.

Huge gastric diospyrobezoars successfully treated by oral intake and endoscopic injection of Coca-Cola.

A diospyrobezoar is a type of phytobezoar that is considered to be harder than any other types of phytobezoars. Here, we describe a new treatment modality, which effectively and easily disrupted huge gastric diospyrobezoars. A 41-year-old man with a history of diabetes mellitus was admitted with lower abdominal pain and vomiting. Upper gastrointestinal endoscopy revealed three huge, round diospyrobezoars in the stomach. He was made to drink two cans of Coca-Cola every 6 h. At endoscopy the next day, the bezoars were partially dissolved and turned to be softened. We performed direct endoscopic injection of Coca-Cola into each bezoar. At repeated endoscopy the next day, the bezoars were completely dissolved.

Radiolabeling of antibodies.

The radiolabeling of antibodies is considered in terms of the choice of radionuclide, the method of conjugation, and the effect of conjugation on plasma clearance. Iodination techniques are reviewed, but the major emphasis is placed on the methods of conjugating metallic radionuclides using bifunctional chelating agents. The technique of producing clinically useful radiolabeled antibodies by balancing altered substrate specificity caused by radiolabeling against accelerated plasma clearance is discussed.

Differences of biodistribution, pharmacokinetics, and tumor targeting between interleukins 2 and 15.

Interleukin-2 (IL-2) and interleukin-15 (IL-15) are T-cell tropic factors that share beta and gammac subunits of their receptors on T/NK-cells. Although these two cytokines share receptor components, the IL-15Ralpha molecule is expressed constitutively by various tissue cells, whereas the IL-2Ralpha expression is mostly restricted to activated mononuclear cells. Consequently, we postulated that the biodistribution of IL-15 might be different from that of IL-2 and that individual alpha chains play an important role in this respect. This study investigated the differences between IL-2 and IL-15 in pharmacokinetics, biodistribution, and their tumor-targeting abilities. It found that only IL-2 showed specific binding to a protein, alpha2-macroglobulin, which may be the reason that IL-2 displays longer blood clearance than IL-15. Upon injection of these cytokines into mice, we observed that IL-15 accumulated significantly more than IL-2 in kidney, spleen, and bone. These are all tissues that express IL-15 receptor alpha but not IL-2 receptor alpha. To evaluate the tumor-targeting ability of each cytokine, we used nude mice xenografted with three A431 tumors, parental and cells transfected with alpha subunit of the receptor for either IL-2 or IL-15. When examined using radioiodinated IL-2 or IL-15, each cytokine accumulated on the target cells, expressing its respective alpha chain, suggesting that the expression of the alpha chains is sufficient to define specific biodistribution of IL-2 and IL-15, although these cytokines share the beta and yc molecules of their receptors. IL-15 displayed better target-specific accumulation and more rapid clearance from the circulation than did IL-2, and thus it can be considered to be a novel and unique therapeutic agent.

Two-step targeting of experimental lung metastases with biotinylated antibody and radiolabeled streptavidin.

Two-step monoclonal antibody tumor targeting using an avidin-biotin system has unique characteristics because of the high-affinity binding (10(15) M-1) and the lower molecular weight ligands (avidin, streptavidin, or biotin) used as carriers of radioisotopes, toxins, or drugs. The distribution of radiolabeled streptavidin in a two-step targeting strategy was investigated in lung metastases of line 10 carcinoma in guinea pigs. The microdistribution of administered D3 monoclonal antibody and 125I-labeled streptavidin in metastatic nodules was examined by immunohistochemistry and autoradiography, and the uptake was quantitated. With monoclonal antibody pretargeting, streptavidin was found mainly at the periphery of metastatic nodules 1.5 h after injection; it had penetrated deeper at 4 h and was approaching homogeneity in many of the tumor nodules at 24 h. These results indicate that streptavidin can penetrate into metastatic nodules more rapidly than can the antibody. The concentration of streptavidin in metastatic nodules 4 h after injection was 5.6 times higher for the pretargeted group than for the nonpretargeted group, and the pretargeting index was 4.7. Although the absolute uptake of streptavidin had decreased between 4 and 24 h, the metastasis:blood ratio had increased from 1.2 to 2.4. When compared with the animals injected with 125I-labeled D3 antibody alone, the pretargeted group achieved higher tumor:blood and tumor:lung ratios and a higher localization index at early times after injection of the radiolabeled species.

