Biodistribution of recombinant factor IX, extended half-life recombinant factor IX Fc fusion protein, and glycoPEGylated recombinant factor IX in hemophilia B mice.

Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX.

Dosimetry of [212Pb]VMT01, a MC1R-Targeted Alpha Therapeutic Compound, and Effect of Free 208Tl on Tissue Absorbed Doses.

[212Pb]VMT01 is a melanocortin 1 receptor (MC1R) targeted theranostic ligand in clinical development for alpha particle therapy for melanoma. 212Pb has an elementally matched gamma-emitting isotope 203Pb; thus, [203Pb]VMT01 can be used as an imaging surrogate for [212Pb]VMT01. [212Pb]VMT01 human serum stability studies have demonstrated retention of the 212Bi daughter within the chelator following beta emission of parent 212Pb. However, the subsequent alpha emission from the decay of 212Bi into 208Tl results in the generation of free 208Tl. Due to the 10.64-hour half-life of 212Pb, accumulation of free 208Tl in the injectate will occur. The goal of this work is to estimate the human dosimetry for [212Pb]VMT01 and the impact of free 208Tl in the injectate on human tissue absorbed doses. Human [212Pb]VMT01 tissue absorbed doses were estimated from murine [203Pb]VMT01 biodistribution data, and human biodistribution values for 201Tl chloride (a cardiac imaging agent) from published data were used to estimate the dosimetry of free 208Tl. Results indicate that the dose-limiting tissues for [212Pb]VMT01 are the red marrow and the kidneys, with estimated absorbed doses of 1.06 and 8.27 mGyRBE = 5/MBq. The estimated percent increase in absorbed doses from free 208Tl in the injectate is 0.03% and 0.09% to the red marrow and the kidneys, respectively. Absorbed doses from free 208Tl result in a percent increase of no more than 1.2% over [212Pb]VMT01 in any organ or tissue. This latter finding indicates that free 208Tl in the [212Pb]VMT01 injectate will not substantially impact estimated tissue absorbed doses in humans.

Therapy of Myeloid Leukemia using Novel Bispecific Fusion Proteins Targeting CD45 and 90Y-DOTA.

Pretargeted radioimmunotherapy (PRIT) has been investigated as a multi-step approach to decrease relapse and toxicity for high-risk acute myeloid leukemia (AML). Relevant factors including endogenous biotin and immunogenicity, however, have limited the use of PRIT with an anti-CD45 antibody streptavidin conjugate and radiolabeled DOTA-biotin. To overcome these limitations we designed anti-murine and anti-human CD45 bispecific antibody constructs using 30F11 and BC8 antibodies, respectively, combined with an anti-yttrium (Y)-DOTA single-chain variable fragment (C825) to capture a radiolabeled ligand. The bispecific construct targeting human CD45 (BC8-Fc-C825) had high uptake in leukemia HEL xenografts [7.8 ± 0.02% percent injected dose/gram of tissue (% ID/g)]. Therapy studies showed that 70% of mice with HEL human xenografts treated with BC8-Fc-C825 followed by 44.4 MBq (1,200 µCi) of 90Y-DOTA-biotin survived at least 170 days after therapy, while all nontreated controls required euthanasia because of tumor progression by day 32. High uptake at sites of leukemia (spleen and bone marrow) was also seen with 30F11-IgG1-C825 in a syngeneic disseminated SJL murine leukemia model (spleen, 9.0 ± 1.5% ID/g and bone marrow, 8.1 ± 1.2% ID/g), with minimal uptake in all other normal organs (<0.5% ID/g) at 24 hours after 90Y-DOTA injections. SJL leukemia mice treated with the bispecific 30F11-IgG1-C825 and 29.6 MBq (800 µCi) of 90Y-DOTA-biotin had a survival advantage compared with untreated leukemic mice (median, 43 vs. 30 days, respectively; P < 0.0001). These data suggest bispecific antibody-mediated PRIT may be highly effective for leukemia therapy and translation to human studies.

Optimizing the radiosynthesis of [68Ga]DOTA-MLN6907 peptide containing three disulfide cyclization bonds - a GCC specific chelate for clinical radiopharmaceuticals.

