Genetically engineered tetravalent single-chain Fv of the pancarcinoma monoclonal antibody CC49: improved biodistribution and potential for therapeutic application.

Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacokinetics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors. Using genetic engineering, antitumor MAbs can be optimized for desirable clinical applications. In the present study, we report the generation of a tetravalent single-chain Fv [[sc(Fv)2]2] of the murine MAb CC49 that recognizes the tumor-associated glycoprotein, TAG-72. [Sc(Fv)2]2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore. The binding affinity constant (K(A)) for the [sc(Fv)2]2 and CC49 IgG were similar, i.e., 1.02 x 10(8) M(-1) and 1.14 x 10(8) M(-1), respectively, and were 4-fold higher than its divalent scFv [sc(Fv)2; 2.75 x 10(7) M(-1)]. At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3+/-1.3, 9.8+/-1.3, and 17.3+/-1.1 for radioiodinated [sc(Fv)2]2, sc(Fv)2, and IgG, respectively. Pharmacokinetic analysis of blood clearance studies showed the elimination half-life for [sc(Fv)2]2, sc(Fv)2, and IgG as 170, 80, and 330 min, respectively. The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.

Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct.

Progress in the use of monoclonal antibodies (MAbs) for the treatment of solid tumors is limited by a number of factors, including poor penetration of the labeled IgG molecule into the tumors, their inability to reach the tumor in sufficient quantities without significant normal tissue toxicity, and the development of a human antimouse antibody response to the injected MAb. One possible way to alter the pharmacology of antibodies is via the use of smaller molecular weight antibody fragments called single-chain Fvs (scFvs). A divalent construct of MAb CC49, CC49 (scFv)2, composed of two noncovalently associated scFvs, was generated and shown to bind a tumor-associated antigen (TAG-72) epitope with a similar binding affinity to that of the murine IgG. The therapeutic potential of this construct after labeling with 131I was examined in athymic mice bearing established s.c. human colon carcinoma (LS-174T) xenografts. Treatment groups (n = 10) received a single dose of 131I-labeled CC49 (scFv)2 (500-2000 microCi) or 131I-labeled CC49 IgG (250 and 500 microCi). The group of mice treated with the lowest dose of 131I-(scFv)2 (500 microCi) showed statistically significant prolonged survival, compared with controls (P = 0.036). Complete tumor regression was observed in 20% of mice given 1500 microCi of labeled (scFv)2 and 30 and 60% of mice treated with 250 and 500 microCi of labeled IgG, respectively. In conclusion, the CC49 (scFv)2 construct provides a promising delivery vehicle for therapeutic applications.

Single-Dose versus fractionated radioimmunotherapy of human colon carcinoma xenografts using 131I-labeled multivalent CC49 single-chain fvs.

The prospects of radiolabeled antibodies in cancer detection and therapy remain promising. However, efforts to achieve cures, especially of solid tumors, with the systemic administration of radiolabeled monoclonal antibodies (MAbs) have met with limited success. Using genetic engineering techniques, MAbs have been tailored to improve the therapeutic index (tumor:normal tissue ratio) in clinical radioimmunotherapy. In the present study, we investigated the potential of tetravalent ([sc(Fv)2]2) and divalent [sc(Fv)2] single chain Fvs of MAb CC49 for therapy in athymic mice bearing s.c. LS-174T human colon carcinoma xenografts. Mice received 1,000 microCi of 131I-labeled [sc(Fv)2]2 or 131I-labeled sc(Fv)2, either as a single injection on day 6 or as four injections (250 microCi each) on days 6, 7, 8, and 9; the day of tumor implantation was taken as day 0. The median survival for the control group was 26 days. Comparisons of single and fractionated therapeutic regimens showed median survival as 32 (P < 0.001) and 53 days (P < 0.0001), respectively for [sc(Fv)2]2 and 26 (P > 0.5) and 38 days (P < 0.0001), respectively for sc(Fv)2 when compared with the control groups. The time for the quadrupling of tumor volume for single and fractionated therapeutic treatments were: 9.0 +/- 0.8 and 21.1 +/- 2.9 days respectively for sc(Fv)2; 16.6 +/- 1.9 and 32.9 +/- 2.7 days respectively for [sc(Fv)2]2; and 8.3 +/- 0.7 and 8.4 +/- 0.6 days respectively for the control group. No 131I-labeled systemic toxicity was observed in any treatment groups. The results show that radioimmunotherapy delivery for sc(Fv)2 and [sc(Fv)2]2 in a fractionated schedule clearly presented a therapeutic advantage over single administration. The treatment group receiving tetravalent scFv showed a statistically significant prolonged survival with both single and fractionated administrations suggesting a promising prospect of this reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Antihuman growth hormone (GH) antibodies cross-react with the GH-like factor from plerocercoids of the tapeworm Spirometra mansonoides.