Biodistribution of 125I-labeled des(1-3) insulin-like growth factor I in tumor-bearing nude mice and its in vitro catabolism.

Insulin-like growth factor I (IGF-I) is a potent mitogen for many tumor cell lines, and IGF-I receptors are overexpressed in many tumors. Specific IGF-binding proteins (IGFBPs) modulate the interaction of IGF and its receptors. Consequently, radiolabeled IGF-I has been considered for tumor imaging. In the present study, we investigated the biodistribution of 125I-labeled des(1-3)IGF-I, a truncated analogue of IGF-I, in tumor-bearing nude mice. Additional studies included its catabolism by tumor cells in vitro and its binding to serum IGFBPs in vivo in nude mice. We also compared groups that were and were not injected with unlabeled peptide analogue. Our data showed that 125I-labeled des(1-3)IGF-I catabolized very fast, with a rapid appearance of nonprecipitable iodine, when incubated at 37 degrees C, but it was not catabolized at 4 degrees C incubation. 125I-labeled des(1-3)IGF-I was bound to serum-binding proteins, mainly in a complex with a molecular weight of M(r) 150,000. The uptake of radioactivity in normal tissues decreased quickly with time, particularly in the kidneys. In mice receiving higher doses of des(1-3)IGF-I, the radioactivity in all normal tissues was lower than in the mice with no carrier-added des(1-3)IGF-I, except in the stomach and spleen. These data suggest that 125I-labeled des(1-3)IGF-I is rapidly internalized after binding to the IGF receptor and is rapidly catabolized with release of breakdown products. Lower specific activity of 125I-labeled des(1-3)IGF-I resulted in altered biodistribution, including faster blood clearance and higher tumor uptake, by decreasing the formation of complexes with IGFBPs.

Biodistribution of 18F- and 125I-labeled anti-Tac disulfide-stabilized Fv fragments in nude mice with interleukin 2 alpha receptor-positive tumor xenografts.

We evaluated the biodistribution, pharmacokinetics, and generation of catabolites of an 18F- and 125I-labeled anti-Tac disulfide-stabilized Fv fragment (dsFv) in tumor-bearing nude mice. This dsFv is genetically engineered from a murine monoclonal antibody that recognizes the alpha subunit of the interleukin 2 (IL-2 alpha) receptor. Labeling was performed with 18F using N-succinimidyl 4-([18F]fluoromethyl)benzoate or with 125I using the Iodo-Gen method. The immunoreactivities of the radiolabeled anti-Tac dsFv were > 82%. The biodistribution was evaluated (at 15, 45, and 90 min and 6 h) in athymic nude mice (approximately five/group) bearing s.c. tumor xenografts. Cell line A431 served as the IL-2 receptor-negative control tumor, whereas the ATAC4 cell line served as our IL-2 receptor-positive tumor. Animals received injections of 18F-labeled anti-Tac dsFv (0.7-1.4 megabecquerels/1.5-3 micrograms) and 125I-labeled anti-Tac dsFv (0.1-0.4 megabecquerels/0.9-1 microgram). Blood clearance for both preparations was rapid, with < 10% retained in the blood by 15 min. Maximum accumulation in ATAC4 tumors occurred between 45 and 90 min and peaked at a mean of 4.2% injected dose/g (18F) and 5.6% of injected dose/g (125I). At 6 h, the ATAC4 tumors contained 11 times more 18F and 3 times more 125I than did the A431 tumors. The ATAC4 tumor:blood ratios for the 18F and 125I were > 12:1 and > 1.4:1 at 6 h, respectively, whereas the ratios for the antigen-negative A431 tumor were less than 1. The kidneys were the major route of elimination. Catabolites appeared quickly and were identified as [125I]iodide and predominantly N-epsilon-[18F]4-fluoromethylbenzoyl(alpha-N-acetyl) lysine. This is the first study to evaluate the biodistribution of an 18F-labeled Fv fragment in vitro and in vivo. In vivo, the dsFv was taken up rapidly by the kidneys, producing lysine-containing catabolites for 18F-labeled dsFv and [125I]iodide for 125I-labeled dsFv.