In the present study, the effect of radiolabeling conditions on radiolabeling efficiency and achievable specific activity of a DOTA-conjugated highly-lipophilic peptide containing three disulfide cyclization bonds was examined. The peptide is designed to bind specifically (with high affinity) to cell-surface receptor guanylyl cyclase C (GCC), which is universally expressed by colorectal cancer cells. The effect of systematic variation of chemical parameters pH, mass of peptide, acetate buffer concentration (ionic strength), and inclusion of ethanol in the radiolabeling reaction vessel on achievable specific activity and labeling efficiency was examined. In addition, a unique approach to acetone-based elution of 68Ga from an initial cation-exchange pre-concentration column is introduced, which improved radiochemical yield and radiochemical purity. For the evaluation of the acetone-based method, two different post-radiolabeling reverse-phase (C18) approaches to purify the final radiolabeled peptide were tested. These results revealed the potential for peptide degradation via the cleavage of disulfide cyclization bonds to form free thiols when using one of these C18 cartridges. The final optimized procedure enabled radiolabeling efficiency of greater than 99% and specific activity greater than 35 MBq/nmole in less than 30 min. The optimized parameters were amenable to the use of an automated 68Ge/68Ga generator and fluid-handling system for clinical production of the GCC receptor-specific [68Ga]DOTA-MLN6907 peptide. The chemical characteristics of individual peptides govern the most appropriate radiolabeling conditions for the preparation of radiopharmaceuticals.

Dual-Isotope Cryoimaging Quantitative Autoradiography: Investigating Antibody-Drug Conjugate Distribution and Payload Delivery Through Imaging.

In vitro properties of antibody-drug conjugates (ADCs) such as binding, internalization, and cytotoxicity are often well characterized before in vivo studies. Interpretation of in vivo studies might be significantly enhanced by molecular imaging tools. We present here a dual-isotope cryoimaging quantitative autoradiography (CIQA) methodology combined with advanced 3-dimensional imaging and analysis allowing for the simultaneous study of both antibody and payload distribution in tissues of interest in a preclinical setting. Methods: TAK-264, an investigational ADC targeting anti-guanylyl cyclase C (GCC), was synthesized using tritiated monomethyl auristatin E. The tritiated ADC was then conjugated to diethylenetriaminepentaacetic acid, labeled with 111In, and evaluated in vivo in animals bearing GCC-positive and GCC-negative tumors. Results: CIQA revealed the time course of drug release from ADC and its distribution into various tumor regions that are less accessible to the antibody. For GCC-positive tumors, a representative section obtained 96 h after tracer injection showed only 0.8% of the voxels to have colocalized signal, versus over 15% of the voxels for a GCC-negative tumor section, suggesting successful and specific cleaving of the toxin in the GCC-positive lesions. Conclusion: The combination of a veteran established autoradiography technology with advanced image analysis methodologies affords an experimental tool that can support detailed characterization of ADC tumor penetration and pharmacokinetics.

CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies.

Pretargeted radioimmunotherapy (PRIT) has demonstrated remarkable efficacy targeting tumor antigens, but immunogenicity and endogenous biotin blocking may limit clinical translation. We describe a new PRIT approach for the treatment of multiple myeloma (MM) and other B-cell malignancies, for which we developed an anti-CD38-bispecific fusion protein that eliminates endogenous biotin interference and immunogenic elements. In murine xenograft models of MM and non-Hodgkin lymphoma (NHL), the CD38-bispecific construct demonstrated excellent blood clearance and tumor targeting. Dosimetry calculations showed a tumor-absorbed dose of 43.8 Gy per millicurie injected dose of 90Y, with tumor-to-normal organ dose ratios of 7:1 for liver and 15:1 for lung and kidney. In therapy studies, CD38-bispecific PRIT resulted in 100% complete remissions by day 12 in MM and NHL xenograft models, ultimately curing 80% of mice at optimal doses. In direct comparisons, efficacy of the CD38 bispecific proved equal or superior to streptavidin (SA)-biotin-based CD38-SA PRIT. Each approach cured at least 75% of mice at the highest radiation dose tested (1200 µCi), whereas at 600- and 1000-µCi doses, the bispecific outperformed the SA approach, curing 35% more mice overall (P < .004). The high efficacy of bispecific PRIT, combined with its reduced risk of immunogenicity and endogenous biotin interference, make the CD38 bispecific an attractive candidate for clinical translation. Critically, CD38 PRIT may benefit patients with unresponsive, high-risk disease because refractory disease typically retains radiation sensitivity. We posit that PRIT might not only prolong survival, but possibly cure MM and treatment-refractory NHL patients.