A factor produced by the plerocercoid stage of S. mansonoides mimics some, but not all, of the actions reported for hGH. The biological actions of plerocercoid growth factor (PGF) suggest structural similarity to human GH (hGH). Plerocercoid membranes were solubilized, and PGF was purified more than 1000-fold by hGH receptor affinity chromatography. The ability of purified PGF to displace [125I]hGH from monoclonal antibodies specific for four distinct nonoverlapping antigenic determinants of hGH and from an anti-hGH polyclonal antibody was tested in liquid phase RIA. All of the hGH antibodies cross-reacted with PGF, with potencies ranging from more than 60% to less than 1% that of the hGH standard. Of the four major epitopes of hGH defined by the monoclonal antibodies used in this study, only one is not represented to a significant extent in PGF. The epitope of hGH that is only marginally present in PGF is highly conformationally dependent, and a minor difference in the structure of PGF (compared to hGH) could result in a significant conformational change. The dramatic cross-reactivity between anti-hGH antibodies and PGF suggests that the similarities in biological activities between these two substances are based in significant molecular homology.

Reduction of lactogenic receptors in female hamster liver due to the human growth hormone analog produced by plerocercoids of the tapeworm, Spirometra mansonoides.

The inductive effect of GH on hepatic lactogenic receptors is suspected of being due to a direct somatogenic action. Plerocercoid larvae of the tapeworm, Spriometra mansonoides, produce a factor that stimulates body growth, suppresses endogenous GH, and specifically displaces [125I]human (h) GH from hepatic receptors. Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but it has not been shown to duplicate all of the activities reported for GH. An important function of GH is its role in the maintenance of liver receptors for lactogenic hormones. This study was undertaken to determine if treatment of female hamsters with PGF would increase, decrease, or have no effect on liver receptors that bind hGH. Since hGH binds to somatogenic as well as lactogenic receptors, it was necessary to demonstrate the specificity of PGF's effects on [125I]hGH binding. PGF-treated (15 pleocercoids sc) hamsters had accelerated body growth, suppressed serum GH, and a marked reduction in [125I]hGH and [125I]ovine PRL binding to hepatic microsomes. Specific binding of [125I] bGH was unaltered by PGF treatment. The difference in [125I] hGH binding was due to a reduction in receptor number and not to receptor occupancy or reduced affinity. Serum GH was normalized after 10 days of estradiol benzoate (25 micrograms/day) injections, but the binding capacity for [125I]hGH of the PGF-treated group was less than half that of the control group. The fact that estrogen injections normalized serum GH, but not hGH binding, indicates that down-regulation of these receptors by PGF cannot be entirely explained on the basis of reduced levels of serum GH. The lack of any effect of PGF treatment on [125I]bGH binding suggests that the hepatic somatogenic receptors were not involved and that the reduction in receptors for [125I]hGH was associated with the lactogenic component of hGH.

Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts.

UNLABELLED: Monoclonal antibodies (MAbs) have been proven useful in clinical studies for both diagnostic and therapeutic applications. The single-chain Fv (scFv) construct made from MAbs has potential applications for improved cancer diagnosis and therapy. A new CC49 scFv construct recognizing a tumor-associated mucin, TAG-72, was engineered and evaluated by immunological, pharmacokinetic and biodistribution analysis. METHODS: The CC49 scFv construct was generated in which the V(L) and V(H) variable region genes were joined together with a 25-amino acid helical linker (205C). The new CC49 scFv(205C) was expressed as a monomer as well as a stable noncovalent dimer ([scFv]2). The pharmacokinetic, biodistribution and tumor targeting characteristics of radiolabeled CC49 scFv were compared with CC49 IgG and enzymatically derived fragments F(ab')2 and Fab', using the athymic mice bearing human colon cancer xenografts. RESULTS: The association constant (K(A)) for the intact CC49, dimeric scFv (scFv)2 and monomeric scFv were 1.7 x 10(9), 1.99 x 10(9) and 0.52 x 10(9) M(-1) by Scatchard analysis and 1.14 x 10(8), 4.46 x 10(7) and 1.5 x 10(7) M(-1), respectively, by BIAcore analysis. Pharmacokinetic studies showed that more than 50% of monomeric scFv (approximately 27 kDa) was cleared from the blood in less than 10 min. The CC49 Fab' generated enzymatically from the parent murine Mab' (50 kDa) had a blood clearance that was faster than that of the (scFv)2 (60 kDa) with half of the activity cleared from the serum within 30 and 50 min, respectively. The CC49 dimeric scFv(205C) showed a two-fold higher tumor uptake (than scFv or Fab') reaching 10 %ID/g at 60 min after injection. The scFv dimer also showed an excellent stability and increased avidity in vivo compared with the monomer, as demonstrated by the longer retention in tumor with 3%ID/g remaining at 48 h. CONCLUSION: The rapid clearance from the blood, higher tumor uptake and longer retention of the stable dimer of CC49 scFv make it an important agent for potential imaging and therapeutic applications.

