Effect of nesiritide in patients with acute decompensated heart failure.

BACKGROUND: Nesiritide is approved in the United States for early relief of dyspnea in patients with acute heart failure. Previous meta-analyses have raised questions regarding renal toxicity and the mortality associated with this agent. METHODS: We randomly assigned 7141 patients who were hospitalized with acute heart failure to receive either nesiritide or placebo for 24 to 168 hours in addition to standard care. Coprimary end points were the change in dyspnea at 6 and 24 hours, as measured on a 7-point Likert scale, and the composite end point of rehospitalization for heart failure or death within 30 days. RESULTS: Patients randomly assigned to nesiritide, as compared with those assigned to placebo, more frequently reported markedly or moderately improved dyspnea at 6 hours (44.5% vs. 42.1%, P=0.03) and 24 hours (68.2% vs. 66.1%, P=0.007), but the prespecified level for significance (P≤0.005 for both assessments or P≤0.0025 for either) was not met. The rate of rehospitalization for heart failure or death from any cause within 30 days was 9.4% in the nesiritide group versus 10.1% in the placebo group (absolute difference, -0.7 percentage points; 95% confidence interval [CI], -2.1 to 0.7; P=0.31). There were no significant differences in rates of death from any cause at 30 days (3.6% with nesiritide vs. 4.0% with placebo; absolute difference, -0.4 percentage points; 95% CI, -1.3 to 0.5) or rates of worsening renal function, defined by more than a 25% decrease in the estimated glomerular filtration rate (31.4% vs. 29.5%; odds ratio, 1.09; 95% CI, 0.98 to 1.21; P=0.11). CONCLUSIONS: Nesiritide was not associated with an increase or a decrease in the rate of death and rehospitalization and had a small, nonsignificant effect on dyspnea when used in combination with other therapies. It was not associated with a worsening of renal function, but it was associated with an increase in rates of hypotension. On the basis of these results, nesiritide cannot be recommended for routine use in the broad population of patients with acute heart failure. (Funded by Scios; ClinicalTrials.gov number, NCT00475852.).

Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant III monoclonal antibody.

The mutant epidermal growth factor receptor variant III (EGFRvIII) has been found on gliomas and other tumors but not on normal tissues, including those that express the wild-type receptor. Monoclonal antibodies (mAbs) specific for EGFRvIII are rapidly internalized and degraded after binding to EGFRvIII-expressing cells. If anti-EGFRvIII mAbs are to be useful for radioimmunotherapy, then methods for trapping radionuclides in target cells after mAb processing are required. Because lysosomes are known to retain positively charged molecules, we have evaluated a new reagent for this purpose that uses a polycationinc peptide composed of D-amino acids (D-Lys-D-Arg-D-Tyr-D-Arg-D-Arg; D-KRYRR). D-KRYRR was first labeled using lodogen and then coupled to the murine anti-EGFRvIII mAb L8A4 via maleimido bond formation in 60% yield. In vitro assays with the U87deltaEGFR cell line indicated that internalized and total cell-associated activity for the 125I-labeled D-KRYRR-L8A4 conjugate were up to 4 and 5 times higher, respectively, than for L8A4 labeled with 131I using Iodogen. Paired-label comparisons in athymic mice with s.c. U87deltaEGFR xenografts demonstrated up to 5-fold higher tumor uptake for mAb labeled using D-KRYRR. Higher levels of radioiodine activity also were observed in kidney when L8A4 was labeled using D-KRYRR. Another paired-label study directly compared L8A4 labeled using radioiodinated D-KRYRR and L-KRYRR, and confirmed the role of D-amino acids in enhancing tumor uptake. These results suggest that D-KRYRR is a promising reagent for the radioiodination of internalizing mAbs, such as the anti-EGFRvIII mAb L8A4.

125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody using N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate.

Monoclonal antibodies (MAbs) such as the anti-epidermal growth factor variant III (EGFRvIII) MAb L8A4 are rapidly internalized, which can lead to rapid loss of radioactivity from the tumor cell. The aim of this study was to evaluate the potential utility of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate ([211At]SAPC) for labeling murine L8A4 with 211At. SAPC was synthesized by astatodestannylation of N-succinimidyl 5-tri-n-butylstannyl 3-pyridinecarboxylate and then coupled to L8A4 in approximately 50% yield. The affinity and immunoreactive fraction for 211At-labeled L8A4 were comparable to those obtained when the MAb was labeled with 131I via N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). Paired-label comparisons of the 211At- and 131I-labeled MAbs demonstrated similar internalization and catabolism by EGFRvIII-positive cells in vitro, and with the exception of the stomach, similar tissue distribution in athymic mice with EGFRvIII-expressing U87MGdeltaEGFR xenografts. These results suggest that SAPC may be a useful reagent for labeling L8A4, and possibly other internalizing proteins, with 211At.