L-lysine effectively blocks renal uptake of 125I- or 99mTc-labeled anti-Tac disulfide-stabilized Fv fragment.

In this study, we investigated the ability of L-lysine to block renal uptake of 125I- or 99mTc- labeled Fv fragments. Anti-Tac disulfide-stabilized Fv fragment (dsFv) was derived from a murine monoclonal antibody that recognizes the alpha subunit of the interleukin-2 receptor (IL-2R alpha). The 125I- or 99mTc-labeled dsFv was injected i.v. into non-tumor-bearing nude mice or into nude mice bearing SP2/Tac (IL-2R alpha positive) and SP2/0 (IL-2R alpha negative) tumor. We then evaluated the pharmacokinetics of L-[3H]lysine and the effect of L-lysine dose, timing of administration, and route of delivery on catabolism and biodistribution of i.v. dsFv. Peak renal uptake of i.v. or i.p. injected L-[3H]lysine occurred within 5 and 15 min, respectively. The kidney uptake of L-lysine exhibited a dose-response effect. When L-lysine was coinfused or injected shortly before dsFv, renal uptake of dsFv was blocked to < 5% of the control, but longer intervals were less effective. Aminosyn II and Travasol 10% (parenteral amino acid solutions) also blocked renal uptake of radiolabeled dsFv. Administration of L-lysine did not alter the blood kinetics and slightly increased the tumor uptake of dsFv, but it did prevent catabolism in the kidney and resulted in lower amounts of catabolites in the serum and urine. In conclusion, we have shown that a blocking dose of lysine, injected with or immediately before the injection of radiolabeled dsFv, is most effective in blocking the renal uptake of dsFv. This is consistent with the rapid uptake of L-[3H]lysine by the kidney and is further substantiated by the relative ineffectiveness of lysine injected immediately after the radiolabeled dsFv injection.

Preclinical evaluation of 111In-labeled B3 monoclonal antibody: biodistribution and imaging studies in nude mice bearing human epidermoid carcinoma xenografts.

Biodistribution and imaging characteristics of monoclonal antibody B3 were evaluated in nude mice bearing A431 human epidermoid carcinoma xenografts. B3 is a murine IgG1k, recently isolated, reacting with a carbohydrate epitope abundantly and uniformly expressed by most carcinomas. B3 was conjugated to a new backbone-substituted derivative of diethylenetriaminepentaacetic acid, 2-(p-isothiocyanato benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid, and labeled with 111In. Tumor-bearing mice were given i.v. injections of approximately 5 microCi of either 111In-B3 or 111In-MOPC-21, an isotype-matched control, and sacrificed in groups of five at 6 h and daily up to 168 h. Imaging was performed at 24, 72, and 144 h. Significant differences were observed in tumor uptake at all time points with peak values at 48 h (25 +/- 5.2% versus 6.3 +/- 0.4% of the injected dose/g tissue) (mean +/- SD) for 111In-B3 and 111In-MOPC-21, respectively (P < 0.001). All tumor to organ ratios increased with time for 111In-B3. In particular, tumor:liver ratios rose from 3.2 +/- 0.6 at 24 h to 6.3 +/- 1.2 at 168 h. Imaging results showed selective and progressive accumulation of 111In-B3 at the tumor site, whereas 111In-MOPC-21 did not show specific localization. In summary, 111In-labeled B3 demonstrated good and specific tumor targeting, which warrants its future clinical evaluation.