99mTc-labeled divalent and tetravalent CC49 single-chain Fv's: novel imaging agents for rapid in vivo localization of human colon carcinoma.

UNLABELLED: Radioimmunopharmaceutical agents enabling rapid high-resolution imaging, high tumor-to-background ratios, and minimal immunogenicity are being sought for cancer diagnosis and imaging. Genetic engineering techniques have allowed the design of single-chain Fv's (scFv's) of monoclonal antibodies (mAbs) recognizing tumor-associated antigens. These scFv's show good tumor targeting and biodistribution properties in vivo, indicating their potential as imaging agents when labeled with a suitable radionuclide. METHODS: Divalent (sc(Fv)(2)) and tetravalent ([sc(Fv)(2)](2)) scFv's of mAb CC49 were evaluated for radioimmunolocalization of LS-174T colon carcinoma xenografts in athymic mice. scFv's were radiolabeled with (99m)Tc by way of the bifunctional chelator succinimidyl-6-hydrazinonicotinate hydrochloride using tricine as the transchelator. The immunoreactivity and in vitro stability of the scFv's were analyzed after radiolabeling. Biodistribution and pharmacokinetic studies were performed to determine the tumor-targeting potential of the radiolabeled scFv's. Whole-mouse autoradiography illustrated the possible application of these (99m)Tc-labeled multivalent scFv's for imaging. RESULTS: The radiolabeling procedure gave > or =95% radiometal incorporation, with a specific activity of >74 MBq/mg scFv. In solid-phase radioimmunoassay, both sc(Fv)(2) and [sc(Fv)(2)](2) exhibited 75%-85% immunoreactivity, with nonspecific binding between 0.8% and 1.2%. Size-exclusion high-performance liquid chromatography showed sc(Fv)(2) as a 60-kDa protein and [sc(Fv)(2)](2) as a 120-kDa protein. Blood clearance studies showed the elimination half-life of (99m)Tc-labeled sc(Fv)(2) as 144 min and that of [sc(Fv)(2)](2) as 307 min. Whole-body clearance studies confirmed the rapid elimination of scFv's, with half-lives of 184 +/- 19 min for sc(Fv)(2) and 265 +/- 39 min for [sc(Fv)(2)](2) (P < 0.001). At 6 h after administration, the tumor localization was 7.2 +/- 0.7 percentage injected dose per gram of tumor (%ID/g) for (99m)Tc-sc(Fv)(2). (99m)Tc-[sc(Fv)(2)](2) showed a tumor uptake of 19.1 +/- 1.1 %ID/g at the same time; the amount of radioactivity in the tumors was 4-fold higher than in the spleen and kidneys and 2-fold higher than in the liver. Macroautoradiography performed at 6 and 16 h after administration clearly detected the tumor with both scFv's. CONCLUSION: (99m)Tc-labeled multivalent scFv's show good tumor-targeting characteristics and high radiolocalization indices (tumor-to-background ratio). These reagents, therefore, have the potential for use in clinical imaging studies of cancer in the field of nuclear medicine.

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization.

Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.

Single-chain antibodies in pancreatic cancer.

