Differential localization of PD-L1 and Akt-1 involvement in radioresistant and radiosensitive cell lines of head and neck squamous cell carcinoma.

Immunotherapy by blockade of the PD-1/PD-L1 checkpoint demonstrated amazing tumor response in advanced cancer patients including head and neck squamous cell carcinoma (HNSCC). However, the majority of HNSCC patients still show little improvement or even hyperprogression. Irradiation is currently investigated as synergistic treatment modality to immunotherapy as it increases the number of T-cells thereby enhancing efficacy of immunotherapy. Apart from this immunogenic context a growing amount of data indicates that PD-L1 also plays an intrinsic role in cancer cells by regulating different cellular functions like cell proliferation or migration. Here, we demonstrate opposing membrane localization of PD-L1 in vital and apoptotic cell populations of radioresistant (RR) and radiosensitive (RS) HNSCC cell lines up to 72 h after irradiation using flow cytometry. Moreover, strong PD-L1 expression was found in nuclear and cytoplasmic cell fractions of RR. After irradiation PD-L1 decreased in nuclear fractions and increased in cytoplasmic fractions of RR cells. In contrast, RS cell lines did not express PD-L1, neither in the nucleus nor in cytoplasmic fractions. Additionally, overexpression of PD-L1 in RS cells led to a proportional increase of vital PD-L1 positive cells after irradiation. Moreover, co-immunoprecipitation experiments revealed an interaction between Akt-1 and PD-L1, mostly in irradiated RR cells compared to RS cells suggesting a differential influence of PD-L1 on cell signaling. In summary, our data imply the need for different therapeutic strategies dependent on the molecular context in which PD-L1 is embedded.

Pharmacokinetic and safety study of subcutaneously administered weekly ING-1, a human engineere monoclonal antibody targeting human EpCAM, in patients with advanced solid tumors.

BACKGROUND: ING-1 is a high-affinity, human engineeredtrade mark monoclonal antibody that recognizes a 40 kilodalton epithelial cell adhesion molecule (EpCAM) glycoprotein that is expressed in high levels on most adenocarcinomas and is an attractive target for immunotherapy. METHODS: ING-1 was administered subcutaneously weekly at doses between 0.1 and 2 mg/kg/week. Pharmacokinetic samples were drawn during weeks 1 and 6. RESULTS: Fourteen patients with advanced refractory cancer received a median of 6 (range 1-9) doses of ING-1. At 1 mg/kg, a 62-year-old man with colon cancer developed reversible grade 3 pancreatitis after the third dose. His plasma ING-1 levels were similar to the other two patients dosed at 1 mg/kg. Two patients dosed at 0.6 mg/kg experienced stable disease at 6 weeks. Peak drug levels increased with dose and time, suggesting drug accumulation with repeated dosing. Low human anti-human antibody response was noted in three of the 13 patients assessed and was directed towards the variable region of ING-1. CONCLUSIONS: Weekly ING-1 administered subcutaneously was well tolerated at 0.6 mg/kg/week and further experience at this dose is warranted to demonstrate safety. The risk of pancreatitis and the marginal anti-tumor effect may preclude further monotherapy studies; however, combination studies with chemotherapy are warranted.

A current 10-year retrospective survey of 199 surgically treated orbital floor fractures in a nonurban tertiary care center.

This study characterizes the surgically treated patient population suffering from orbital floor fractures by use of current data from a large series consisting of 199 cases taken from a nonurban setting. Data were gathered through a retrospective chart review of patients surgically treated for orbital floor fractures at the University of Michigan Health System, collected over a 10-year period. Data regarding patient demographics, signs and symptoms of presentation, cause of injury, nature of injury, associated facial fractures, ocular injury, and associated nonfacial skeleton trauma were collected. In total, there were 199 cases of orbital floor fractures among 189 patients. Male patients outnumbered female patients by a 2:1 ratio and were found to engage in a wider range of behaviors that resulted in orbital floor fractures. Motor vehicle accidents were the leading cause of orbital floor fractures, followed by physical assault and sports-related mechanisms. The ratio of impure to pure orbital floor fracture was 3:1. The most common signs and symptoms associated with orbital floor fractures, in descending order, were periorbital ecchymosis, diplopia, subconjunctival hemorrhage, and enophthalmos. Associated facial fractures were found in 77.2 percent of patients, the most prevalent of which was the zygoma-malar fracture. Serious ocular injury occurred in 19.6 percent of patients, with globe rupture being the most prevalent, accounting for 40.5 percent of those injuries. There was a 38.1 percent occurrence of associated nonfacial skeletal trauma; skull fracture and intracranial injury were the most prevalent manifestations. Associated cervical-spine fractures were rare (0.5 percent). Statistical examination, using odds ratios and chi-squared analysis, demonstrated significant associations that have not previously been reported. Impure and pure orbital floor fractures revealed striking differences in several demographic aspects, including mechanism of injury, signs and symptoms of presentation, spectrum of associated trauma, and the severity of concomitant trauma.