Dosimetry of 131I-labeled 81C6 monoclonal antibody administered into surgically created resection cavities in patients with malignant brain tumors.

UNLABELLED: The objective of this study was to perform the dosimetry of 131I-labeled 81C6 monoclonal antibody (MAb) in patients with recurrent malignant brain tumors, treated by direct injections of MAb into surgically created resection cavities (SCRCs). METHODS: Absorbed dose estimates were performed for nine patients. Dosimetry was performed retrospectively using probe counts (during patient isolation) and whole-body and SPECT images thereafter. Absorbed doses were calculated for the SCRC interface and for regions of interest (ROIs) 1 and 2 cm thick, measured from the margins of cavity interface. Also, mean absorbed doses were calculated for normal brain, liver, spleen, thyroid gland, stomach, bone marrow and whole body. The average residence time for the SCRC was 111 h (65-200h). RESULTS: The average absorbed dose per unit injected activity (range) to the SCRC interface and ROIs 1 and 2 cm thick from the cavity interface were 31.9 (7.8-84.2), 1.9 (0.7-3.6) and 1.0 (0.4-1.8) cGy/MBq, respectively. Average absorbed doses per unit administered activity to brain, liver, spleen, thyroid, stomach, bone marrow and whole body were 0.18, 0.03, 0.08, 0.05, 0.02, 0.02 and 0.01 cGy/MBq, respectively. The high absorbed dose delivered to the SCRC interface may have produced an increase in cavity volume independent of tumor progression. CONCLUSION: At the maximum tolerated dose of 3700 MBq 131I-labeled 81C6 MAb, the absorbed doses to the SCRC interface and ROIs of 1 and 2 cm thickness were estimated to be 1180, 71 and 39 Gy, respectively. The estimated average absorbed dose to the brain was 6.5 Gy. There was no neurological toxicity and minimal hematologic toxicity at this maximum tolerated administration level.

Human IgG2 constant region enhances in vivo stability of anti-tenascin antibody 81C6 compared with its murine parent.

The in vivo properties of radiolabeled chimeric monoclonal antibodies (mAbs) with human IgG1 and IgG3 constant regions generally are similar to those of their corresponding murine construct. In contrast, we have observed that chimeric anti-tenascin mAb 81C6, which contains IgG2 constant regions, exhibits significantly higher localization in s.c. D-54 MG xenografts and prolonged retention in most normal tissues compared with its IgG2b murine parent. The purpose of the present study was to determine whether substitution of the murine IgG2b constant region domains in mAb 81C6 with those from human IgG2 enhanced the in vivo stability of the 81C6 mAb. Both mAbs were radioiodinated using Iodogen and administered to athymic mice bearing s.c. D-54 MG human glioma xenografts. The nature of the labeled species present in tumor and normal tissues over a 144-h period was investigated by trichloroacetic acid precipitation and SDS PAGE. In tumor and most normal tissues, a greater fraction of chimeric compared with murine 81C6 was present as intact IgG. For example, in tumor at 144 h, the fraction of radioactivity present as intact IgG was twice as high for chimeric compared with murine 81C6. A substantial fraction of murine but not chimeric 81C6 was present as a Mr 70,000-90,000 molecule, which could represent the generation of Fab/Fc monomers through the reduction of the interchain disulfide bonds in the murine IgG2b molecule. These results suggest that the higher tumor and normal tissue levels of chimeric compared with murine 81C6 can be attributed in part to the enhanced in vivo stability of the IgG2 chimeric mAb. The chimeric construct also was demonstrated to be more stable than murine after incubation with cyst fluid obtained from glioma resection cavity patients. Chimeric mAbs containing human IgG2 constant region domains could be of particular value for certain radioimmunotherapeutic applications.

The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target.

Any immunotherapeutic approach to cancer cell eradication is based upon the specific recognition of neoplastic cells and the sparing of surrounding normal tissue; perhaps nowhere is this distinction more important than within the central nervous system, due to the diffuse infiltrative nature of primary glial tumor cell growth. Whether ultimate effect moieties are immunoglobulins, fragments and/or their constructs with drugs, toxins, radionuclides, or immune cells, the specificity of effector: cell surface marker is crucial. This review describes the identification, immunologic characterization, and biologic behavior of a transmembrane tumor-specific altered growth factor receptor molecule which may well serve as a mediator of multiple immunotherapeutic approaches: the class III variant of the epidermal growth factor receptor, EGFRvIII.