Molecular Simulation of Receptor Occupancy and Tumor Penetration of an Antibody and Smaller Scaffolds: Application to Molecular Imaging.

PURPOSE: Competitive radiolabeled antibody imaging can determine the unlabeled intact antibody dose that fully blocks target binding but may be confounded by heterogeneous tumor penetration. We evaluated the hypothesis that smaller radiolabeled constructs can be used to more accurately evaluate tumor expressed receptors. PROCEDURES: The Krogh cylinder distributed model, including bivalent binding and variable intervessel distances, simulated distribution of smaller constructs in the presence of increasing doses of labeled antibody forms. RESULTS: Smaller constructs <25 kDa accessed binding sites more uniformly at large distances from blood vessels compared with larger constructs and intact antibody. These observations were consistent for different affinity and internalization characteristics of constructs. As predicted, a higher dose of unlabeled intact antibody was required to block binding to these distant receptor sites. CONCLUSIONS: Small radiolabeled constructs provide more accurate information on total receptor expression in tumors and reveal the need for higher antibody doses for target receptor blockade.

Dynamic dual-isotope molecular imaging elucidates principles for optimizing intrathecal drug delivery.

The intrathecal (IT) dosing route offers a seemingly obvious solution for delivering drugs directly to the central nervous system. However, gaps in understanding drug molecule behavior within the anatomically and kinetically unique environment of the mammalian IT space have impeded the establishment of pharmacokinetic principles for optimizing regional drug exposure along the neuraxis. Here, we have utilized high-resolution single-photon emission tomography with X-ray computed tomography to study the behavior of multiple molecular imaging tracers following an IT bolus injection, with supporting histology, autoradiography, block-face tomography, and MRI. Using simultaneous dual-isotope imaging, we demonstrate that the regional CNS tissue exposure of molecules with varying chemical properties is affected by IT space anatomy, cerebrospinal fluid (CSF) dynamics, CSF clearance routes, and the location and volume of the injected bolus. These imaging approaches can be used across species to optimize the safety and efficacy of IT drug therapy for neurological disorders.

Comparative Analysis of Bispecific Antibody and Streptavidin-Targeted Radioimmunotherapy for B-cell Cancers.

Streptavidin (SA)-biotin pretargeted radioimmunotherapy (PRIT) that targets CD20 in non-Hodgkin lymphoma (NHL) exhibits remarkable efficacy in model systems, but SA immunogenicity and interference by endogenous biotin may complicate clinical translation of this approach. In this study, we engineered a bispecific fusion protein (FP) that evades the limitations imposed by this system. Briefly, one arm of the FP was an anti-human CD20 antibody (2H7), with the other arm of the FP an anti-chelated radiometal trap for a radiolabeled ligand (yttrium[Y]-DOTA) captured by a very high-affinity anti-Y-DOTA scFv antibody (C825). Head-to-head biodistribution experiments comparing SA-biotin and bispecific FP (2H7-Fc-C825) PRIT in murine subjects bearing human lymphoma xenografts demonstrated nearly identical tumor targeting by each modality at 24 hours. However, residual radioactivity in the blood and normal organs was consistently higher following administration of 1F5-SA compared with 2H7-Fc-C825. Consequently, tumor-to-normal tissue ratios of distribution were superior for 2H7-Fc-C825 (P < 0.0001). Therapy studies in subjects bearing either Ramos or Granta subcutaneous lymphomas demonstrated that 2H7-Fc-C825 PRIT is highly effective and significantly less myelosuppressive than 1F5-SA (P < 0.0001). All animals receiving optimal doses of 2H7-Fc-C825 followed by 90Y-DOTA were cured by 150 days, whereas the growth of tumors in control animals progressed rapidly with complete morbidity by 25 days. In addition to demonstrating reduced risk of immunogenicity and an absence of endogenous biotin interference, our findings offer a preclinical proof of concept for the preferred use of bispecific PRIT in future clinical trials, due to a slightly superior biodistribution profile, less myelosuppression, and superior efficacy. Cancer Res; 76(22); 6669-79. ©2016 AACR.