Analysis of pilus adhesins from Haemophilus influenzae biotype IV strains.

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.

Amplitude histograms can identify positively but not negatively coupled channels.

We investigated the ability of amplitude distributions to determine if the gating of a pair of channels is coupled. These distributions are expressed as probability density amplitude histograms with peaks corresponding to zero, one, or two open channels. If the channels gate independently, the areas under these peaks (A, B, and C, respectively) determine the open probabilities of the two channels (p(1) and p(2)). Manivannan et al. (Biophys J 1994;61:216) showed that if Delta=B(2)/AC was less than 4 then the channel gating is coupled. We defined a similar parameter, D=(B(2)/4)-AC. If D<0 then channel gating is coupled. However, amplitude histograms with D0 are consistent with both independent and coupled gating. We further present a simple model in which channels are assumed to be identical and can be positively or negatively coupled. Here, amplitude histograms determine q=(B+2C)/2 (open probability of the coupled channels) and r=-D (the coupling parameter). Thus, positively coupled channels (r0) produce amplitude histograms with D<0 whereas negatively coupled channels (r<0) produce amplitude histograms with D0.

Pharmacokinetics of a recombinant modified amino terminal fragment of bactericidal/permeability-increasing protein (rBPI21) in healthy volunteers.

Phase I pharmacokinetic and safety studies were conducted in healthy volunteers with rBPI21, a recombinant protein derived from the amino terminal domain of human bactericidal/permeability-increasing protein. rBPI21 was administered as a 30-minute infusion at doses of 0.25 to 4 mg/kg or as a 24- to 48-hour infusion at doses of 2 to 8 mg/kg. For the 30-minute infusions, the clearance of rBPI21 decreased with increasing dose from 8.4 mL/min/kg at 0.25 mg/kg to 3.3 mL/min/kg at 4 mg/kg. For rBPI21 infused over 24 to 48 hours the clearance was 10 to 11 mL/min/kg. The concentration-time profile of rBPI21 was well described by a three-compartmental model with parallel first-order and Michaelis-Menten (saturable) elimination. This model for the clearance of rBPI21 has been useful in estimating starting doses for therapeutic clinical trials.

Population pharmacokinetics and pharmacodynamics of the anti-CD11a antibody hu1124 in human subjects with psoriasis.

The pharmacokinetics of hu1124, a human anti-CD11a antibody, were investigated in human subjects with psoriasis. CD11a is a subunit of LFA-1, a cell surface molecule involved in T cell mediated immune responses. Subjects received a single dose of 0.03, 0.1, 0.3, 0.6, 1, 2, 3, or 10 mg/kg of hu1124 intravenously over 1-3 hr. Blood samples were collected at selected times from 60 min to 72 days after administration. Plasma samples were assayed for hu1124 by ELISA, and pharmacokinetic analyses were performed on the drug plasma concentrations. As the dose of hu1124 was increased, the clearance decreased from 322 ml/day per kg at 0.1 mg/kg to 6.6 ml/day per kg at 10 mg/kg of hu1124. The plasma hu1124 concentration-time profile suggested that the clearance of hu1124 was saturable above 10 micrograms/ml. In addition, treatment with hu1124 caused a rapid reduction in the level of CD11a expression on CD3-positive lymphocytes (T cells) to about 25% of pretreatment levels. Regardless of the hu1124 dose administered, cell surface CD11a remained at this reduced level as long as hu1124 was detectable (> 0.025 microgram/ml) in the plasma. When hu1124 levels fell below 3 micrograms/ml, the drug was rapidly cleared from the circulation and expression of CD11a returned to normal within 7-10 days thereafter. In vitro, half-maximal binding of hu1124 to lymphocytes was achieved at about 0.1 microgram/ml and saturation required more than 10 micrograms/ml. One of the receptor-mediated pharmacokinetic/pharmacodynamic models which was developed describes the dynamic interaction of hu1124 binding to CD11a, resulting in the removal of hu1124 from the circulation and reduction of cell surface CD11a. The model accounts for the continually changing number of CD11a molecules available for removing hu1124 from the circulation based on prior exposure of cells expressing CD11a to hu1124. In addition, the model also accounts for saturation of CD11a molecules by hu1124 at drug concentrations of approximately 10 micrograms/ml, thereby reducing the clearance rate of hu1124 with increasing dose.

