Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use.

PURPOSE: ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. PROCEDURES: Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. RESULTS: Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. CONCLUSION: Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Low dose of protein A pretreatment can alleviate the inflammatory reaction and the bio-safety was evaluated in vivo.

BACKGROUND: Staphylococcal protein A (SPA) is a protein of Staphylococcus aureus. Up to now, there have been many studies on the biological activities of SPA. Some reported effects of SPA pretreatment on septic shock in mouse models but there is no study which reports the role of SPA pretreatment on the infected incision. METHODS: According to count results, bacterial suspension was set at a density of ∼1.8 × 10(9) colony forming units/mL. BALB/c mice were anesthetized via intraperitoneal injection with pentobarbital sodium. A longitudinal skin incision was made on the medial side of the right thigh. The length of the incision was 5 mm, and the depth was ∼3 mm. The bacterial suspension was gradually dripped and embrocated onto the incision surface to make the wound infection model. Before making the wound infection model for 48 hours and 24 hours, mice were retreated with SPA via intraperitoneal injection. Rats were intraperitoneally injected with SPA 1 mg/kg and the control group was injected with sterile saline to evaluate the biological safety of the best pretreatment dose. RESULTS: A 1-mL bacterial suspension can be utilized to make the wound infection model of BALB/c mouse lower limb. SPA pretreatment can reduce the inflammatory reactions in wound methicillin-resistant Staphylococcus aureus infection mouse model and the best pretreatment dose is 1 mg/kg. Intraperitoneal injection 1 mg/kg SPA does not destroy the functions of the organs. A 1-mg/kg SPA pretreatment can also reduce the inflammatory reactions in wound various bacterial infection mouse models. CONCLUSION: SPA pretreatment can effectively decrease the infected severity of a wound infected by various bacteria in a BALB/c mouse model. The best pretreatment dose is 1 mg/kg, and this dose does not damage organ function in rats up to a point.

Studies of the safety, pharmacokinetics and immunogenicity of repeated doses of intravenous staphylococcal protein A in cynomolgus monkeys.

Three Good Laboratory Practice safety studies were performed with intravenous injections of highly purified staphylococcal protein A (SPA) in cynomolgus monkeys, in support of a clinical development programme utilizing this protein as an immunomodulator. These studies established a no-observable-adverse-effect level (NOAEL) for up to 12 weekly doses of SPA, as well as toxicokinetic profiles for SPA, evaluation of antiproduct antibodies and biomarkers to better characterize the pharmacodynamic response to SPA. Biomarkers included neopterin, C-reactive protein (CRP), troponin I and the change in the blood absolute lymphocyte count (ALC) 24 hr after SPA dosing. The transient decrease in ALC noted at 24 hr after dosing was similar to that seen in human Phase 1 trials. The majority of active-treated monkeys developed antibodies against SPA. Cmax was not affected by development of antidrug antibodies (ADAs), and after the first dose was 87 (SD 19) ng/mL, 330 (SD 84) ng/mL and 1191 (SD 208) ng/mL for 5, 25 and 100 µg/kg doses, respectively. The development of ADAs increased plasma clearance of SPA. By the sixth weekly dose, the AUC was decreased by 76%, 54% and 66% for the 5, 25 and 100 µg/kg dose groups, respectively. These results indicate that SPA can be administered intravenously to non-human primates without observable toxicity at weekly doses of up to 100 µg/kg.

Staphylococcal protein A, Panton-Valentine leukocidin and coagulase aggravate the bone loss and bone destruction in osteomyelitis.

BACKGROUND/AIMS: Osteomyelitis is a debilitating infectious disease of the bone which is predominantly caused by Staphylococcus aureus (S. aureus). The purpose of this study was to investigate the role of the S. aureus virulence factors, i.e. protein A (SpA), Panton-Valentine leukocidin (PVL) and coagulase (Coa) on osteomyelitis. METHODS: The effect of SpA, PVL and Coa on osteoblasts was studied through the following aspects including osteoblast proliferation, apoptosis, bone formation, bone mineralization and RANK-L expression. S. aureus overexpressing PVL, SpA or Coa was constructed and used to study the role of PVL, SpA and Coa, respectively. S. aureus silencing PVL, SpA or Coa was also constructed and used for reversing verification. Osteoblast proliferation was detected by MTT tetrazolium dye reduction assay. Apoptosis was determined by Annexin V-FITC staining. The levels of pro-caspase 3, cleaved-caspase 3, pro-caspase 9 and cleaved-caspase 9 were detected by western blot. Bone formation markers including collagen I, osteopontin and osteocalcin were detected by real time RT-PCR. Alkaline phosphatase activity was measured by adding p-nitrophenyl phosphate as a phosphatase substrate. Von kossa stain and alizarin red stain were applied for determining phosphate and calcium deposition, respectively. The RANK-L expression was tested by ELISA. RESULTS: PVL, SpA and Coa inhibited osteoblast proliferation, induced osteoblast apotosis, prohibited bone formation and mineralization and upregulated RANK-L expression. CONCLUSIONS: PVL, SpA and Coa play a critical role on bone loss and bone destruction of osteomyelitis.