The pharmacokinetic characteristics of glycolated humanized anti-Tac Fabs are determined by their isoelectric points.

To evaluate a method for preventing the nephrotoxicity caused by the high renal accumulation of radiolabeled or toxin-conjugated small immunoproteins used for cancer therapy, we conjugated humanized anti-Tac Fab fragments with various numbers of glycolate molecules [glycolated Fab fragments (glyco-Fabs)] and separated the conjugates by means of ion-exchange columns into three fractions, depending on their isoelectric points (pIs). We evaluated the biodistribution, pharmacokinetics, and catabolism in normal nude mice of nonglycolated Fab (pI > or = 9.3) and three different preparations of glyco-Fab, including strongly anionic glyco-Fab (sa-glyco-Fab: pI < or = 4.5), mildly anionic glyco-Fab (pI = 4.5-7), and mildly cationic glyco-Fab (pI = 7-9.3). In addition, the biodistributions of 125I-labeled sa-glyco-Fab and 131I-labeled nonglycolated Fab were evaluated in normal nude mice coinjected with 50 mg of L-lysine and/or 1 microg of furosemide and in a control group without coinjection. We then evaluated the serial biodistribution of 125I-labeled sa-glyco-Fab (4 microCi/1 microg) and 131I-labeled nonglycolated Fab (5 microCi/1 microg) in Tac antigen-positive (ATAC4) and -negative (A431) tumor-bearing nude mice with s.c. tumor xenografts derived from Tac antigen-positive ATAC4 cells and receptor-negative A431 cells. These animals were coinjected with 30 mg of lysine i.v. and 30 mg of lysine i.p. 15 min after the radiolabeled Fab injection. To evaluate the biodistribution data and study scintigraphic imaging, we performed serial scintigraphy on normal and tumor-bearing mice with all four 131I-labeled preparations. 125I-labeled mildly cationic glyco-Fab and 131I-labeled nonglycolated Fab had similar distributions, except in the kidney. However, both 125I-labeled anionic glyco-Fab preparations showed significantly different distributions from both cationic Fabs in the blood, liver, lung, and spleen. Renal accumulation of all four radiolabeled Fab preparations increased significantly as the pI increased (P < 0.01). In addition, the intact fraction of Fab excreted into urine increased as pI decreased. Therefore, the glomerular filtration depended on whether the charge on the Fab was positive or negative. The proportion of Fab reabsorbed by the proximal tubules increased as pI increased. 125I-labeled sa-glyco-Fab and 125I-labeled mildly anionic glyco-Fab showed a similar distribution in the blood and all organs except the kidney. Lysine led to an additional blocking effect on proximal tubular uptake of both sa-glyco-Fab and nonglycolated Fab. Addition of furosemide yielded only a small effect when used with lysine. With lysine, the sa-glyco-Fab:nonglycolated Fab estimated integral radioactivity ratios were 4.7 and 0.7 in the ATAC4 tumor and in the kidney, respectively. The use of anionic fragments, which may be used in conjunction with lysine, represents a promising approach that may help decrease the renal toxicity of other small fragments, the molecular weights of which range from Mr 40,000 to 70,000, and, thereby, allow higher doses of radiation to the tumor.

Improved biodistribution of 125I-labeled anti-Tac disulfide-stabilized Fv fragment by blocking its binding to the alpha subunit of the interleukin 2 receptor in the circulation with preinjected humanized anti-Tac IgG.