Pharmacokinetics of a recombinant amino terminal fragment of bactericidal/permeability increasing protein (rBPI21) after liver surgery in rats and humans.

Major liver resections are associated with considerable morbidity and mortality. Gut-derived bacteria and bacterial endotoxin (LPS) are considered to play a central role in the pathophysiology of these complications. Like human BPI, rBPI21 binds to LPS from Gram-negative bacteria. By binding and clearing of LPS, rBPI21 can inhibit a number of endotoxin-induced humoral and cellular responses. Because of this capacity, rBPI21 could partially compensate for the loss of hepatic mononuclear phagocytic system function after liver resection. However, the liver is also thought to be an important organ for the clearance of BPI, and reduction of liver mass could result in a decreased clearance and exceedingly high plasma levels of rBPI21. In this study we therefore investigated the pharmacokinetics of rBPI21 in rats and in patients undergoing a major liver resection. Rats were administered an intravenous (i.v.) bolus of rBPI21 after undergoing a 60% or 80% hepatectomy (with sham-operated controls). Patients undergoing a hemihepatectomy and healthy volunteers received rBPI21 or placebo by continuous i.v. infusion for 48 h. Plasma concentrations were measured by sandwich ELISA. In rats, 60% hepatectomy did not consistently change the clearance of rBPI21, whereas 80% hepatectomy decreased the clearance of rBPI21 severalfold. In hemihepatectomized patients, the clearance of rBPI21 after major hepatectomy was also slower, when compared with healthy volunteers, but this difference had disappeared within 24 h. Our data indicate that the administration of rBPI21 in patients undergoing liver resection is well tolerated and does not result in exceedingly high plasma levels. Additional studies on the efficacy of rBPI21 in the prevention of complications after hepatectomy are needed.

Long-term effectiveness of a new alpha-glucosidase inhibitor (BAY m1099-miglitol) in insulin-treated type 2 diabetes mellitus.

In a double-blind, randomized study, miglitol (BAY m 1099), an alpha-glucosidase inhibitor, 100 mg tds or placebo was given orally with meals for a period of 24 weeks in 117 patients with Type 2 (non-insulin-dependent) diabetes mellitus (DM) treated with insulin. Fasting and 1 h postprandial plasma glucose and C-peptide were measured at the beginning and at the end of each 4-week interval and glycosylated haemoglobin was determined at day 0 and at the end of the 12th and 24th week. One hour postprandial plasma glucose was significantly lower in the miglitol group at the end of the 24th week (placebo: 11.6 +/- 1.5 vs miglitol: 8.2 +/- 1.5 mmol l-1, mean +/- SD, p = 0.001). Diabetes control improved in the same group as the HbA1 was lowered by 16% (p = < 0.0001) at the end of the treatment. Mild reversible adverse effects were observed in 37 patients of the miglitol group (mainly flatulence and mild hypoglycaemia) and 2 of the placebo group. Urinary glucose was rendered negative in 41 patients in the miglitol group only. Thus miglitol appears to be a safe and effective adjunct in the management of Type 2 DM, in association with insulin.

The role of liver and kidney on the pharmacokinetics of a recombinant amino terminal fragment of bactericidal/permeability-increasing protein in rats.