Synthesis, expression and purification of a type of chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, and its acute toxicity analysis.

A gene, rBmK Cta, encoding a chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, was synthesized according to the sequence optimized for codon usage in Escherichia coli and was expressed in E. coli BL21 (DE3) using a pExSecI expression system in which the IgG-binding domain-ZZ of protein A is fused to the N-terminal of rBmK CTa. The fusion protein, ZZ-rBmK CTa, was expressed in soluble form (7.8 mg l(-1)) and was purified to give a single band on SDS-PAGE. The domain-ZZ of fusion protein ZZ-rBmK CTa was removed by cleavage of an Asn-Gly peptide bond with hydroxylamine. The rBmK CTa was separated from the IgG-binding moiety by a second passage through the IgG affinity column. Western blot analysis demonstrated that this protein was rBmK CTa. Acute toxicity assay in mice demonstrated that the rBmK CTa had an LD(50) value of 4.3 mg kg(-1).

[Modulation of molecular signaling mechanisms in excitable cells by toxins and bacterial substances].

The mechanisms of bacterial substances (protein A, peptidoglican Staphylococcus aureus), bacterium toxins (St. aureus, Corynebacterium diphtheriae, Shigella dysenteriae, Clostridium botulinum, Clostridium tetani, Vibrio choleral), transfer factor of immune reactivity to Staphylococcus aureus upon the key link (acetylcholine-, ATP-, inositol-1,4,5-triphosphate-, ryanodin-sensitive receptors, G-proteins, Ca2+, K(+)-transporting systems, second messengers) in the chain of signal conduction of excitatory and inhibitory agonists in excitable cells were examined. The action of these immune-active substances upon contractile proteins ATP-ase activity and protein synthesis was also discussed.

Staphylococcal toxin induced preferential and prolonged in vivo deletion of innate-like B lymphocytes.

Contributing to host defenses from the adaptive immune system, splenic marginal zone (MZ) B cells, with their preactivated state and special topographical location, serve essential roles as primary defenders from blood-borne microbes. From studies designed to define the immunologic impact of protein A of Staphylococcus aureus (SpA), a virulence factor with targeted B cell antigen receptor-binding properties, we found that within minutes of in vivo exposure, SpA became surface associated with B lymphocytes and induced trafficking. Within several hours, MZ were completely effaced of affected B cells. This was rapidly followed by massive B cell apoptosis, with accelerated preferential deletion of targeted MZ B cells and impaired responsiveness to T independent immunogens. Subsequently, the temporal recovery of MZ B cells was significantly delayed compared to peripheral follicular B cells (B-2 cells). These studies elucidate the cellular program induced by a natural toxin that is shown to be highly efficient at depleting innate-like B cells important for defense from systemic infection.

Protein A-activated macrophages induce apoptosis in Ehrlich's ascites carcinoma through a nitric oxide-dependent pathway.

Protein A (PA) of Staphylococcus aureus has been demonstrated to possess anti-tumor activity against a wide variety of tumors. In the current study we endeavored to obtain a mechanistic insight into PA-mediated Ehrlich's ascites carcinoma (EAC) killing. Our results indicate that PA stimulates generation of nitric oxide (NO) from murine peritoneal macrophages. Nitric oxide in turn induces cytotoxic damage to the tumor cells. Analysis of the morphological features and cell cycle phase distribution pattern of nuclear DNA revealed an induction of apoptosis (appearance of sub-G0/G1 population) in EAC after PA treatment. We have further elaborated the alterations in the expressions of the proto-oncoproteins p53 and Bax, together with a change in the ratio of Bcl-2/Bax in the treated tumor cells, which favor apoptosis. PA-induced apoptosis and changes in the expression of oncoproteins in the tumor cells was prevented by the suppression of NO release by the addition of L-NAME, the competitive NOS inhibitor, suggesting a possible mechanism by which PA exerts its anti-tumor activities involving nitric oxide through the alteration in the expressions of pro-apoptotic proteins.

