CAR-T Engager proteins optimize anti-CD19 CAR-T cell therapies for lymphoma.

B cell lymphoma therapy has been transformed by CD19-targeting cellular therapeutics that induce high clinical response rates and impressive remissions in relapsed and refractory patients. However, approximately half of all patients who respond to CD19-directed cell therapy relapse, the majority within 6 months. One characteristic of relapse is loss or reduction of CD19 expression on malignant B cells. We designed a unique therapeutic to prevent and reverse relapses due to lost or reduced CD19 expression. This novel biologic, a CAR T Engager, binds CD20 and displays the CD19 extracellular domain. This approach increases the apparent CD19 antigen density on CD19-positive/CD20-positive lymphoma cells, and prevents antigen-loss induced relapse, as CD19 bound to CD20 remains present on the cell surface. We demonstrate that this novel therapeutic prevents and reverses lymphoma relapse in vitro and prevents CD19-negative lymphoma growth and relapse in vivo.

Engineered protein-small molecule conjugates empower selective enzyme inhibition.

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

Anti-CD19 CAR T Cells That Secrete a Biparatopic Anti-CLEC12A Bridging Protein Have Potent Activity Against Highly Aggressive Acute Myeloid Leukemia In Vitro and In Vivo.

Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-anti-CLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.

Anti-CD19 CAR T cells potently redirected to kill solid tumor cells.

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design.

The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity-stability trade-offs. These fundamental insights into CD19 sequence-function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.

Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins.

CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2+ CD19- tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.

Targeting the interleukin-11 receptor α in metastatic prostate cancer: A first-in-man study.

BACKGROUND: Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors ("vascular zip codes") within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS: The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα-based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS: BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m(2) ). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS: These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients.

Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer.

Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells.

Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.

Interferon-beta reduces proteinuria in experimental glomerulonephritis.

Interferon-beta (IFN-beta) is a multifunctional cytokine with immunomodulatory properties. We examined the effect of IFN-beta in three separate rat models of glomerular injury and in cultured human glomerular endothelial cells and podocytes. In nephrotoxic nephritis in WKY rats, recombinant rat IFN-beta started either at induction or after establishment of disease significantly reduced 24-h proteinuria by up to 73% and 51%, respectively, but did not affect serum creatinine. There was a slight reduction in numbers of glomerular macrophages, but no difference in glomerular or tubulointerstitial scarring. In Thy-1 nephritis in Lewis rats, IFN-beta started at induction of disease reduced proteinuria by up to 66% with no effect on numbers of glomerular macrophages, but a reduced number of proliferating cells. In puromycin nephropathy in Wistar rats, IFN-beta started at induction of disease reduced proteinuria by up to 93%, but had no effect on glomerular histology. In cultured cells, human IFN-beta-1a had a dramatic effect on barrier properties, increasing electrical resistance across monolayers of either glomerular endothelial cells or podocytes and decreasing trans-monolayer passage of albumin. In conclusion, these results show that IFN-beta reduces proteinuria in three different rat models of glomerular injury and that its anti-proteinuric action may result from direct effects on cells that comprise the glomerular filtration barrier. These data indicate that IFN-beta may have potential as a therapeutic agent in proteinuric renal disease.

Alphav beta6 integrin regulates renal fibrosis and inflammation in Alport mouse.

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Anti-alpha4 integrin monoclonal antibody inhibits multiple myeloma growth in a murine model.

In a syngeneic murine model of multiple myeloma with many of the characteristics of the human disease, a monoclonal antibody (mAb) to the integrin very late antigen-4 (VLA-4), given after the myeloma has already homed to and begun to establish itself within the bone marrow compartment, produces statistically significant effects on multiple disease variables. These include reductions in circulating levels of IgG2b; percentage of IgG2b-positive myeloma cells circulating in blood; spleen weight; and myeloma cell burden in spleen, bone marrow, and liver. mAb therapy had no effect on nonmalignant hematopoietic cells. An acute 6-day regimen of mAb treatment, initiated very late in disease to avoid mAb elimination in the immunocompetent animals, still significantly reduced spleen and blood myeloma cell burden. The ability of the (VLA-4) mAb to affect multiple variables in this model, even as monotherapy, suggests this pathway plays a central role in disease progression.

Rational design of potent and selective VLA-4 inhibitors and their utility in the treatment of asthma.