In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent.

The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MG delta EGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MG delta EGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4.

Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate.

Monoclonal antibody (mAb) L8A4, specific for the tumor-associated mutant epidermal growth factor receptor variant III (EGFRvII), is internalized and degraded after cell binding. Four paired-label experiments were performed in athymic mice bearing EGFRvIII-positive xenografts to determine the suitability of N-succinimidyl 3-iodo-5-pyridinecarboxylate (SIPC) for labeling this internalizing mAb. In mice with HC2 20 d2 xenografts, tumor uptake reached a maximum of 32.7 +/- 2.0% injected dose/g when labeled using SIPC, a value significantly higher (P < 0.05, paired t test) than that observed when L8A4 was labeled using lodogen (24.4 +/- 2.2% injected dose/g). The specificity of mAb uptake in HC2 20 d2 and U87MG(delta)EGFR xenografts was measured in separate experiments by coadministration of L8A4 and nonspecific, isotype-matched P3X63Ag8 mAb, both radioiodinated using SIPC. Tumor localization indices were approximately 10 or more by 72 h, a degree of specificity 3-4 times higher than that reported previously when labeling was performed using the tyramine cellobiose (TCB) method. In a final study directly comparing L8A4 labeled using SIPC and TCB, similar tumor levels were obtained (SIPC, 33.7 +/- 6.1% injected dose/g at 24 h; TCB, 37.8 +/- 6.7% injected dose/g at 24 h); however, tumor-to-tissue ratios for the liver, spleen, and kidneys were 3 times higher with SIPC at later time points. These results suggest that SIPC is a promising method for labeling this anti-EGFRvIII mAb and possibly other mAbs that internalize after binding.

L-amino acid oxidase (LOX) modulation of melphalan activity against intracranial glioma.

These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P < 0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 micrograms administered intravenously. Polyclonal anti-LOX antibody was successfully produced in mice, rabbits, and goats subsequent to immunization with LOX. Staphylococcal protein A-purified rabbit anti-LOX serum inhibited approximately 50% of LOX activity in vitro relative to control samples. This antiserum was used in vivo to inactivate LOX after it had depleted the large neutral amino acids, thereby preventing LOX-mediated catabolism of melphalan. Inoculation of three mice with rabbit anti-LOX serum after the treatment with LOX (100 micrograms) reduced LOX activity by 100%, 89%, and 100% at 6 h compared with reductions of 80%, 59%, and 52% over the same period in animals receiving LOX alone. In three separate studies using groups of eight to ten mice bearing intracranial human glioma xenografts, pretreatment with LOX followed by anti-LOX serum increased the antitumor activity of melphalan as compared with treatments with melphalan plus LOX, melphalan plus anti-LOX serum, or melphalan alone.

Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate.

Monoclonal antibodies (mAbs) that internalize following binding to cell-surface receptors require radiolabeling approaches that minimize loss of radioactivity from the cell after intracellular processing. One class of internalizing mAbs of great interest for imaging and radioimmunotherapy are those specific for EGFRvIII, a truncated form of the epidermal growth factor receptor found on gliomas, non-small cell lung carcinomas, breast carcinomas, and ovarian carcinomas. Because lysosomes are known to retain positively charged compounds, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) might be ideal for radioiodination of these mAbs because of the positive charge on its pyridine ring. To investigate this hypothesis, the anti-EGFRvIII mAb L8A4 was labeled using SIPC, and internalization assays were performed using the EGFRvIII-positive cell lines HC2 20 d2 and NR6M. Compared with L8A4 labeled using Iodogen or N-succinimidyl 3-iodobenzoate, SIPC increased intracellular retention of activity by up to 65%. Reverse-phase high-performance liquid chromatography analyses indicated that a significantly higher fraction of the low molecular weight catabolites from mAbs labeled via SIPC were retained within cells (SIPC, 28.1%; Iodogen, 7.6% at 1 h). With SIPC, the primary labeled species in cell lysates was the 5-iodonicotinic acid (INA)-lysine conjugate, whereas in the supernatant, both INA-lysine and INA were seen. A 3-4-fold higher percentage of these catabolites were charged at lysosomal pH in comparison with those from mAb labeled using N-succinimidyl 3-iodobenzoate, in concert with the differences in cellular retention observed between these two labeling methods. In mice bearing HC2 20 d2 xenografts, a significant improvement in tumor retention of radioiodine and tumor:normal tissue ratios was seen when L8A4 was labeled using SIPC instead of the Iodogen method. These results suggest that SIPC is a promising reagent for the radioiodination of anti-EGFRvIII L8A4 and, possibly, other internalizing mAbs.

Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts.

Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene are characteristics of many types of tumors. One class of EGFR mutations, EGFRvIII, is characterized by an in-frame deletion resulting in a truncated external domain of the receptor. EGFRvIII was first identified in a subset of gliomas and has since been found in some non-small cell lung carcinomas and breast carcinomas. mAbs specific for this variant form of EGFR but unreactive with the wild-type EGFR have been reported from our laboratory. This study further characterizes three of these antibodies. We determined, via radiolabeling techniques and immunofluorescence microscopy, that, after cell binding in vitro, the anti-EGFRvIII-specific mAbs internalize at 37 degrees C. Furthermore, subsequent to internalization, the antibodies were processed intracellularly, presumably by lysosomal degradation. We also examined the use of an alternative radiolabeling procedure that uses nonmetabolizable radio-iodinated tyramine cellobiose. Our results show that the tyramine cellobiose labeling method allows for greater tumor cell retention of radiolabel in vitro (76% for tyramine cellobiose and 27% for Iodo-Gen after 24 h). Paired-label biodistribution studies in athymic mice indicate that anti-EGFRvIII mAb L8A4 localizes specifically to EGFRvIII-expressing tumor xenografts with a maximum of 34.3 +/- 7.6% injected dose/g when labeled using tyramine cellobiose compared with a maximum of 14.9 +/- 4.3% injected dose/g using Iodo-Gen; similar results were obtained with mAb H10. These results suggest that the anti-EGFRvIII mAbs may serve as potential carriers for radioconjugate- and immunotoxin-based therapies for tumors expressing EGFRvIII.

Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.

Despite molecular biological advances in understanding human cancers, translation into therapy has been less forthcoming; targeting neoplastic cells still requires that tumor-specific markers, preferably those on the cell surface, be identified. The epidermal growth factor receptor (EGFR) exists in a deletion-mutant form, EGFRvIII, which has been identified by genetic and immunological means in a subset of gliomas and non-small cell lung carcinomas. Specific polyvalent antisera to the extracellular portion of the variant were readily induced, but immunization using a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We report here five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic peptide and the intact variant in different formats that maintained secondary and tertiary conformation. These mAbs identify the EGFRvIII on the cell surface with relatively high affinity (KA range, 0.13 to 2.5 x 10(9) M-1) by live cell Scatchard analysis. These mAbs are specific for EGFRvIII as determined by RIA, ELISA, Western blot, analytical flow cytometry, autophosphorylation, and immunohistochemistry. Isolating specific mAbs enabled us to analyze normal and neoplastic human tissue and establish that EGFRvIII is truly tumor specific for subsets of breast carcinomas and for previously reported non-small cell lung carcinomas and gliomas. Also, this receptor is not expressed by any normal human tissues thus far examined, including elements of the peripheral, central nervous, and lymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII positivity in gliomas than previously described and identified an EGFRvIII-positive subset of breast tumors; also, we observed that the EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumors expressing this epitope. Our observations strongly warrant development of this mAb-antigen system as therapy for breast, lung, and central nervous system tumors.

Complement-independent binding of microorganisms to primate erythrocytes in vitro by cross-linked monoclonal antibodies via complement receptor 1.

Under certain circumstances, soluble antigens, particulate antigens, and/or microorganisms have been shown to bind to primate erythrocytes via complement receptor 1 (CR1) in the presence of specific antibodies and complement. This immune adherence reaction, specific for CR1, can lead to neutralization of antigens in the circulation and their subsequent clearance from the blood. The present experiments utilized cross-linked monoclonal antibody complexes (heteropolymers) with specificity for both CR1 and either 35S-labeled herpes simplex virus capsid or Haemophilus influenzae as prototype viral and bacterial particulate antigens, respectively. In each case, the respective specific heteropolymers facilitated binding of the target antigens (> or = 70 to 90%) in vitro to erythrocytes in the absence of complement. Several experimental protocols were employed to demonstrate that heteropolymers mediate specific, rapid (> or = 30 s), and quantitative binding of prototypical particulate pathogens to human and monkey erythrocytes but not to sheep erythrocytes, which lack CR1. These results extend the potential use of the erythrocyte-heteropolymer system to the neutralization and clearance of particulate viral and bacterial pathogens from the blood.

Antigen-based heteropolymers. A potential therapy for binding and clearing autoantibodies via erythrocyte CR1.