The B-cell targeted CTA1-DD vaccine adjuvant is highly effective at enhancing antibody as well as CTL responses.

A novel immunomodulating gene-fusion protein, CTA1-DD, has been developed which combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant is non-toxic and greatly augmented T-cell-dependent and T-cell-independent responses to soluble admixed antigens after systemic as well as mucosal immunizations. CTL and antibody responses of all classes were increased by 10- to 100-fold above those observed in control mice immunized without adjuvant. CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injection, but rather it binds directly to B-cells of all isotypes, including naïve IgD+ cells. No binding was observed to macrophages or dendritic cells, and immunizations in Fc gamma R-deficient mice demonstrated unaltered enhancing effects. As shown by inactive mutants, the CTA1-DD adjuvant is dependent on ADP-ribosyltransferase activity and requires the binding to Ig- via the DD moiety. The enhancing effect is associated with enlarged germinal centers, and binding of CTA1-DD to the B-cells strongly upregulates co-stimulatory molecules and counteracts apoptosis by inducing intracellular Bcl-2.

Glutathione reduces the toxicity associated with antitumor therapy of ascites fluid adsorbed over Staphylococcus aureus Cowan I in tumor bearing mice.

It has been well documented in the literature that the removal of circulatory immune complexes (CICs) from the host circulation leads to the immunopotentiation as well as generation of antitumor responses in a variety of tumors in rats, cats, dogs and human patients. CICs are the major immunosuppressive factors in tumor bearing host. Protein A (PA) has been extensively used for the removal of these CICs from the sera/plasma of tumor bearers, because PA has the ability to bind with the Fc portion of mammalian immunoglobulins. Previously, we reported for the first time a potent antitumor response by the inoculation of cell free Ehrlich's ascites fluid adsorbed in vitro over PA containing Staphylococcus aureus Cowan I (SAC) in Ehrlich's ascites tumor model. However, there was toxicity associated with this form of therapy in terms of early death of treated animals and the depletion of hepatic glutathione pool as well as phase I biotransformation enzyme and increase in glutathione-S-transferase (GST) activities. In the present investigation, tumor bearing animals were treated intraperitoneally (i.p.) on alternate days for 15 days with adsorbed ascites fluid (ad-ASF) (0.1 ml) and glutathione (GSH) (250 mg/kg body weight) separately. We found that GSH supplementation increases mean survival time of GSH and ad-ASF treated mice up to 37.2 days in comparison with 19.9 days for only ad-ASF treated animals, while percent increase in body weight was found to be not affected by the GSH substitution, which remains significantly lower (P < 0.01) in comparison to the tumor control animals. GSH supplementation causes a significant decrease (P < 0.05) of glutathione-S-transferase and restoration of aniline hydroxylase activity (P < 0.05) and aminopyrine-N-demethylase activity. We have also observed that GSH supplementation does not alter the tumor cell viability and tumor cell counts in ad-ASF treated animals in comparison to only ad-ASF treated animals, which indicates that GSH supplementation does not alter the antitumor effect of the therapy. Treatment of Ehrlich's ascites tumor bearing mice with ad-ASF and glutathione increased their survival, but did not reduce the mortality of animals because of tumor.

Depletion of tumor necrosis factor-alpha in mice by Nyctanthes arbor-tristis.

The effect of the water soluble fraction of the ethanol extract of Nyctanthes arbor-tristis (NAT) on tumor necrosis factor-alpha (TNF-alpha) level in plasma of arthritic and soluble protein A (SpA)-treated Balb/c mice has been studied. Oral administration of this fraction in arthritic mice showed a consistent depletion of TNF-alpha from the host plasma. A similar depletion of TNF-alpha in the plasma of SpA-treated mice has been observed. The extract also reduces plasma interferon-gamma level but the plasma IgM and IgG levels are not affected. The implications of these observations are discussed in the light of management of TNF-alpha in clinical disorders.

Effects of bleomycin and Staphylococcus protein-A on lungs of mice.

