Properties of a general PK/PD model of antibody-ligand interactions for therapeutic antibodies that bind to soluble endogenous targets

Antibodies that target endogenous soluble ligands are an important class of biotherapeutic agents. While much focus has been placed on characterization of antibody pharmacokinetics, less emphasis has been given to characterization of antibody effects on their soluble targets. We describe here the properties of a generalized mechanism-based PK/PD model used to characterize the in vivo interaction of an antibody and an endogenous soluble ligand. The assumptions and properties of the model are explored and situations are described when deviations from the basic assumptions may be necessary. This model is most useful for in vivo situations where both antibody and ligand levels are available following drug administration. For a given antibody exposure, the extent and duration of suppression of free ligand is impacted by the apparent affinity of the interaction, as well as by the rate of ligand turnover. The applicability of the general equilibrium model of in vivo antibody-ligand interaction is demonstrated with an anti-Aβ antibody.Key words: monoclonal antibody, ligand, PK/PD modeling, mechanism-based, antigen     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

Imaging of Fas-FasL membrane microdomains during apoptosis in a reconstituted cell-cell junction

The Fas death receptor interacts with its ligand FasL and induces apoptosis. The Fas-FasL interaction occurs at the cell-cell interface in vivo, since both proteins are expressed in cell membranes. However, most studies on the Fas signal pathway have been performed in a nonphysiological manner by using soluble molecules (antibody or crosslinked FasL proteins) to stimulate Fas. The Fas-FasL interaction at the cell-cell contact site has only been studied recently, but the information derived from cell-cell interaction studies is still rather limited and not necessarily consistent with the past results obtained by using soluble Fas-stimulatory molecules. Therefore, we develop a novel reconstituted system that mimics the Fas-FasL interaction at cell-cell contact sites for further examination of the physiological Fas-FasL signaling system. By conjugating FasL extracellular domains to planar lipid bilayers, we created a model cell membrane to activate Fas-expressing cells. Using this system, we generated an image of Fas-FasL interactions at the cell-membrane interface at high resolution. We observed that the Fas-FasL interaction between two membranes creates submicron membrane microdomains. Shortly after microdomain formation, the cells exhibit various features of apoptosis. These results suggest that our reconstituted system provides a useful platform to dissect Fas-FasL apoptosis signaling at near physiological conditions.     

Specific allowance for antibody bivalence in the determination of dissociation constants by kinetic exclusion assay

Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants (K_d) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium–ethylenediaminetetraacetic acid (Cd–EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen–antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads—a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based.     

Characterization and acceptor preference of a soluble meningococcal group C polysialyltransferase

Vaccines against Neisseria meningitidis group C are based on its α-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation α-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the α-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of α-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight α-2,9-polysialic acid in a nonprocessive fashion when initiated with an α-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the α-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.     

A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa.

The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.     

Binding isotherms for soluble immobilized affinity ligands from spectral titration

The method of spectral titration has been applied to binding equilibria between proteins and soluble immobilized ligands and evaluated using the interaction between Cibacron blue-dextran conjugates and lysozyme. The method is both simple and rapid and provides a convenient screening technique for characterization of soluble adsorbents designed for use in aqueous two-phase affinity extraction or as liquid-phase models for affinity chromatography systems. The results indicate that regardless of ligand density a constant 28% of the total coupled dye is available for high-affinity protein binding at saturation. The dissociation constant for the dye-protein interaction, however, decreases with dye loading. The potential for kinetic investigations has been demonstrated using a stopped-flow apparatus. The results indicate that a simple rate equation is inadequate to describe the data for lysozyme binding to dye-dextran conjugates. A modified model, which better describes the data, was developed by including a second rate limiting process, the transition from stacked to unstacked dye ligands on the dextran backbone. This effect could have practical significance for protein binding kinetics in affinity chromatography, especially in high-performance liquid affinity chromatography applications where mass transfer is rapid. (c) 1992 John Wiley & Sons, Inc.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997     

Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry

BACKGROUND: The kinetics of protein-protein interactions can be monitored with optical biosensors based on the principles of either surface plasmon resonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane. METHODS: We monitored with an IASys biosensor system, based on a resonant mirror: (1) the binding of cells to an immobilized ligand, (2) the binding of a soluble ligand to immobilized cells, and (3) the binding of a soluble ligand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic flow cytometry. RESULTS: With an optical biosensor, the useful configuration is the one based on immobilized plasma membrane vesicles. However, signals can be detected only for very abundant binding sites (>10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor. CONCLUSIONS: Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode.     

CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.     

FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development

Latrophilins (LPHNs) are a small family of G protein-coupled receptors known to mediate the massive synaptic exocytosis caused by the black widow spider venom α-latrotoxin, but their endogenous ligands and function remain unclear. Mutations in LPHN3 are strongly associated with attention deficit hyperactivity disorder, suggesting a role for latrophilins in human cognitive function. Using affinity chromatography and mass spectrometry, we identify the FLRT family of leucine-rich repeat transmembrane proteins as endogenous postsynaptic ligands for latrophilins. We demonstrate that the FLRT3 and LPHN3 ectodomains interact with high affinity in trans and that interference with this interaction using soluble recombinant LPHN3, LPHN3 shRNA, or FLRT3 shRNA reduces excitatory synapse density in cultured neurons. In addition, reducing FLRT3 levels with shRNA in vivo decreases afferent input strength and dendritic spine number in dentate granule cells. These observations indicate that LPHN3 and its ligand FLRT3 play an important role in glutamatergic synapse development.     

Multimeric soluble CD40 ligand and GITR ligand as adjuvants for human immunodeficiency virus DNA vaccines

For use in humans, human immunodeficiency virus (HIV) DNA vaccines may need to include immunostimulatory adjuvant molecules. CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily (TNFSF), is one candidate adjuvant, but it has been difficult to use because it is normally expressed as a trimeric membrane molecule. Soluble trimeric forms of CD40L have been produced, but in vitro data indicate that multimeric, many-trimer forms of soluble CD40L are more active. This multimerization requirement was evaluated in mice using plasmids that encoded either 1-trimer, 2-trimer, or 4-trimer soluble forms of CD40L. Fusion with the body of Acrp30 was used to produce the 2-trimer form, and fusion with the body of surfactant protein D was used to produce the 4-trimer form. Using plasmids for secreted HIV-1 antigens Gag and Env, soluble CD40L was active as an adjuvant in direct proportion to the valence of the trimers (1 < 2 < 4). These CD40L-augmented DNA vaccines elicited strong CD8(+) T-cell responses but did not elicit significant CD4(+) T-cell or antibody responses. To test the applicability of the multimeric fusion protein approach to other TNFSFs, a 4-trimer construct for the ligand of glucocorticoid-induced TNF family-related receptor (GITR) was also prepared. Multimeric soluble GITR ligand (GITRL) augmented the CD8(+) T-cell, CD4(+) T-cell, and antibody responses to DNA vaccination. In summary, multimeric CD40L and GITRL are new adjuvants for DNA vaccines. Plasmids for expressing multimeric TNFSF fusion proteins permit the rapid testing of TNFSF molecules in vivo.     

The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity.

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti-CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39-expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39-expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co-stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39-CD40 to drive proliferation and that soluble gp39 alone in a non-membrane bound form is able to provide co-stimulatory signals to B cells.     

Nucleic acids exert a sequence-independent cooperative effect on sequence-dependent activation of Toll-like receptor 9

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect.     

Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.     

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.     

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.     

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.     

Homogeneous time-resolved G protein-coupled receptor–ligand binding assay based on fluorescence cross-correlation spectroscopy

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor–ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.     

Antibody-directed liposomes Determination of affinity constants for soluble and liposome-bound antifluorescein

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.