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Dive into the research topics where Kevin Lindquist is active.

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Featured researches published by Kevin Lindquist.


Journal of Biological Chemistry | 2012

Increasing Serum Half-life and Extending Cholesterol Lowering in Vivo by Engineering Antibody with pH-sensitive Binding to PCSK9

Javier Chaparro-Riggers; Hong Liang; Rachel M. DeVay; Lanfang Bai; Janette Sutton; Wei Chen; Tao Geng; Kevin Lindquist; Meritxell Galindo Casas; Leila Marie Boustany; Colleen Brown; Jeffrey R. Chabot; Bruce Charles Gomes; Pamela D. Garzone; Andrea Rossi; Pavel Strop; Dave Shelton; Jaume Pons; Arvind Rajpal

Background: An antagonistic anti-PCSK9 antibody exhibits target-mediated clearance, resulting in a dose-dependent PK. Results: Engineering of an antibody with pH-sensitive binding to PCSK9 decreases target-mediated clearance, resulting in increased PK and efficacy in vivo. Conclusion: pH-sensitive anti-PCSK9 antibodies are excellent candidates for therapeutic development. Significance: pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load. Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5–6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.


Journal of Molecular Biology | 2012

Generating Bispecific Human IgG1 and IgG2 Antibodies from Any Antibody Pair

Pavel Strop; Wei-Hsien Ho; Leila Marie Boustany; Yasmina Noubia Abdiche; Kevin Lindquist; Santiago E. Farias; Mathias Rickert; Charles Takeshi Appah; Edward Derrick Pascua; Teresa Radcliffe; Janette Sutton; Javier Chaparro-Riggers; Wei Chen; Meritxell Galindo Casas; Sherman Michael Chin; Oi Kwan Wong; Shu-Hui Liu; German J. Vergara; Dave Shelton; Arvind Rajpal; Jaume Pons

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.


Journal of Molecular Biology | 2011

Synthetic Antibodies Designed on Natural Sequence Landscapes

Wenwu Zhai; Jacob Glanville; Markus Fuhrmann; Li Mei; Irene Ni; Purnima Sundar; Thomas Van Blarcom; Yasmina Noubia Abdiche; Kevin Lindquist; Ralf Strohner; Dilduz Telman; Guido Cappuccilli; William J. J. Finlay; Jan Van den Brulle; David R. Cox; Jaume Pons; Arvind Rajpal

We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V(H)) and two kappa (V(κ)) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V(H) and V(κ) sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×10(10) transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×10(5) clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.


Journal of Immunology | 2013

High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Igκ/Igλ Loci Bearing the Rat CH Region

Michael J. Osborn; Biao Ma; Suzanne Avis; Ashleigh Binnie; Jeanette Dilley; Xi Yang; Kevin Lindquist; Séverine Ménoret; Anne-Laure Iscache; Laure-Hélène Ouisse; Arvind Rajpal; Ignacio Anegon; Michael S. Neuberger; Roland Buelow; Marianne Brüggemann

Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human VHs, all human D and JH segments in natural configuration linked to the rat CH locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.


mAbs | 2015

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

Yasmina Noubia Abdiche; Yik Andy Yeung; Javier Chaparro-Riggers; Ishita Barman; Pavel Strop; Sherman Michael Chin; Amber Pham; Gary Louis Bolton; Dan McDonough; Kevin Lindquist; Jaume Pons; Arvind Rajpal

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgGs variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgGs serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.


Nature Biotechnology | 2015

Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading

Pavel Strop; Kathy Delaria; Davide Foletti; Jody Melton Witt; Adela Hasa-Moreno; Kris Poulsen; Meritxell Galindo Casas; Magdalena Grazyna Dorywalska; Santiago E. Farias; Ariel Pios; Victor Lui; Russell Dushin; Dahui Zhou; Thayalan Navaratnam; Thomas-Toan Tran; Janette Sutton; Kevin Lindquist; Bora Han; Shu-Hui Liu; David L. Shelton; Jaume Pons; Arvind Rajpal

Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading


Analytical Biochemistry | 2011

Expanding the ProteOn XPR36 biosensor into a 36-ligand array expedites protein interaction analysis

Yasmina Noubia Abdiche; Kevin Lindquist; Alanna Pinkerton; Jaume Pons; Arvind Rajpal

Here we demonstrate methods to expand the throughput of the ProteOn XPR36 biosensor allowing for the simultaneous kinetic characterization of several multiplexed formats, such as 36 disparate antibodies targeting the same antigen, and facilitating detailed epitope binning and mapping studies. The kinetic rate constants determined by these methods correlated with those obtained on Biacore 2000 and the absolute parameter values obtained on the ProteOns alginate-based GLC chip agreed closer with those from Biacores flat C1 chip than Biacores dextran-based CM4 chip. Pairwise epitope binning data from the ProteOn 36-ligand array format and those generated on an orthogonal array-based biosensor, the Octet QK384, gave similar results. In an epitope mapping study using biotinylated peptides, all three biosensor platforms were similar in their ability to identify antibodies that bound to linear epitopes. We apply alternative formats of the ProteOn array that enable a significantly higher number of assays to be conducted simultaneously than previously anticipated on this platform.


PLOS ONE | 2012

Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

Christine Bee; Yasmina Noubia Abdiche; Donna Marie Stone; Sierra Jones Collier; Kevin Lindquist; Alanna Pinkerton; Jaume Pons; Arvind Rajpal

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.


Nature Communications | 2016

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Y.A. Yeung; Davide Foletti; X. Deng; Y. Abdiche; Pavel Strop; Jacob Glanville; Steven J. Pitts; Kevin Lindquist; Purnima Sundar; Marina Sirota; Adela Hasa-Moreno; A. Pham; J. Melton Witt; I. Ni; Jaume Pons; David L. Shelton; Arvind Rajpal; Javier Chaparro-Riggers

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.


Journal of Hematology & Oncology | 2017

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia

Manoj Kumar Kashyap; Carlos I. Amaya-Chanaga; Deepak Kumar; Brett H. Simmons; Nanni Huser; Yin Gu; Max Hallin; Kevin Lindquist; Rolla Yafawi; Michael Y. Choi; Ale-Ali Amine; Laura Z. Rassenti; Cathy Zhang; Shu-Hui Liu; Tod Smeal; Valeria R. Fantin; Thomas J. Kipps; Flavia Pernasetti; Januario E. Castro

BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

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