Fibrosis of organs and tissues are major causes of morbidity and mortality in human. The currently available pharmacologically based treatments are unsatisfactory. As an experimental animal model antitumor antibiotic drug bleomycin (BLM) is widely used to produce lung fibrosis. The present study has been undertaken to investigate the possible role of a potent immunomodulator Staphylococcus protein-A (SpA) in the modulation of lung lesions caused by treatment of BLM. In mice BLM, 0.5 mg in 200 microliters of normal saline and SpA, 6 micrograms in 200 microliters of normal saline was administered singly or in combination twice a week for 4 weeks. The fibrotic lesions in the lungs were observed after 4 weeks of BLM treatment. After 4 weeks treatment of SpA, the hyperreactive changes in bronchi and bronchioles were observed. In the co-treatment group of BLM and SpA, the effects observed were in the form of enhanced lesions in the lung parenchyma. Moreover, the pleural lesions were also observed in co-treatment group (BLM + SpA). Opposite to the assumption, SpA being a potent immunomodulator was not able to reduce the lung lesions produced by BLM.

Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and protein A in pathogenesis.

Staphylococcus aureus produces a variety of proteins, including alpha-toxin and protein A, that could contribute to corneal tissue damage during keratitis. We examined corneal infections produced by intrastromal injection of four S. aureus strains--three isogenic mutants, one lacking alpha-toxin (Hly- Spa+), one lacking protein A (Hly+ Spa-), and one lacking both alpha-toxin and protein A (Hly- Spa-), and the wild type (Hly+ Spa+)--in a rabbit model of experimental keratitis. Rabbit corneas were injected intrastromally with 100 CFU of one of the four strains, and the eyes were examined by slit lamp biomicroscopy over a 25-h period. Corneal homogenates were used for determination of CFU and neutrophil myeloperoxidase activity at 5-h intervals. All strains had the same logarithmic growth curve from 0 to 10 h postinfection, after which CFU remained constant at 10(7) CFU per cornea. By 15 h postinfection, slit lamp examination scores were significantly higher for eyes infected with Hly+ strains than for Hly(-)-infected eyes. At this time, distinct epithelial erosions were seen in Hly(+)-infected eyes but not in Hly(-)-infected eyes. Myeloperoxidase activity was significantly greater for Hly(+)-infected corneas than for Hly(-)-infected corneas at both 20 and 25 h postinfection. Spa(+)- and Spa(-)-infected eyes showed no differences in slit lamp examination scores or myeloperoxidase activities. These results suggest that alpha-toxin, but not protein A, is a major virulence factor in staphylococcal keratitis, mediating the destruction of corneal tissue in eyes infected with this bacterial pathogen.

Effects of staphylococcal alpha-toxin on the ultrastructure of the rat hypothalamo-neurohypophyseal system.

The influence of staphylococcal alpha-toxin on the ultrastructure of hypothalamo-neurohypophysical system in the brain (nucleus supraopticus, nucleus paraventricularis, neurohypophysis) was studied in the rat. In neurohypophysis, an area lacking blood-brain barrier, alpha-toxin damaged both neuronal endings and capillary vessels. On the other hand in hypothalamus, where blood-brain barrier is present structural alterations were much less pronounced. Reactive gliosis, accordant with cell damage, was observed in the entire neurosecretory system. Putative mechanisms leading to brain damage after systemic administration of alpha-toxin, including direct disruption of cell membrane and induction of nitric oxide synthesis, are discussed.

Staphylococcus aureus proteins that bind to human endothelial cells.

The adherence of Staphylococcus aureus to human endothelial cells is saturable in both dose- and time-dependent assays. Staphylococcal surface components which bound to endothelial cells in vitro were identified by using biotin-labeled, solubilized staphylococcal proteins. Four trypsin-sensitive components with molecular sizes of 30, 55 to 57, 70, and 85 kDa were recognized. These proteins did not label with the glycan detection system. When staphylococci were harvested during the exponential phase of growth, staphylococcal adherence to endothelial cells was significantly increased and increased expression of the S. aureus binding proteins was observed. Preincubation of endothelial cells with protein A did not reduce S. aureus adherence in an in vitro infection assay. Four S. aureus surface components whose expression is growth phase dependent adhere to human endothelial cells in vitro.

Entry of diphtheria toxin-protein A chimeras into cells.

Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.

[Cytotoxic properties of a recombinant hybrid of the A protein of Staphylococcus aureus with a fragment of exotoxin A of Pseudomonas aeruginosa]. / Tsitotoksicheskie svoistva rekombinantnogo gibrida A-belka Staphylococcus aureus s fragmentom ékzotoksina A Pseudomonas aeruginosa.

By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and . Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.