Asthma, a chronic inflammatory disease of the airways, is a significant burden on our healthcare system. There is high unmet need for treatments directed towards the underlying causes of the disease. The cell surface integrin VLA-4 (very late antigen-4; alpha4beta1; CD49d/CD29) plays an important role in the trafficking of white blood cells to sites of inflammation and represents an exciting target for the development of novel anti-inflammatory drugs for the treatment of asthma. Here, we review our efforts to use rational design to identify potent, selective inhibitors of VLA-4. We describe the discovery of a series of potent VLA-4 inhibitors through the addition of a novel N-terminal organic cap to a tetrapeptide VLA-4 binding motif 4-((N'-2-methylphenyl)uriedo)phenylacetyl-Leu-Asp-Val-Pro ; Kd = 70 pM), and rationalize their structure-activity relationships using 3D-QSAR. Also, we show our rational peptidomimetic design strategy using "template hopping" from the gpIIb/IIIa integrin antagonist field, and also a novel virtual screening strategy. Two series have been developed, one that has high selectivity for the activated over the non-activated state of the receptor, and the other which is non-selective inhibiting both activated and non-activated VLA-4. Both series are highly selective for VLA-4 versus against other integrin family members. These inhibitors show promise in the treatment of asthma, based upon efficacy in a sheep model of asthma, where they inhibit both the early and late-phase responses to asthma and also block hypersensitivity.

Direct evidence of leukocyte adhesion in arterioles by angiotensin II.

Although leukocytes adhere in arteries in various vascular diseases, to date no endogenous proinflammatory molecule has been identified to initiate leukocyte adhesion in the arterial vasculature. This study was undertaken to assess angiotensin II (Ang II)-induced leukocyte adhesion in arterioles in vivo. Rats received intraperitoneal injections of Ang II; 4 hours later, leukocyte recruitment in mesenteric microcirculation was examined using intravital microscopy. Ang II (1 nM) produced significant arteriolar leukocyte adhesion of mononuclear cells. Using function-blocking monoclonal antibodies (mAbs) against different rat cell adhesion molecules (CAMs), we discovered that this effect was dependent on P-selectin and beta(2)-integrin. In postcapillary venules, Ang II also induced leukocyte infiltration, which was reduced by P-selectin and by beta(2)- and alpha(4)-integrin blockade. Interestingly, neutrophils were the primary cells recruited in venules. Although beta(2)-integrin expression in peripheral leukocytes of Ang II-treated animals was not altered, it was increased in peritoneal cells. Immunohistochemical studies revealed increased P-selectin, E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression in response to Ang II in arterioles and venules. These findings provide the first evidence that Ang II causes leukocyte adhesion to the arterial endothelium in vivo at physiologically relevant doses. Therefore, Ang II may be a key molecule in cardiovascular diseases in which leukocyte adhesion to the arteries is a characteristic feature.

A monoclonal antibody to alpha1beta1 blocks antigen-induced airway responses in sheep.

The integrin alpha1beta1 (very late antigen-1; CD49a/CD29) is a major adhesion receptor for collagen I, IV, and VI, and its induced expression on activated monocytes and lymphocytes plays a central role in their retention and activation at inflammatory sites in autoimmune pathologies. However, the role of alpha1beta1 in allergic settings has not been explored. In this study, we show that a single 45-mg dose of aerosolized monoclonal antibody AQC2 to the alpha1 chain of human and sheep very late antigen-1, given 30 minutes before challenge, blocks both the allergen-induced late response and the associated airway hyperresponsiveness, functional indicators of allergen-induced inflammation, in sheep. AQC2 does not affect the early response. Consistent with these effects, AQC2 tended to reduce the cell response associated with local antigen instillation. An isotype-matched control antibody had no protective effects. Two humanized versions of AQC2, a wild-type IgG1 and an aglycosyl form of the same monoclonal antibody, which has reduced Fc receptor-mediated effector functions, are equally effective in blocking the antigen-induced late response and airway hyperresponsiveness in the sheep model. These data suggest that mononuclear leukocyte adhesion-dependent pathologies contribute to allergic lung disease and provide proof-of-concept that antagonists of alpha1 integrins may be useful in preventing these events.

Blocking VLA-4 prevents progression of experimental crescentic glomerulonephritis.

