Radioimmunodetection of human colon cancer in nude mice by a new monoclonal antibody A7 against human colorectal cancer

The in vivo localization of a monoclonal antibody A7 against a human colorectal cancer was studied in nude mice bearing human solid carcinomas, to evaluate potential applications of this antibody for radioimmunodetection of cancer. The tissue distribution of¹²⁵I-labeled A7 MoAb at 3 days after i.v. injection into mice bearing five different kinds of human solid tumors revealed a high uptake ratio by colon cancer, mammary cancer, and glioblastoma. In contrast, the uptake ratio by murine colorectal cancer (Colon-38) was extremely low. In immunoscintigraphic studies, HCT-15, one of the human colon cancer, was clearly visualized with ¹¹¹In-DTPA-A7 MoAb. Glioblastoma was also imaged with the same extent. These results suggest that A7 MoAb would be applicable to the in vivo radioimmunodetection of colon- and mammary-cancer, and of glioblastoma.     

Localization of an anti-CEA monoclonal antibody in colo-rectal carcinoma xenografts

Summary A mouse monoclonal anti-CEA antibody (11.285.14) has been examined for tumour localization potential by assessing its distribution in immunodeprived mice with xenografts of human colon carcinoma cell lines HCT-8, HRT-18, HT-29, and LS174T and a xenograft (HRVB) established from a primary rectal carcinoma. With four carcinomas (HCT-8, HT-29, LS174T, and HRVB) preferential tumour localization of ¹²⁵I anti-CEA was seen. Compared with ¹³¹I normal IgG₁ localization indices of up to 4.4:1 were achieved. Up to 10% of the injected dose of ¹²⁵I anti-CEA was present/g of tumour tissue and with the largest xenografts examined (3–4 g) up to 40% of the total body reactivity was localized in tumour tissue. The tumour localization of ¹³¹I labelled antibody was visualized by external gamma camera imaging. Overall antibody localization correlated with the CEA content of the xenografts and the fourth colon carcinoma xenograft (HRT-18) and an osteogenic sarcoma xenograft (791T), both with very low CEA levels, showed no localization of the monoclonal antibody.     

Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

In this report, we have employed macroautoradiography to compare the tumor targeting of ¹²⁵I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to ¹²⁵I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab′)₂ and Fab′ fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3–7 days post-treatment. Treatment with F(ab′)₂ and Fab′ fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background ¹²⁵I-radioactivity which was well advanced by 6–24 h and complete by 24–48 h after injection. Localization of ¹²⁵I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab′)₂ and Fab′ fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments.     

Antibody-directed enzyme prodrug therapy (ADEPT)

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with¹²⁵I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab′)₂ fragment showed localization in these xenografts. The AB57-F(ab′)₂ fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with¹²⁵iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon). A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab′)₂-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress.     

A comparison of 131I-labeled antibodies, 2-deoxy-2-[18F]fluoro-d-glucose and 68Ga-EDTA in the imaging of human tumor xenografts in nude mice

Three different biological properties—glucose metabolism, gallium imaging and antigen-antibody interaction—have been targeted to image human tumor xenografts implanted in nude mice. Seventy-two experiments were performed in 25 nude mice. Two types of human tumors were used: colorectal carcinoma SW 1116 and melanoma WM 9. Immunoscintigraphic studies produced the highest tumor sampling and confirmed earlier findings that F(ab′)₂ fragments generate better tumor images than whole antibodies.     

A potential complication in the use of monoclonal antibodies: inhibition of apoB-mediated receptor binding by an anti-apoE antibody

Monoclonal antibody (Mab) 1D7 is specific for human apolipoprotein (apo) E and blocks binding of lipid-associated apoE to the low density lipoprotein (LDL) receptor. We report here that 1D7 can also block the binding of apoE-free LDL to the LDL receptor. The inhibition of LDL-receptor binding is not due to immunological cross-reactivity between the anti-apoE Mab and apoB, the ligand responsible for the interaction of LDL with the LDL receptor: 1) Mab 1D7 did not react with apoE-depleted LDL; 2) the LDL receptor binding inhibitory activity of 1D7 immunoglobulin G (IgG) preparations could be dissociated from the anti-apoE activity; 3) the inhibition was maintained when the fibroblasts were preincubated with the 1D7 IgG, extensively washed, and only then exposed to 125I-labeled LDL. Rather, it appears that 1D7 recognizes mouse apoE, that mouse apoE-1D7 immune complexes contaminate 1D7 IgG preparations and that the contaminating mouse apoE can compete with 125I-labeled LDL for the LDL receptor. We have demonstrated mouse apoE in IgG preparations of 1D7 but not in those of other anti-apoE Mabs that do not influence LDL-receptor binding. Precipitation of 1D7 IgG with NH4SO4 eliminates both apoE and the capacity of 1D7 to block LDL receptor binding. Finally, mouse apoE can be isolated by immunoaffinity chromatography of mouse serum on immobilized 1D7 Mab. As this is probably not a unique case, the observation has important implications for the use of Mabs as structural probes.     