A gradient-loadable (64)Cu-chelator for quantifying tumor deposition kinetics of nanoliposomal therapeutics by positron emission tomography.

Effective drug delivery to tumors is a barrier to treatment with nanomedicines. Non-invasively tracking liposome biodistribution and tumor deposition in patients may provide insight into identifying patients that are well-suited for liposomal therapies. We describe a novel gradient-loadable chelator, 4-DEAP-ATSC, for incorporating (64)Cu into liposomal therapeutics for positron emission tomographic (PET). (64)Cu chelated to 4-DEAP-ATSC (>94%) was loaded into PEGylated liposomal doxorubicin (PLD) and HER2-targeted PLD (MM-302) with efficiencies >90%. (64)Cu-MM-302 was stable in human plasma for at least 48h. PET/CT imaging of xenografts injected with (64)Cu-MM-302 revealed biodistribution profiles that were quantitatively consistent with tissue-based analysis, and tumor (64)Cu positively correlated with liposomal drug deposition. This loading technique transforms liposomal therapeutics into theranostics and is currently being applied in a clinical trial (NCT01304797) to non-invasively quantify MM-302 tumor deposition, and evaluate its potential as a prognostic tool for predicting treatment outcome of nanomedicines.

Regional, kinetic [(18)F]FDG PET imaging of a unilateral Parkinsonian animal model.

Positron emission tomography (PET) imaging with the glucose analog 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) has demonstrated clinical utility for the monitoring of brain glucose metabolism alteration in progressive neurodegenerative diseases. We examined dynamic [(18)F]FDG PET imaging and kinetic modeling of atlas-based regions to evaluate regional changes in the cerebral metabolic rate of glucose in the widely-used 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. Following a bolus injection of 18.5 ± 1 MBq [(18)F]FDG and a 60-minute PET scan, image-derived input functions from the vena cava and left ventricle were used with three models, including Patlak graphical analysis, to estimate the influx constant and the metabolic rate in ten brain regions. We observed statistically significant changes in [(18)F]FDG uptake ipsilateral to the 6-OHDA injection in the basal ganglia, olfactory bulb, and amygdala regions; and these changes are of biological relevance to the disease. These experiments provide further validation for the use of [(18)F]FDG PET imaging in this model for drug discovery and development.

A series of anti-CEA/anti-DOTA bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance.

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.

Effect of small-molecule-binding affinity on tumor uptake in vivo: a systematic study using a pretargeted bispecific antibody.

Small-molecule ligands specific for tumor-associated surface receptors have wide applications in cancer diagnosis and therapy. Achieving high-affinity binding to the desired target is important for improving detection limits and for increasing therapeutic efficacy. However, the affinity required for maximal binding and retention remains unknown. Here, we present a systematic study of the effect of small-molecule affinity on tumor uptake in vivo with affinities spanning a range of three orders of magnitude. A pretargeted bispecific antibody with different binding affinities to different DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-based small molecules is used as a receptor proxy. In this particular system targeting carcinoembryonic antigen, a small-molecule-binding affinity of 400 pmol/L was sufficient to achieve maximal tumor targeting, and an improvement in affinity to 10 pmol/L showed no significant improvement in tumor uptake at 24 hours postinjection. We derive a simple mathematical model of tumor targeting using measurable parameters that correlates well with experimental observations. We use relations derived from the model to develop design criteria for the future development of small-molecule agents for targeted cancer therapeutics.

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging.

INTRODUCTION: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. METHODS: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). RESULTS: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2±1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a (111)In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. CONCLUSIONS: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

Assessing antibody pharmacokinetics in mice with in vivo imaging.