Pancreatic cancer is a therapeutic challenge for surgical and medical oncology. Development of specific molecular tracers for the diagnosis and treatment of this lethal cancer has been one of our major goals. Monoclonal antibodies (MAbs) have been successfully used as selective carriers for delivering radionuclides, toxins or cytotoxic drugs to malignant cell populations; therefore, monoclonal antibody technology has led to a significant amount of research into optimizing targeted therapy. This targeted therapy results in the selective concentration of cytotoxic agents or radionuclides in tumors and should lessen the toxicity to normal tissues, which would normally limit the dosage and effectiveness of systemically administered drugs. The MAb CC49 reacts with a unique disaccharide, Sialyl-Tn, present on tumor-associated mucin (TAG-72) expressed by a majority of human adenocarcinomas. The unique Sialyl-Tn epitope has provided a potential target for immunotherapy of cancer. A single chain Fv (scFv) recombinant protein from CC49 MAb was prepared by engineering the DNA fragments for coding heavy-chain and light-chain variable regions with an appropriate oligonucleotide linker. scFv molecules, when compared to intact MAbs and the more conventional enzymatically derived F(ab')2 and Fab' fragments, offer several advantages as carriers for the selective delivery of radionuclides to tumors. The divalent antibody fragments (sc(Fv)2 or (scFv)2) display an affinity constant similar to that of the intact CC49 IgG and are stable with storage, and after radiolabeling. In preclinical studies, both the covalent and the non-covalent dimeric scFvs exhibit excellent tumor targeting properties with characteristics similar to those of the monomer, e.g., the rapid blood clearance, low kidney uptake and small size suitable for rapid penetration through tumor tissue. Increased tumor targeting of the dimers are probably due to their increased functional affinity attributable to valency, coupled with their higher molecular weight and fewer interactions with normal organs. These properties make these constructs superior to monovalent CC49 scFv. The relatively high tumor uptake, the in vitro and in vivo targeting specificity, and the stability in storage demonstrated by the dimeric CC49 sc(Fv)2 makes it a promising delivery vehicle for therapeutic applications in pancreatic cancer.

Bitterness of sweeteners as a function of concentration.

Sixteen trained tasters provided sweetness and bitterness intensity ratings for 19 compounds including: acesulfame-K, alitame, aspartame, fructose, glucose, glycine, lactitol, maltitol, monoammonium glycyrrhizinate, neohesperidin dihydrochalcone, neosugar (fructo-oligosaccharide), palatinit (isomalt), rebaudioside-A, sodium cyclamate, sodium saccharin, stevioside, sucralose, sucrose, and thaumatin. With increasing concentration, high-potency sweeteners including acesulfame-K, neohesperidin dihydrochalcone, sodium saccharin, rebaudioside-A, and stevioside tended to become more bitter. Low-potency sweeteners including fructose, sucrose, and lactitol tended to become less bitter with increasing concentration.

Investigation of synergism in binary mixtures of sweeteners.

The purpose of the present study was to determine the presence and degree of synergism among all binary mixtures of 14 sweeteners varying in chemical structure. A trained panel evaluated binary combinations of the following sweeteners: three sugars (fructose, glucose, sucrose), two polyhydric alcohols (mannitol, sorbitol), two diterpenoid glycosides (rebaudioside-A, stevioside), two dipeptide derivatives (alitame, aspartame), one sulfamate (sodium cyclamate), one protein (thaumatin), two N-sulfonyl amides (acesulfame-K, sodium saccharin), and one dihydrochalcone (neohesperidin dihydrochalcone). Each sweetener was tested at three concentrations that were isosweet with 3%, 5%, and 7% sucrose. Two methods of analysis were performed to determine synergistic effects. In Method I, an ANOVA was performed for each intensity level to determine if the mean sweetness intensity ratings of each binary mixture were equal to nominal sweetness (i.e., additivity) or not equal to nominal sweetness (i.e., synergism or suppression). In Method II, an additional ANOVA was performed to determine if the sweetness intensity ratings of any given mixture were equal to or greater than the average of the sweetness ratings of the two pure components in that blend.

Charge-modified single chain antibody constructs of monoclonal antibody CC49: generation, characterization, pharmacokinetics, and biodistribution analysis.

A novel strategy was developed in which an antibody scFv fragment of the monoclonal antibody (MAb) CC49 was modified by engineering DNA coding sequences to lower its isoelectric point. Negatively charged amino acids were added to the carboxy terminus of the CC49 VH region by adding nucleotide sequences in a polymerase chain reaction (PCR) amplification of the coding sequence of CC49 scFv. Two new DNA constructs coding for CC49 scFv with lower isoelectric points of 5.8 and 5.2 were engineered. These novel strategy-generated, charge-modified antibody constructs were compared for their immunological, pharmacokinetic, and biodistribution properties in athymic mice bearing LS-174T human colon carcinoma xenografts.

Adaptation of sweeteners in water and in tannic acid solutions.