Animal studies using radiolabeled anti-Tac disulfide-stabilized Fv (dsFv) monoclonal antibody have shown formation of complexes in serum with the soluble alpha subunit of the interleukin 2 receptor alpha (sIL-2R alpha). In this study, we improved the targeting of 125I-labeled anti-Tac dsFv to receptor-positive tumors in the presence of circulating receptor by preinjecting unlabeled humanized anti-Tac IgG antibody (HuTac IgG). We used mice bearing SP2/Tac tumor xenografts that express the IL-2R alpha. A positive correlation was seen between tumor size and the concentration of circulating receptor. Tumor-bearing mice were injected with 125I-labeled anti-Tac dsFv (400 ng), either alone or 15 min after injection of HuTac IgG. The 125I-labeled anti-Tac dsFv formed high molecular weight complexes with the sIL-2R alpha. The fraction of the dsFv present in the complexes increased as tumor size increased (greater sIL-2R alpha levels). The fractions of dsFv in the complexes were 9.9- to 11.6-fold higher when sIL-2R alpha was not blocked with preinjected HuTac IgG. The administration of a 12-fold molar excess of HuTac IgG over sIL-2R alpha resulted in >80% of the 125I activity present as the dsFv rather than in the complexes. Furthermore, the biodistribution of 125I-labeled anti-Tac dsFv was improved by blocking its binding to sIL-2R alpha by preinjecting HuTac IgG. Specifically, in the preinjected group, at 15 min postinjection, the 125I-labeled anti-Tac dsFv levels in tumor increased to 10.8% compared to 5.6% injected dose per gram in the non-preinjected group. In summary, our studies showed that preinjection of HuTac IgG can block the formation of complexes of circulating sIL-2R alpha and 125I-labeled anti-Tac dsFv. This blockade is associated with faster blood clearance, higher tumor uptake, and greater tumor:nontumor ratios of the radiolabeled antibody fragment.

Phase I study of the pharmacokinetics of a radioimmunoconjugate, 90Y-T101, in patients with CD5-expressing leukemia and lymphoma.

Ten patients with advanced or refractory CD5-expressing hematologic neoplasms [two with chronic lymphocytic leukemia and eight with cutaneous T-cell lymphoma (CTCL)] were treated in a Phase I study with the radioimmunoconjugate 90Y-T101, which targets CD5+ lymphocytes. Prior imaging studies using 111In-T101 demonstrated uptake in involved lymph nodes and skin in patients with CTCL, and Phase I studies with unmodified T101 demonstrated transient responses. In this study, patients were treated with 5 or 10 mCi of 90Y chelated to T101 via isothiocyanatobenzyl diethylenetriamine pentaacetic acid, along with tracer doses of 111In-T101 for imaging. The biodistribution of the radioimmunoconjugate was determined by measuring 90Y and 111In blood clearance, urine excretion, and accumulation in bone marrow and in involved skin lesions. The intravascular pharmacokinetics of 90Y were predicted by 111In-labeled T101. The greatest differences in biodistribution between 111In and 90Y were in the higher bone accumulation of 90Y and its lower urinary excretion. Imaging studies demonstrated targeting of skin lesions and involved lymph nodes in CTCL patients. The predominant toxicity was bone marrow suppression. Rapid antigenic modulation of CD5 on circulating T and B cells was observed. Recovery of T-cell populations occurred within 2-3 weeks; however, suppression of B-cell populations persisted after 5+ weeks. All CTCL patients developed human antimouse antibody after one cycle and thus were not retreated; one patient with chronic lymphocytic leukemia received a second cycle of therapy. Partial responses occurred in five patients, two with chronic lymphocytic leukemia and three with CTCL. The median response duration was 23 weeks. One CTCL patient who subsequently received electron beam irradiation to a residual lesion is disease-free after 6 years.

Prevention of radiolysis of monoclonal antibody during labeling.