OBJECTIVE: To determine if complexes containing monoclonal antibodies to CR1 cross-linked with antigen (antigen-based heteropolymers [AHP]) can bind the corresponding autoantibody to primate erythrocyte CR1 and promote autoantibody clearance from the circulation. METHODS: AHP were constructed by cross-linking double-stranded DNA (dsDNA) to monoclonal antibodies to CR1. The ability of AHP to facilitate binding of human anti-dsDNA antibodies to primate erythrocytes was studied in vitro using a variety of radioimmunoassays (including Farr assays), enzyme immunoassays, and fluorescence-activated cell sorting. In addition, we used a monkey model to study in vivo the AHP-mediated clearance of passively infused human anti-dsDNA antibodies. RESULTS: Large amounts of lupus IgG anti-dsDNA antibodies can be specifically bound to human erythrocytes via the complexes, and studies in 2 rhesus monkeys indicate that the erythrocyte-bound antibodies are rapidly cleared from the circulation. CONCLUSION: This methodology may allow for development of a new therapy to facilitate autoantibody clearance in autoimmune disease.

Cross-linked bispecific monoclonal antibody heteropolymers facilitate the clearance of human IgM from the circulation of squirrel monkeys.

We have previously demonstrated that cross-linked bispecific monoclonal antibodies (mAb) heteropolymers (HP), specific for primate erythrocyte (E) complement receptor type 1 (CR1) and target antigen (Ag), facilitate the binding of these target Ag to human and non-human primate E. Once bound in vitro to rhesus monkey E, upon re-infusion these HP/Ag complexes are recognized in vivo by cells of the reticuloendothelial system (RES) and removed from the circulation without loss of the E. We now show, in squirrel monkeys, that an HP specific for E CR1 and human IgM (anti-CR1 x anti-IgM) can be used to facilitate in vivo E binding and clearance from the circulation of a previously injected and circulating model protein pathogen, human IgM. Approximately 70-80% of 125I-labeled human IgM is cleared from the circulation of each of five squirrel monkeys via the HP system. We observe, in experiments analogous to previous studies on immune complex (IC) clearance, that subsequent to HP/Ag clearance there is a decrease in the number of CR1 epitopes per E which is manifested when we use both monoclonal and polyclonal anti-CR1 probes. Our results indicate that the primary organs responsible for uptake of the complexes are the liver and spleen. This work strongly suggests that the HP/Ag complexes, bound to E, function as IC prototypes and are recognized and processed as such in vivo. Thus, the HP-E system may eventually serve as a viable immunotherapy for the clearance of blood-borne pathogens from the circulation.

Antigens pre-bound to the primate erythrocyte complement receptor via cross-linked bispecific monoclonal antibody heteropolymers are rapidly cleared from the circulation.

We have used cross-linked, bispecific monoclonal antibody heteropolymers, specific for primate erythrocyte (E) complement receptor (CR1) and target antigen (Ag), to attach 125I-labeled Ag to 51Cr-labeled monkey erythrocytes (E) in vitro. Injection of these sensitized E into monkeys leads to rapid clearance from the circulation of several different 125I-labeled Ag with little, if any sequestration, lysis, or clearance of the E. It should now be possible, in principle, to use the heteropolymer-E system to facilitate the clearance of blood-borne pathogens from the circulation.

Human IgG in immune complexes bound to human erythrocyte CR1 is recognized differently than human IgG bound to an erythrocyte surface antigen.

We have developed several methods, including a new use of magnetic cell separation techniques, to examine how human IgG bound to human erythrocytes can be recognized. We find that human IgG in complement-opsonized immune complexes bound to erythrocyte CR1 is less accessible to several probes than human IgG bound to the rhesus antigen on erythrocytes. These findings suggest that the mechanism by which immune complexes are removed and cleared from erythrocyte CR1 in the circulation is different from the mechanisms which lead to splenic clearance of IgG sensitized erythrocytes.

In vivo binding and clearance of circulating antigen by bispecific heteropolymer-mediated binding to primate erythrocyte complement receptor.

We have used the avidin/biotin system to construct soluble, cross-linked bispecific heteropolymers containing mAb to both the primate E C receptor and the DNP group. These heteropolymers facilitate in vitro binding of DNP-bovine gamma-globulin (DNP-BGG) to both human and squirrel monkey E. Intravenous injection in squirrel monkeys of DNP-BGG followed by heteropolymer leads to E binding and clearance from the circulation of a significant fraction of both heteropolymer and DNP-BGG, without lysis or clearance of the E. This methodology may potentially be used to treat a variety of infectious diseases and other syndromes associated with blood-borne pathogens.

Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: a potential therapeutic treatment.

We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine gamma globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37 degrees C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.