Development of a new thiol-reactive prosthetic group for site-specific labeling of biomolecules with radioactive iodine

In this report, we describe the radiosynthesis of a new thiol-targeting prosthetic group for efficient radioactive iodine labeling of biomolecules. Radioiodination using the precursor 3 was performed to obtain ¹²⁵I-labeled tetrazole 4b with high radiochemical yield (73%) and radiochemical purity. Using the radiolabeled 4b, a single free cysteine containing peptide and human serum albumin were labeled with ¹²⁵I in modest-to-good radiochemical yields (65–99%) under mildly reactive conditions. A biodistribution study of [¹²⁵I]7 in normal ICR mice exhibited lower thyroid uptake values than those of ¹²⁵I-labeled human serum albumin prepared via a traditional radiolabeling method. Thus, [¹²⁵I]7 could be employed as an effective radiotracer for molecular imaging and biodistribution studies. The results clearly demonstrate that 4b has the potential to be effectively implemented as a prosthetic group in the preparation of radiolabeled biomolecules.     

Pharmacokinetics of a radioiodinated monoclonal antibody F(ab′)2 fragment in a xenograft model with circulating antigen

The pharmacokinetics of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment were investigated in athymic mice bearing OVCAR-3 ovarian carcinoma xenografts, a model in which the CA 125 antigen is present in serum. Nine antibody doses between 0.1 and 650 μg were studied. Optimal tumor to normal tissue ratios were obtained at 100–200 μg of F(ab′)₂. At most antibody doses, the pre-injection level of circulating CA 125 appeared to influence the localization of ¹³¹I activity in tumor, liver and spleen.     

Comparative Binding of 125I-and 99mTc-Labeled Native and Glycated Low-Density Lipoprotein to Human Microvascular Endothelial Cells-Potential for Atherosclerosis Imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with ¹²⁵I or ^99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-γ receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the K_D s using either ¹²⁵I-or ^99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that ^99mTc–g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for ^99mTc-nLDL and all the ¹²⁵I-labeled LDL forms. Binding studies using both ¹²⁵I-and ^99mTc-nLDL indicated a weak binding affinity (K_D 10^{? 7}mol/L) to human microvascular endothelial cells. The binding affinity of ¹²⁵I-g/goLDL to these cells was significantly higher (K_D 10^{? 9}mol/L) and could be increased further by preactivation of the endothelial cells using TNFα. Incubation with ^99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with ^99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with ¹²⁵I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only ¹²⁵I-labeled goLDL should be used, whereas nLDL may be applied as ¹²⁵I-or ^99mTc-labeled ligand.     

In vivo Molecular Imaging and Radionuclide (131I) Therapy of Human Nasopharyngeal Carcinoma Cells Transfected with a Lentivirus Expressing Sodium Iodide Symporter

Introduction

Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using ¹²⁵I and ¹³¹I therapy in vivo.

Methods

We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; ¹²⁵I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with ¹³¹I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors.

Results

qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more ¹²⁵I than CNE-2Z cells and xenografts. In vitro, ¹³¹I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, ¹³¹I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of ¹³¹I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67.

Conclusion

Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.     

Detection of Rapalog-Mediated Therapeutic Response in Renal Cancer Xenografts Using 64Cu-bevacizumab ImmunoPET

The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of ⁶⁴Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. ⁶⁴Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated ⁶⁴Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, ⁶⁴Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.     

Clinical value of (18)F-FDG PET/CT in postoperative monitoring for patients with colorectal carcinoma

Objective: To evaluate the clinical value of ¹⁸F-FDG PET/CT in postoperative monitoring for patients with colorectal carcinoma. Methods: 66 postoperative patients with colorectal carcinoma underwent whole-body FDG PET/CT. The final histopathological and formal clinical follow-up findings were used as gold standard to determine the sensitivity and specificity of FDG PET/CT and enhanced CT of the same periods. Results: The sensitivity and specificity of FDG PET/CT in detecting recurrence are 96.30%, 94.87% (while enhanced CT are 70.37% and 87.18% respectively). The sensitivity and specificity in detecting metastasis are 95.35%, 82.61% (enhanced CT are 61.90%, 75.00%). SUVmax was significantly higher in malignant lesions [range 4.16–22.00, mean ± standard deviation (x ± s) 8.06 ± 4.30] than in benign ones (range1.18–6.25, x ± s 2.82 ± 1.02). Conclusion: At present, whole-body ¹⁸F-FDG PET/CT is an advanced diagnostic imaging technique in detecting loco-regional recurrence and metastasis in postoperative patients with colorectal carcinoma for its higher sensitivity and specificity.     

Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts

Summary We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11–13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an¹²⁵I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4, using a high or a low protein dose (500 µg or 5 µg). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as thecoefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-µg protein doses had substantially lower CV values, 0.327±0.027, than did tumors from animals given 5-µg protein doses, 0.458±0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.     

Synthesis and biological evaluation of novel radioiodinated imidazopyridine derivatives for amyloid-β imaging in Alzheimer’s disease

Non-invasive detection for amyloid-β peptide (Aβ) deposition has important significance for the early diagnosis and medical intervention for Alzheimer’s disease (AD). In this study, we developed a series of imidazopyridine derivatives as potential imaging agents for single-photon emission computed tomography (SPECT). Two of them, compounds DRK092 and DRM106, showed higher affinity for synthetic human Aβ 1–40 fibrils than did the well-known amyloid-imaging agent IMPY. A metabolite analysis revealed brain-permeable radioactive metabolites of ¹²⁵I-labeled DRK092 and IMPY; no radioactive metabolites from ¹²⁵I-labeled DRM106 ([¹²⁵I]DRM106) were detected. In addition, in vitro autoradiography clearly demonstrated specific binding of [¹²⁵I]DRM106 in the hippocampal region of AD enriched with Aβ plaques. Thus, our results strongly suggested that compound DRM106 can be used as an imaging agent for SPECT to detect Aβ deposition in AD brain.     

Biodistributions of intact monoclonal antibodies and fragments of BLCA-38, a new prostate cancer directed antibody

Background: Monoclonal antibodies (MAbs) are used for targeting agents to tumours while minimizing normal tissue exposure. Methods: A new anti–prostate cancer MAb, BLCA-38, was radioiodinated (I¹²⁵) and assessed for its ability to target subcutaneous human prostate cancer (DU-145) xenografts after systemic intraperitoneal administration. For comparison, the profile of J591 MAb (now in clinical trial) against LNCaP-LN3 tumours was examined. Biodistribution profiles were obtained at various times, by assessing injected dose/gram (%ID/g) and xenograft to blood (X/B) ratios. Microautoradiography of xenografts was performed. After conjugation with a melittin peptide toxin, the profiles of BLCA-38 and J591 were compared with that of an irrelevant antibody, DS-1. Results: Xenograft localization by ¹²⁵I-labeled BLCA-38 and J591 MAbs to their relevant antigen-positive tumors was comparable, and there was no unusual localization in nontumour tissues. F(ab)₂ and Fab fragments gave improved X/B ratios, but the %ID/g xenograft was decreased and they accumulated in kidneys, bladder and stomach. In contrast, the conjugates of irrelevant antibody showed no tumour targeting. Microautoradiography showed more tumour accumulation of MAbs than F(ab)₂s or Fabs. Conclusions: BLCA-38 can target prostate cancer in vivo almost as effectively as J591. Given that J591 is used clinically, BLCA-38, which targets a different antigen, has potential for radioimmunoscintigraphy and for therapeutic targeting of prostate cancer.     

Imaging Axl expression in pancreatic and prostate cancer xenografts

The receptor tyrosine kinase Axl is overexpressed in and leads to patient morbidity and mortality in a variety of cancers. Axl–Gas6 interactions are critical for tumor growth, angiogenesis and metastasis. The goal of this study was to investigate the feasibility of imaging graded levels of Axl expression in tumors using a radiolabeled antibody. We radiolabeled anti-human Axl (Axl mAb) and control IgG1 antibodies with ¹²⁵I with high specific radioactivity and radiochemical purity, resulting in an immunoreactive fraction suitable for in vivo studies. Radiolabeled antibodies were investigated in severe combined immunodeficient mice harboring subcutaneous CFPAC (Axl^high) and Panc1 (Axl^low) pancreatic cancer xenografts by ex vivo biodistribution and imaging. Based on these results, the specificity of [¹²⁵I]Axl mAb was also validated in mice harboring orthotopic Panc1 or CFPAC tumors and in mice harboring subcutaneous 22Rv1 (Axl^low) or DU145 (Axl^high) prostate tumors by ex vivo biodistribution and imaging studies at 72 h post-injection of the antibody. Both imaging and biodistribution studies demonstrated specific and persistent accumulation of [¹²⁵I]Axl mAb in Axl^high (CFPAC and DU145) expression tumors compared to the Axl^low (Panc1 and 22Rv1) expression tumors. Axl expression in these tumors was further confirmed by immunohistochemical studies. No difference in the uptake of radioactivity was observed between the control [¹²⁵I]IgG1 antibody in the Axl^high and Axl^low expression tumors. These data demonstrate the feasibility of imaging Axl expression in pancreatic and prostate tumor xenografts.     