UNLABELLED: Monoclonal antibody may undergo loss of immunoreactivity due to radiation damage when labeled with large amounts of 131I or 90Y for therapy. Our aim was to develop a method to protect an antibody during the labeling procedure. METHODS: As a model we used T101, a murine monoclonal antibody directed against CD5 antigen. Iodine-125-T101 (100 micrograms, 1 ml) was mixed with 90Y-DTPA (0.64 MBq to 165.9 MBq) for 24 hr in order to deliver doses of 5 Gy to 1280 Gy to the solution. In separate experiments, 125I-T101 solutions were irradiated with 60Co external beam delivering radiation doses of 40 Gy to 1280 Gy. The effect of radiation on T101 immunoreactivity was tested by using the CCRF-CEM cell line, and the bound T101 radioactivity was determined. In a final experiment, we directly labeled a DTPA conjugated T101 using 561 MBq of 90Y under conditions delivering approximately 640 Gy to the solution. Previously used radioprotectants including human serum albumin, cysteamine and glycerol were evaluated. We focused on ascorbic acid because it is an FDA approved drug that does not interfere with the radiolabeling process. RESULTS: The immunoreactivity of 125I-T101 was approximately 83%, but at 640 Gy the immunoreactivity decrease to 7%. In contrast, in the presence of radioprotectants this decrease could be abrogated. External irradiation also showed a dose dependent decrease in immunoreactivity to as low as 0.3% at 1280 Gy. Adding ascorbic acid (5.5 mg/ml) to the solutions prior to the irradiation largely abrogated this decrease. The immunoreactivity of T101 labeled with 90Y without protectant showed 46% immunoreactivity whereas, in presence of ascorbic acid (11 mg/ml) full retention of immunoreactivity was observed. CONCLUSION: Various radioprotectants can successfully prevent the loss of immunoreactivity or breakdown as a result of radiolysis. Ascorbic acid is an effective radioprotectant that can be used to prevent loss of antibody immunoreactivity during the labeling process.

Optimization of the DTPA mixed-anhydride reaction with antibodies at low concentration.

Diethylenetriaminepentaacetic acid (DTPA) was conjugated with antibody to human serum albumin (Ab) at low concentration (300 micrograms/ml, 2.0 microM) via the DTPA carboxycarbonyl mixed-anhydride method. To study parameters determining the balance between the degree of conjugation and the antibody-binding activity of Ab, a known concentration of the anhydride prepared at isobutylchloroformate (IBC)-to-DTPA ratios of 1, 2.1, or 4.2 was reacted with Ab. The percentage yields of the anhydride were determined by spectrophotometric and gravimetric titration. By the former method the percentage yields, based on DTPA concentration, were 18, 24, and 220, respectively, when the IBC-to-DTPA ratios were 1, 2.1, and 4.2. The corresponding percentage yields were 17, 39, and 262 when determined by the latter method. When the anhydride was prepared at an IBC-to-DTPA ratio of 2.1, an optimum conjugation giving three indium atoms per Ab was obtained, with 64% retention of antibody-binding activity. For an IBC-to-DTPA ratio of 1, the antibody retained almost 100% binding activity but the number of indium atoms incorporated (0.2) was too small. For an IBC-to-DTPA ratio of 4.2, up to 22 indium atoms were incorporated but antibody-binding activity was completely destroyed.

Relative reactivity of DTPA, immunoreactive antibody-DTPA conjugates, and nonimmunoreactive antibody-DTPA conjugates toward indium-111.

Anti-human serum albumin antibody (Ab) was reacted with cyclic DTPA dianhydride (cDTPAA) at various cDTPAA/Ab molar ratios between 1 and 40. Using a carrier In titration method for DTPA and DTPA-antibody conjugate (Ab-DTPA), we determined that the above reactions produced between 0.1 and 11 DTPA molecules per either immunoreactive antibody (sAb) or nonimmunoreactive antibody (nAb). The percentage of sAb remaining after the above reactions was between 88 and 62%. The reaction of no-carrier-added 111In with the reaction mixture from cDTPAA/Ab molar ratios of 1 to 40 gave radiochemical yields less than or equal to 25% for the respective Ab-DTPA. The rest of the 111In activity was associated with free DTPA. Our results indicate that Ab-DTPA containing greater than 1 DTPA molecule per Ab is more reactive than that containing less than 1 DTPA but is about as reactive as free DTPA. This allows us to label in the presence of free DTPA and consequently prevent colloid formation. The percentage of 111In activity incorporated into sAb-DTPA from the reactions at these molar ratios was similar to that found from the uv analysis. This indicates that the reactivity of sAb-DTPA and nAB-DTPA from the same conjugation reaction is similar. As a result, we were able to conjugate about one DTPA molecule to the Ab without causing deactivation of the Ab and label it with 111In in the presence of excess DTPA. We obtained a specific activity of 6 muCi 111In per microgram of Ab using research grade 111In without further purification.