Repeated exposure to a tastant often leads to a decrease in magnitude of the perceived intensity; this phenomenon is termed adaptation. The purpose of this study was to determine the degree of adaptation of the sweet response for a variety of sweeteners in water and in the presence of two levels of tannic acid. Sweetness intensity ratings were given by a trained panel for 14 sweeteners: three sugars (fructose, glucose, sucrose), two polyhydric alcohols (mannitol, sorbitol), two terpenoid glycosides (rebaudioside-A, stevioside), two dipeptide derivatives (alitame, aspartame), one sulfamate (sodium cyclamate), one protein (thaumatin), two N-sulfonyl amides (acesulfame-K, sodium saccharin), and one dihydrochalcone (neohesperidin dihydrochalcone). Panelists were given four isointense concentrations of each sweetener by itself and in the presence of two concentrations of tannic acid. Each sweetener concentration was tasted and rated four consecutive times with a 30 s interval between each taste and a 2 min interval between each concentration. Within a taste session, a series of concentrations of a given sweetener was presented in ascending order of magnitude. Adaptation was calculated as the decrease in intensity from the first to the fourth sample. The greatest adaptation in water solutions was found for acesulfame-K, Na saccharin, rebaudioside-A, and stevioside. This was followed by the dipeptide sweeteners, alitame and aspartame. The least adaptation occurred with the sugars, polyhydric alcohols, and neohesperidin dihydrochalcone. Adaptation was greater in tannic acid solutions than in water for six sweeteners. Adaptation of sweet taste may result from the desensitization of sweetener receptors analogous to the homologous desensitization found in the beta adrenergic system.

Effect of temperature, pH, and ions on sweet taste.

The purpose of this experiment was to determine the effects of temperature (50 degrees C and 6 degrees C), pH (pH 3.0, 4.0, 5.0, 6. 0, and 7.0) and the addition of monovalent and divalent cations (5 mM Na(+), 5 mM K(+), and 5 mM Ca(2)+ ) on the sweetness intensity ratings of sweeteners ranging widely in chemical structure. A trained panel provided intensity evaluations for prototypical tastes (sweet, bitter, sour, and salty) as well as aromatic and mouth-feel attributes. The following sweeteners were included in this experiment: three sugars (fructose, glucose, sucrose), three terpenoid glycosides (monoammonium glycyrrhizinate, rebaudioside-A, stevioside), two polyhydric alcohols (mannitol, sorbitol), two dipeptide derivatives (alitame, aspartame), two N-sulfonylamides (acesulfame-K, sodium saccharin), one sulfamate (sodium cyclamate), one protein (thaumatin), one dihydrochalcone (neohesperidin dihydrochalcone), and one chlorodeoxysugar (sucralose). Two to five levels of each sweetener reflecting a range of sweetness intensities were tested, using formulae developed by DuBois et al. The main finding from this three-part study was that temperature, pH, and ions had little effect on perceived sweetness intensity. Even when significant differences were found in the temperature study, the effects were very small.

Serial casting for the management of spasticity in the head-injured adult.

Soft tissue contractures resulting from spasticity are a frequent sequella of traumatic head injuries. This article identifies rationale for the use of serial casting to manage these deformities, provides guidelines for cast fabrication and application, and reports the results of lower extremity casting on 42 head-injured adults at Rancho Los Amigos Hospital.

Suppression of receptors for prolactin and estrogen in rat liver due to treatment with the growth hormone analogue produced by the tapeworm Spirometra mansonoides.

Somatogenic hormones play an important role in regulation of receptors for prolactin (PRL) and estrogen. Plerocercoids of the tapeworm, S. mansonoides produce a factor which mimics some, but not all of the actions reported for GH. Intact female rats were subjected to a constant infusion of plerocercoid growth factor (PGF) via a subcutaneous infection for two weeks to determine if PGF influences receptors for PRL, GH or estradiol. The rate of weight gain in the PGF-treated rats was accelerated in spite of a marked reduction in serum GH. Partially-purified PGF specifically displaced [125I]hGH from rat liver receptors but microsomes prepared from rats treated with PGF specifically bound significantly less [125I]hGH than microsomes from control rats. The reduction in [125I]hGH binding was not due to occupancy or to a change in affinity but to a suppression in receptor concentration. Scatchard analysis of [3H]estradiol binding in rat liver cytosols shows a 50% reduction in receptor concentration in the PGF-treated group. Specific binding of [3H]estradiol in anterior pituitary was also suppressed by PGF treatment.