Serological evaluation of melanoma patients in a phase I/II trial of vaccinia melanoma oncolysate (VMO) immunotherapy.

Vaccinia melanoma oncolysates (VMO) were tested in a Southeastern Cancer Study Group (SECSG)-sponsored phase I/II multiinstitutional trial. Forty-eight patients with stage I or II disease were placed on study at six different dose levels of VMO and two different dose schedules, immediate or delayed. Patients' sera, obtained before treatment and every 3 months following initiation of treatment, were tested for antimelanoma antibodies using a Staphylococcus protein A (SpA) assay. Pretreatment sera were negative in 46 of 47 patients, and only two of 19 patients on delayed treatment developed reactivity by 6 months. However, 13 of 23 on immediate treatment developed reactivity, including eight of eight at the higher doses (1.5 and 2.0 mg). Neither anti-HLA antibody tested by a standard microcytotoxicity assay nor circulating immune complexes measured by both Clq and conglutinin binding assays were produced as a result of the immunization. The demonstration of immunogenicity of VMO at the 2 mg dose and immediate schedule supported the rationale for the use of this dose and schedule for the ongoing second phase Ia/Ib trial and for the future phase III randomized prospective study.

Further characterization of the in vitro tumoricidal activity of staphylococcal protein A.

Treatment of plasma or serum from leukemic patients with solid phase staphylococcal Protein A induced leukemic blast cell lysis in vitro, but this effect was relatively independent of the amount of immunoglobulin G (IgG) removed. Samples with approximately equal cytotoxic activity contained markedly different IgG levels, while samples with similar IgG levels had a wide range of tumoricidal activity. Assays of plasma samples collected during a perfusion of one plasma volume through a Protein A-Sepharose column indicated that the duration of the procedure had a greater effect on cytotoxic activity than did the amount of IgG removed. Neither added leukemic nor normal IgG significantly improved blast cell viability in treated serum. Cytotoxic activity was not dialyzable and concentrated in the Mr less than 100,000 fraction of samples separated by filtration. Treated cytotoxic serum samples did not have important Clq binding activity. These results suggest that the in vitro tumoricidal activity of solid phase Protein A is probably due to a toxic substance added to serum during immunoadsorption rather than to its immunoadsorptive capacity.

Protein A and staphylococcal products in neoplastic disease.

Tumoricidal responses and tumor regressions have been observed after plasma perfusion over Staphylococcus aureus Cowan I (SAC), or purified protein A immobilized on solid supports. This system was initially studied in a single human patient and then extended to dogs with spontaneous mammary carcinoma, an excellent model of human breast cancer. In the single patient and dogs with mammary tumors, perfusion of plasma over protein A bearing staphylococcus resulted in tumor necrosis and tumor regression. Tumor reduction or growth retardation with similar perfusion systems has been noted in various feline and rodent tumor models. Tumoricidal responses were also observed in canine tumors after perfusion over commercial protein A which was immobilized in a collodion charcoal matrix (PACC). These responses were amplified when a subtherapeutic and nontoxic dose of cytarabine was given after perfusion. Similar tumor reduction in murine and feline tumor models has been noted after perfusion of autologous serum over protein A immobilized on various other solid supports. The PACC perfusion system was extended to five consecutive patients with advanced breast adenocarcinoma. Four of five patients showed tumor regression after perfusion of small volumes of autologous or homologous plasma over PACC. Patients also experienced pyrexia, nausea, vomiting, and significant cardiopulmonary toxicity. Detailed hemodynamic studies of these effects showed that the major pathophysiology involved a decline in total peripheral resistance associated with an increase in cardiac output. With reduction of immobilized protein A quantity and diminution in plasma perfusion rate, the cardiopulmonary toxicity associated with treatments was diminished. Chemotherapy given as FAC to a single patient shortly after concluding perfusion therapy resulted in rapid regression of residual large tumor masses. Studies focusing on the mechanism of the tumoricidal responses have examined changes in sera after incubation or perfusion over immobilized SAC or PACC. Major findings include (1) the identification of protein A leaching from PACC and SAC after serum perfusion and appearing in the effluent as Clq binding oligomers composed predominantly of IgG and protein A but also containing IgA, IgM and C3 with a molecular weight range of 600,000 to 2,000,000; (2) the identification of C3a anaphylatoxins in serum perfused over PACC or SAC; (3) the recognition that several enterotoxins, in particular enterotoxin B are present in commercial protein A preparation.(ABSTRACT TRUNCATED AT 400 WORDS)