Immunoreactivity affects the biodistribution and tumor targeting of radiolabeled anti-P97 Fab fragment.

Hydroxylapatite high performance liquid chromatography was used to prepare two fractions from 125I- Fab 96.5. One fraction (peak 1) had relatively low immunoreactivity (25-38%) and the second fraction (peak 2) had high immunoreactivity (70-81%). Scatchard analysis showed similar affinity constants for the two preparations (2.9 x 10(9) M-1 for peak 1; 3.4 x 10(9) M-1 for peak 2). In biodistribution and imaging studies in athymic mice with human melanoma (FEMX-II) xenografts the high immunoreactivity preparation rapidly cleared from the blood and nontumor organs while retention of radioactivity in the tumor was prolonged. The low immunoreactivity preparation, had slower blood and nontumor organ clearance but faster tumor clearance than the high immunoreactivity fraction. Thus, in these studies highly immunoreactive antibody gave higher tumor to nontumor ratios and enhanced the target to nontarget image contrast.

Evaluation of the serum stability and in vivo biodistribution of CHX-DTPA and other ligands for yttrium labeling of monoclonal antibodies.

UNLABELLED: Serum stability and in vivo biodistribution of both A and B isomers of the 2-(p-isothiocyanatobenzyl) (p-SCN-Bz)-cyclohexyldiethylenetriaminepentaacetic acid ligand (CHX-DTPA), a recently developed backbone-substituted derivative of DTPA, were evaluated and compared to those of 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M-DTPA) and 2-(p-SCN-Bz)-1,4,7,10-tetraazacyclododecane tetra-acetic acid (2B-DOTA). METHODS: Stability of 88Y-labeled ligands (0.1 microM) was evaluated in serum for up to 17 days. For biodistribution, ligands were conjugated to monoclonal antibody (Mab) B3, a murine IgG1k, and labeled with 88Y at 0.1-0.3 mCi/mg. Nontumor-bearing nude mice were injected intravenously with 1-2 microCi/4-10 micrograms of 88Y-labeled B3-conjugates and killed at 6 hr and daily up to 168 hr postinjection. Indium-111-(1B4M)-B3 was co-injected in all mice as internal control. RESULTS: Serum stability of 88Y-DOTA failed to show any significant release of activity, whereas pseudo-first-order dissociation rate constants of 3.97 x 10(-3), 2.54 x 10(-3) and 1.46 x 10(-2) (day-1) were calculated for 88Y-1B4M, 88Y-CHX-A and 88Y-CHX-B, respectively. Accordingly, cortical bone uptake of 88Y was significantly higher for all DTPA-derivative chelates than for DOTA. CONCLUSIONS: While none of the DTPA-derivative chelates could challenge DOTA in its ability to hold the radioytrium, significant differences were observed in the kinetic inertness of the A and B isomers of CHX, indicating that the CHX-B ligand is not as suitable for 90Y-labeling of Mabs.

Pharmacokinetics of 111In- and 125I-labeled antiTac single-chain Fv recombinant immunotoxin.