PURPOSE: The pharmacokinetics of rBPI23, a recombinant amino terminal fragment of bactericidal/permeability-increasing protein that binds to and neutralizes endotoxin, was investigated. METHODS: rBPI23 was administered to rats at doses 0.01-10 mg/kg and plasma rBPI23 levels were measured by ELISA. rBPI23 was also administered to bilaterally nephrectomized rats. In addition, rBPI23 was administered intra-hepatically via the pyloric vein to determine the first-pass effect by the liver. rBPI23 concentrations were also simultaneously measured in the right atrium and aorta to determine the removal of rBPI23 by the lungs. RESULTS: The concentration-time profile of rBPI23 was described by a 3-compartmental model with parallel first order and Michaelis-Menten (saturable) elimination. The clearance of rBPI23 was not altered by bilateral nephrectomy. Clearance of intra-hepatically administered rBPI23 was 4.5 fold lower than intra-femorally administered rBPI23. The concentration difference of rBPI23 between aortic and right atrial blood was no greater than 11%. Clearance of rBPI23 in rats could be reduced up to 10 fold by co-administration of heparin. Uptake by liver of intra-hepatically administered rBPI23 was prevented by co-administration of heparin. CONCLUSIONS: rBPI23 is not significantly cleared by the kidneys, and no more than 11% of the rBPI23 was removed by the lungs with each pass. The liver could remove 78% of the rBPI23 from the hepatic circulation. Studies with heparin suggest rBPI23 is cleared by binding to heparan sulfate sites in the liver.

Alteration of the pharmacokinetics of small proteins by iodination.

The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.

Determination of channel open probabilities from multichannel data.

We developed a method for determining whether channels in a multichannel patch or bilayer have the same or statistically significantly different open probabilities. We use a maximum likelihood method to fit the distribution of (unbinned) current amplitudes and to provide estimates of individual channel open probabilities, single channel currents, and standard deviations of the channel currents. These parameters are used to compare models with increasing constraints on the open probabilities including the model where all channels have different open probabilities and the model where all channels have the same open probability. A chi 2 statistic is used to identify models that are statistically less likely to predict the data. The ability of multichannel data to determine individual open probabilities is limited by two factors: the signal to noise ratio of the record and the fact that changes in amplitude distributions caused by a 0.2 difference in open probabilities are comparable in magnitude to the variations caused by random channel gating. These limitations notwithstanding, we demonstrate the utility of our approach by using it to analyze the open probabilities of 3 large conductance Ca2(+)-activated K+ channels in an artificial lipid bilayer revealing the response of one of those channels to GTP gamma S.

A phase I safety and pharmacokinetic study of a recombinant amino terminal fragment of bactericidal/permeability-increasing protein in healthy male volunteers.

A phase I pharmacokinetic and safety clinical trial of rBPI23, a recombinant amino terminal fragment of bactericidal/permeability-increasing protein, was conducted in healthy male volunteers. rBPI23 was administered as a 5 or 30 min infusion at doses of .1 to 1 mg/kg. The pharmacokinetics of rBPI23 in human subjects were described by a bi-exponential disposition function with evidence of concentration-dependent kinetics. The alpha half-life increased significantly with increasing dose, from 4-5 min at .1 mg/kg to 7-8 min at 1 mg/kg. The beta half-life varied between 18 and 29 min regardless of dose and the clearance varied from 5 to 10 mL/min/kg. Very little, if any, of the administered rBPI23 was excreted intact in the urine. Electrocardiograms, ionized calcium concentration, prothrombin and partial prothrombin times, hematologic parameters, and blood chemistries remained normal. Furthermore, no antibody response to rBPI23 was observed in any of the subjects.

T cell-targeted immunofusion proteins from Escherichia coli.

Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.

Enhanced stability in vitro and in vivo of immunoconjugates prepared with 5-methyl-2-iminothiolane.