Recent advances in small-animal molecular imaging instrumentation combined with well characterized antibody-labeling chemistry have enabled detailed in vivo measurements of antibody distribution in mouse models. This article reviews the strengths and limitations of in vivo antibody imaging methods with a focus on positron emission tomography and single-photon emission computed tomography and a brief discussion of the role of optical imaging in this application. A description of the basic principles behind the imaging techniques is provided along with a discussion of radiolabeling methods relevant to antibodies. Practical considerations of study design and execution are presented through a discussion of sensitivity and resolution tradeoffs for these techniques as defined by modality, signaling probe (isotope or fluorophore) selection, labeling method, and radiation dosimetry. Images and analysis results from a case study are presented with a discussion of output data content and relevant informatics gained with this approach to studying antibody pharmacokinetics.

Biodistribution and clearance of small molecule hapten chelates for pretargeted radioimmunotherapy.

PURPOSE: The favorable pharmacokinetics and clinical safety profile of metal-chelated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) suggests that it might be an ideal hapten for pretargeted radioimmunotherapy. In an effort to minimize hapten retention in normal tissues and determine the effect of various chemical adducts on in vivo properties, a series of DOTA-based derivatives were evaluated. PROCEDURES: Biodistribution and whole-body clearance were evaluated for (177)Lu-labeled DOTA, DOTA-biotin, a di-DOTA peptide, and DOTA-aminobenzene in normal CD1 mice. Kidney, liver, and bone marrow doses were estimated using standard Medical Internal Radiation Dose methodology. RESULTS: All haptens demonstrated similar low tissue and whole-body retention, with 2-4% of the injected dose remaining in mice 4 h postinjection. The kidney is predicted to be dose limiting for all (177)Lu-labeled haptens tested with an estimated kidney dose of approximately 0.1 mGy/MBq. CONCLUSIONS: We present here a group of DOTA-based haptens that exhibit rapid clearance and exceptionally low whole-body retention 4 h postinjection. Aminobenzene, tyrosine-lysine, and biotin groups have minimal effects on the blood clearance and biodistribution of (177)Lu-DOTA.

Intersectional innovation in biomarker development for patient-centric medicine.

The pharmaceutical and healthcare industries are being revolutionized by the use of genomics, proteomics, metabolomics, bioinformatics and molecular imaging. Patient friendly diagnosis, treatment and disease management options that utilize the combination of these technologies are currently in development. New innovations in pharmaceutical advancement are taking place at the intersection of these technologies, and will be coupled with societal changes as we move to a fully networked and individual-centric consumer base. Numerous examples of the combinations of molecular characterization technologies aimed at better preclinical and clinical disease understanding, diagnosis and treatment are highlighted that are ideally situated to generate the intersectional innovation that drives healthcare advancement. The true value in patient-centric medicine will only be realized as the improved molecular characterization of disease provided by these technologies is integrated across platforms that operate directly in the patient and care provider space to provide a comprehensive view of health. Molecular profiling and imaging technologies must become fully integrated and amenable for patient and physician use in a networked environment that can provide a personal health avatar approach to medicine.

A modular IgG-scFv bispecific antibody topology.

Here we present a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. When expressed in mammalian cells and purified by one-step protein A chromatography, the bsAb retains parental affinities of each binding domain, exhibits IgG-like stability and demonstrates in vivo IgG-like tumor targeting and blood clearance. The extension of the C-terminus of the light chain of an IgG with an scFv or even a smaller peptide does appear to disrupt disulfide bond formation between the light and heavy chains; however, this does not appear to affect binding, stability or in vivo properties of the IgG. Thus, we demonstrate here that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. This format serves as a standardized platform for the construction of functional bsAbs.

Highly avid magnetic bead capture: an efficient selection method for de novo protein engineering utilizing yeast surface display.

Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million-fold improvements in binding affinity by alternating rounds of diversification and flow cytometry-based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads-allowing isolation of extremely weak binders from billions of non-binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000-fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin-biotin that do not cross-react to streptavidin alone.