UNLABELLED: The use of immunotoxins for cancer therapy is an attractive strategy that exploits the targeting specificity of monoclonal antibodies and their fragments as well as the exquisite toxicity of the toxins. However, few studies of immunotoxins have evaluated their biodistribution in vivo. Previous studies have used 125I for tracing immunotoxin biodistribution in mice. Because the immunotoxin works only when it is internalized and because of known problems with quick dehalogenation after internalization of antibodies, we decided to use 111In, which has greater intracellular retention than iodine. METHODS: To trace the in vivo pharmacokinetics of the immunotoxin in mice, we labeled the antiTac(Fv)-PE38 with 111ln and compared it with 125I-labeled antiTac(Fv)-PE38. We successfully labeled antiTac(Fv)-PE38 with 111In at up to 2.96 GBq/mg. A 3- to 4-fold decrease in cytotoxicity was observed for both radiolabeled preparations. We evaluated the internalization of 111In- and 125I-labeled antiTac(Fv)PE38 into ATAC4 cells (Tac-positive) as well as their biodistribution and pharmacokinetics in vivo in mice. In addition, some mice receiving these reagents were co-infused with 30 mg L-lysine to inhibit renal accumulation. RESULTS: Significantly more 111In- than 125I-labeled antiTac(Fv)-PE38 accumulated in the ATAC4 cells (20% versus 5% of initial surface-bound radioactivity; P < 0.001). In vivo, significantly more 111In- than 125I-labeled antiTac(Fv)-PE38 accumulated in the kidney (119 versus 31 percentage injected dose per gram [%ID/g]; P < 0.001). The tumor accumulation of 111In-labeled antiTac(Fv)-PE38 at 96 h was 13-fold greater than that of 125I-labeled antiTac(Fv)-PE38 (1.4 versus 0.1 %ID/g; P < 0.001). No antiTac(Fv)-PE38 was excreted into the urine in its intact form unless lysine was co-infused. Co-injected lysine reduced the renal accumulation of 111In-labeled antiTac(Fv)-PE38 by 62%. CONCLUSION: We evaluated the biodistribution, pharmacokinetics, and catabolism of 111In-labeled antiTac(Fv)-PE38 and found that it differed from 125I-labeled antiTac(Fv)PE38. These studies suggest that 111In-labeled antiTac(Fv)-PE38 can be used to trace the fate of antiTac(Fv)-PE38 in humans.

Effects of insulinlike growth factor binding proteins on insulinlike growth factor-I biodistribution in tumor-bearing nude mice.

UNLABELLED: This study evaluated the biodistribution and tumor targeting ability of radiolabeled insulinlike growth factor (IGF)-I. Because IGF binding proteins (IGFBPs) play a critical role in modulating IGF activity, the binding properties of 125I-labeled IGF-I to IGFBPs were investigated in vitro and in vivo. Because a large amount of the IGF-I was catabolized in vivo, we also studied the catabolism of IGF-I by tumor cells in vitro. METHODS: 125I-labeled-IGF-I was prepared using the chloramine T method. The biodistribution of 125I-labeled-IGF-I in tumor-bearing nude mice was compared between groups injected with 125I-labeled IGF-I alone or coinjected with unlabeled peptide. In vitro and in vivo chromatography studies were performed to evaluate the binding profile to IGFBPs and the degree of catabolites in serum as well as urine. RESULTS: Data indicated that the binding of radiolabeled IGF-I to IGFBPs in vitro was dose dependent. However, there was a difference in complex formation between the serum and the heparinized plasma. In heparinized plasma, the radioactivity shifted from a 30- to 50-kDa complex to a 150-kDa complex and to a free ligand, because the binding of heparin with IGFBPs decreased its affinity for IGF-I. In plasma prepared with acid citrate dextrose a binding pattern identical to that of serum was observed. Moreover, there was a binding difference between mouse and rat. The 125I-labeled IGF-I catabolized very quickly when incubated at 37 degrees C but not at all at 4 degrees C. In tumor-bearing nude mice, the uptake of radioactivity in normal tissues decreased quickly, particularly in the kidneys. In mice coinjected with unlabeled carrier, the radioactivity in most normal tissues was lower and the tumor uptake higher than in the mice without carrier. CONCLUSION: These data confirm that 125I-labeled IGF-I is avidly bound to IGFBPs, both in vitro and in vivo. By partially saturating this binding with unlabeled peptides, a favorable biodistribution was achieved, including faster clearance from normal tissue and higher tumor uptake, which resulted in better tumor-to-nontumor ratios. Nevertheless, the rapid catabolism and release of the radiolabel from tumor tissue result in a suboptimal targeting agent.