BACKGROUND/AIMS: Integrins are adhesion molecules of fundamental importance to the recruitment of leucocytes in inflammation. The alpha4beta1 integrin (VLA-4) is a leucocyte ligand for endothelial vascular cell adhesion molecule-1 (VCAM-1), fibronectin and osteopontin. We addressed the role of VLA-4 in mediating progressive renal injury in vivo using a blocking monoclonal antibody (mAb) in a rat model of crescentic glomerulonephritis. METHODS: WKY rats with nephrotoxic nephritis were given anti-VLA-4 or control mAb at 2.5 mg/kg by i.p. injection on alternate days. In separate experiments, antibodies were given from days 5-13, from days 13-21 or from days 14-28. RESULTS: Early treatment with anti-VLA-4 mAb from days 5-13 showed a significant effect on renal function, with a reduction in albuminuria (p < 0.01) and a higher creatinine clearance (p < 0.05). Delayed treatment from days 13-21 also showed a reduction in albuminuria (p < 0.05) and serum creatinine (p < 0.05). However, there was no significant effect on glomerular or interstitial scarring in these two experiments. In the late treatment study, in which anti-VLA-4 mAb was administered from days 14-28, serum creatinine was reduced (p < 0.05), creatinine clearance was improved (p < 0.05), and renal survival was significantly prolonged (p < 0.05). Interstitial scarring was significantly less in treated rats (p < 0.05). Glomerular macrophage and CD8+ cell counts were higher in anti-VLA-4 mAb treated rats (p < 0.05), possibly reflecting greater glomerular scarring in control animals. CONCLUSION: Leucocyte VLA-4 mediates pro-inflammatory and pro-fibrotic effects within the kidney, independent of any role in recruitment of leucocytes into the kidney. Blocking VLA-4 is a promising therapeutic approach in human glomerulonephritis.

Obtustatin: a potent selective inhibitor of alpha1beta1 integrin in vitro and angiogenesis in vivo.

A novel disintegrin, obtustatin, was purified from the venom of the Vipera lebetina obtusa viper. Obtustatin is the shortest disintegrin yet described, containing only 41 amino acids. It contains a similar pattern of cysteines to the short disintegrins echistatin and eristostatin but contains the sequence KTS rather than RGD in its active site loop. Obtustatin is a potent and selective inhibitor of alpha1beta1 integrin. It does not inhibit the closely related integrin alpha2beta1, nor a panel of other integrins tested. It does not inhibit ligand binding to the recombinant alpha1 I-domain. Importantly, obtustatin potently inhibited angiogenesis in vivo in the chicken chorioallantoic membrane assay, and in the Lewis lung syngeneic mouse model, it reduced tumor development by half, confirming and extending previous results on the relevance of alpha1beta1 integrin to angiogenesis and suggesting novel approaches to the generation of angiogenesis inhibitors.

Treatment with an antibody to VLA-1 integrin reduces glomerular and tubulointerstitial scarring in a rat model of crescentic glomerulonephritis.

The alpha 1 beta 1 integrin (VLA-1) is a major collagen/laminin receptor that regulates fibroblast proliferation and mesangial cell migration and cell contraction. We have examined the effect of an antibody to VLA-1 in crescentic glomerulonephritis. Nephrotoxic nephritis was induced in Wistar-Kyoto rats and rats were given monoclonal antibody to VLA-1 (Ha31/8), 2.5 mg/kg, on alternate days. Antibodies were given from day -1 to day 10 or from day 14 to day 28. Treatment from day -1 to day 10, during the early inflammatory phase of nephrotoxic nephritis, had no effect on albuminuria or glomerular crescent formation. In the delayed treatment experiment, all rats developed florid crescentic glomerulonephritis, and control rats showed marked glomerular and tubulointerstitial scarring at day 32. VLA-1 expression, by immunohistochemistry, was increased in glomeruli and around tubules. Proteinuria did not differ between groups. In anti-VLA-1-treated rats, serum creatinine was significantly lower at day 32 (P = 0.002) and renal survival was significantly better (P = 0.045). Both glomerular and interstitial scarring were significantly less at day 32 in rats given anti-VLA-1 (P = 0.002). Deposition of ED(A) fibronectin, a marker of new matrix synthesis, and of type IV collagen, were reduced in glomeruli and interstitium in anti-VLA-1-treated animals (P = 0.0006). Expression of alpha-smooth muscle actin, a marker of myofibroblasts, showed no significant difference. Expression of matrix metalloproteinase-9 was increased in the glomeruli of rats treated with anti-VLA-1. We conclude that VLA-1 mediates both glomerular and interstitial fibrosis in crescentic glomerulonephritis and that neutralization of VLA-1, which enhanced expression of matrix metalloproteinase-9, is a possible therapeutic strategy in progressive renal scarring.

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions.

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.