Substituted 2-iminothiolanes (X2ITs) are new heterobifunctional crosslinking agents designed for the preparation of disulfide-linked conjugates with enhanced resistance to reduction. Based upon 2-IT substituted at the 4 and/or 5 position, these reagents appear to function by sterically protecting the conjugate disulfide bond from attack by thiolate nucleophiles. Here, we have used the X2ITs to prepare and evaluate a series of immunoconjugates (antibody-cytotoxin conjugates) between the murine monoclonal antibody 791/T36, which recognizes a 72-kDa surface antigen present on many human tumor cells, and RTA30, the naturally occurring 30-kDa glycoform of ricin A chain. The X2IT-linked conjugates were also compared to immunoconjugates prepared with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) and 4-[(succinimidyloxy)carbonyl]-alpha-methyl-alpha-(2- pyridyldithio)toluene (SMPT), as well as with methyl- and dimethyl-substituted structural analogs of SPDP. In vitro, 791-(X2IT)-TNB model compounds exhibited a 6000-fold range of stabilities. In contrast, the corresponding 791-(X2IT)-RTA30 immunoconjugates were up to 20-fold more stable than conjugates made with unhindered linkages. These improvements resulted in immunoconjugates with prolonged serum half-lives in animals. Our data indicate that one of the crosslinking agents, 5-methyl-2-iminothiolane (M2IT), has optimal properties for the preparation of disulfide crosslinked immunoconjugates intended for therapeutic use in that (i) it is highly water soluble and reacts rapidly with protein amino groups at neutral pH, preserving the positive charge, (ii) it forms conjugates with RTA30 efficiently, and (iii) its conjugates exhibited enhanced disulfide bond stability in vitro and in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties.

We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates.

Nonlinear pharmacokinetics of recombinant human macrophage colony-stimulating factor (M-CSF) in rats.

The pharmacokinetics and mechanisms of elimination of recombinant human macrophage-colony stimulating factor (M-CSF) were investigated in rats. Intravenous injections of 0.1, 1 or 10 mg/kg M-CSF were administered and plasma samples were measured for M-CSF by bioassay. Systemic clearance decreased and the shape of the concentration-time curve changed with increasing dose, indicating nonlinear pharmacokinetics. At 10 mg/kg, two half-lives were initially observed, but after about 20 hr the plasma M-CSF suddenly declined with a steep slope. The rapidly declining phase suggested a saturable clearance mechanism that was prominent at low plasma concentrations of M-CSF (below 300 ng/ml) and obscured at high plasma concentrations of M-CSF. The rapid decline of plasma M-CSF occurred at earlier times with multiple daily injections of M-CSF, indicating induction of the saturable clearance mechanism. The rapidly declining phase was inhibited by carrageenan, indicating that saturable clearance might be due to metabolism of M-CSF by macrophages. With ligation of either the renal pedicles or ureters, the apparent half-lives of M-CSF increased by a factor of 2- to 3-fold, while the occurrence of the rapidly declining phase was delayed, but not eliminated. Overall, the results are well described by a two-compartment, first-order elimination model with a parallel Michaelis-Menten elimination pathway. First-order elimination is largely performed by the kidneys and the saturable Michaelis-Menten elimination pathway appears to be mediated by cells of the monocyte-macrophage lineage.

Phase I trial of subcutaneous recombinant macrophage colony-stimulating factor: clinical and immunomodulatory effects.

PURPOSE: Recombinant human macrophage colony-stimulating factor (rM-CSF) has been demonstrated to control the growth, differentiation, and function of mononuclear phagocytes. Preclinical studies have indicated antitumor effects, and therefore a phase I trial of rM-CSF in patients with malignancy was initiated. The toxicity and hematologic and immunologic effects were investigated. PATIENTS AND METHODS: rM-CSF was administered as a subcutaneous injection on days 1 through 5 and 8 through 12. Cycles were repeated every 28 days. Cohorts of four to seven patients received rM-CSF at dose levels from 0.1 to 25.6 mg/m2/d. Forty-two patients received 88 cycles of rM-CSF. All patients had metastatic solid tumors refractory to standard therapy. RESULTS: The toxicity of rM-CSF was mild. Dose-limiting toxicity included thrombocytopenia (two patients) and iritis (one patient) occurring at a dose of 25.6 mg/m2/d. Hematologic studies demonstrated dose-related monocytosis occurring routinely at doses > or = 3.2 mg/m2/d, and thrombocytopenia. Immunologic studies demonstrated enhanced secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1 beta) by monocytes after in vitro stimulation with lipopolysaccharide, and increased expression of TNF-alpha mRNA at higher rM-CSF dose levels. Pharmacokinetic studies demonstrated that the systemic clearance rate of M-CSF increases during week 1 of therapy, resulting in lower blood levels of M-CSF during the second week of therapy. CONCLUSION: rM-CSF can be safely administered to patients, and has biologic activity on peripheral-blood monocytes.