Target mediated disposition of T84.66, a monoclonal anti-CEA antibody: Application in the detection of colorectal cancer xenografts

Carcinoembryonic antigen (CEeA) is a glycosylated cell surface antigen known to be highly overexpressed in several adenocarcinomas, including colorectal cancer, while demonstrating limited expression in normal tissues. Prior work has shown that the plasma clearance of T84.66, a monoclonal anti-CEA antibody, is enhanced by several-fold in a CEA-expressing xenograft mouse model, suggesting the presence of a target mediated elimination pathway. purpose of this study is to investigate the influence of tumor volume on the plasma clearance of and test the hypothesis that the plasma pharmacokinetics of T84.66 may be used as a sensitive and selective test for the diagnosis of CEA-positive tumors. plasma pharmacokinetics were studied following intravenous (iv) administration of a 1 mg/kg dose in animals without tumor and mice bearing low (20–75 mm³), medium (400–570 mm³), and high volume (800–1,200 mm³) LS174T xenografts.Based on comparison of the disposition of in non-tumor bearing mice and mice bearing low-volume tumors, it was predicted that a single plasma concentration of obtained seven days after dosing, would provide a sensitive and selective means of determining the presence of tumor in mice. A blinded follow-up study was conducted using athymic mice with or without intraperitoneal LS174T xenografts. 1 mg/kg of ¹²⁵I-T84.66 was administered iv, and plasma samples were collected on day 7. Comparison of the observed concentration of ¹²⁵I-T84.66 to the pre-determined threshold value (7.63 nM) enabled identification of tumor bearing mice with a sensitivity of 93.3% and specificity of 100%.Key words: carcinoembryonic antigen (CEA), target mediated disposition (TMD), T84.66, anti-CEA IgG, screening test, sensitivity, specificityin     

Plasma clearance and liver metabolism of native and asialo-human transcortin in the rat

Plasma kinetics and liver metabolism of iodanated human corticosteroid-binding protein have been studied in ovariectomized female rats. ¹²⁵I-labeled human corticosteroid-binding globulin prepared by a modified chloramine T reaction was shown to be physically intact and biologically active. Intravenously injected ¹²⁵I-labeled human corticosteroid-binding globulin was shown to give a complex clearance pattern from the plasma, with half-lives of 7.5 and 51 min. Estrogen injections had no effect on plasma clearance rate. Direct involvement of liver plasma membrane receptors for asialoglycoproteins in ¹²⁵I-labeled human corticosteroid-binding globulin metabolism was demonstrated in vivo and in vitro using asialofetuin as a competitive inhibitor. ¹²⁵I-labeled human asialo-corticosteroid-binding globulin was cleared from the plasma with a half-life of less than 1 min, while the simultaneous injection of 5 mg asialofetuin maintained the circulating plasma levels. Asialofetuin also slowed the clearance of intact ¹²⁵I-labeled human corticosteroid-binding globulin from the plasma ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=90}\text{min}$). Binding of ¹²⁵I-labeled human asialo-corticosteroid-binding globulin to rat liver plasma membranes in vitro was inhibited in a dose-dependent manner by asialofetuin, but not by intact human corticosteroid-binding globulin or fetuin. ¹²⁵I-labeled human corticosteroid-binding globulin did not bind significantly to the membranes. It is concluded that human corticosteroid-binding globulin clearance from rat plasma is rapid and that the carbohydrate moiety of human corticosteroid-binding globulin is involved in its clearance and catabolism by the liver.     

Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

Concanavalin a binding and cytodifferentiation in pancreatic acinar carcinoma of rat

The binding of concanavalin A to the plasmalemma of acinar carcinoma cells was characterized by electron microscopy utilizing horseradish peroxidase. Heavy labeling due to specific concanavalin A binding was detected on the plasmalemma of undifferentiated carcinoma cells lacking zymogen maturation, neoplastic cells of intermediate differentiation with only occasional zymogen granules, and highly differentiated acinar carcinoma cells containing numerous cytoplasmic zymogen granules. The plasmalemma of acinar carcinoma cells was also compared to the normal pancreatic acinar cell plasmalemma by measurement of specific ¹²⁵I-labeled concanavalin A binding. Although only about one-third of pancreatic acinar carcinoma cells demonstrate mature zymogen differentiation, the acinar carcinoma had a full complement of normal plasmalemma receptors for ¹²⁵I-labeled concanavalin A. It is concluded that, unlike normal pancreas, the presence of concanavalin A receptors on the plasmalemma of acinar carcinoma cells is not a specific membrane marker for differentiated cells containing zymogen granules.