DOCSLIB.ORG

Toward Drug-Like Multispecific Antibodies by Design

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Toward Drug-Like Multispecific Antibodies by Design International Journal of Molecular Sciences Review Toward Drug-Like Multispecific Antibodies by Design 1,2, 1,2,3, 2,4 1,2,4,5, Manali S. Sawant y, Craig N. Streu y, Lina Wu and Peter M. Tessier * 1 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA; [email protected] (M.S.S.); [email protected] (C.N.S.) 2 Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA; [email protected] 3 Department of Chemistry, Albion College, Albion, MI 49224, USA 4 Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA 5 Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA * Correspondence: [email protected]; Tel.: +1-734-763-1486 These authors contributed equally to this work. y Received: 1 September 2020; Accepted: 2 October 2020; Published: 12 October 2020 Abstract: The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic. Keywords: bispecific; polyspecificity; pharmacokinetics; solubility; aggregation; viscosity; developability; stability; affinity; specificity; protein engineering; self-association; non-specific binding; immunogenicity 1. Introduction Antibodies are among the most well-established biologics and are widely employed as therapeutics. Their success as therapeutics is largely due to their unique combination of properties, including their favorable activities, safety profiles, and physical and chemical properties (also known as developability properties). The activity of antibodies is linked to their high binding affinities and specificities as well as their Fc-mediated interactions with receptors that enable extended half-lives and, in some cases, effector functions. The safety of antibodies is due to their low off-target binding, low immunogenicity (for human or humanized antibodies), and non-toxic breakdown products (amino acids). The desirable developability properties of antibodies are due to their high folding stabilities, high solubilities, low viscosities and high chemical stabilities. The combination of these key properties has led to >80 approved antibody drugs and hundreds more in clinical trials [1]. Nevertheless, most lead antibody (IgG) candidates do not have the required combination of activity, safety, and developability properties for therapeutic applications, and must be further engineered Int. J. Mol. Sci. 2020, 21, 7496; doi:10.3390/ijms21207496 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 43 Int. J. Mol.Nevertheless, Sci. 2020, 21, 7496most lead antibody (IgG) candidates do not have the required combination2 ofof 41 activity, safety, and developability properties for therapeutic applications, and must be further toengineered achieve drug-like to achieve molecules. drug-like Overcoming molecules. Overcomi these issuesng these is even issues more is challenging even more forchallenging multispecific for multispecific antibodies, a class of engineered antibodies that seeks to engage either two or more antibodies, a class of engineered antibodies that seeks to engage either two or more targets or two or targets or two or more epitopes on the same target. Generally, multispecifics are chimeric proteins more epitopes on the same target. Generally, multispecifics are chimeric proteins composed of IgGs composed of IgGs and smaller antibody fragments or multiple antibody fragments (Figure 1). Given and smaller antibody fragments or multiple antibody fragments (Figure1). Given their ability to bind their ability to bind more than one target, multispecific antibodies have functional advantages for more than one target, multispecific antibodies have functional advantages for applications in which it is applications in which it is necessary to bring together two targets in close proximity. This is a key necessary to bring together two targets in close proximity. This is a key feature of the two multispecific feature of the two multispecific antibodies blinatumomab and emicizumab that have been approved antibodies blinatumomab and emicizumab that have been approved for use in humans and are for use in humans and are currently being marketed [2–6]. Although the full potential of multispecific currently being marketed [2–6]. Although the full potential of multispecific antibodies is just beginning antibodies is just beginning to be tapped [7], one particularly notable therapeutic application has been to be tapped [7], one particularly notable therapeutic application has been the recruitment of immune the recruitment of immune effector cells, such as T cells [8–11] or natural killer (NK) cells [12–15], to effector cells, such as T cells [8–11] or natural killer (NK) cells [12–15], to tumor cells using multispecific tumor cells using multispecific formats that recognize antigens on both cell types. Still other potential formats that recognize antigens on both cell types. Still other potential advantages of binding multiple advantages of binding multiple targets include synergistic effects, improved specificity, and reduced targets include synergistic effects, improved specificity, and reduced incidence of drug-resistance. incidence of drug-resistance. FigureFigure 1. 1.Antigen-binding Antigen-binding regionsregions of monospecificmonospecific IgGs can be combined into into various various multispecific multispecific formatsformats that that havehave uniqueunique developability concerns, concerns, incl includinguding those those related related to to their their stability, stability, solubility, solubility, aggregation,aggregation, viscosity,viscosity, andand pharmacokinetics.pharmacokinetics. Th Thee abbreviations abbreviations are are scFv-IgG scFv-IgG for for single-chain single-chain variablevariable fragmentfragment immunoglobulin, DVD-Ig DVD-Ig for for du dualal variable variable domain domain immunoglobulin, immunoglobulin, FIT-Ig FIT-Ig for forfabs-in-tandem fabs-in-tandem immunoglobulin, immunoglobulin, BiTE BiTE for bispecific for bispecific T-cell T-cell engager engager and andDART DART for dual-affinity for dual-affi nityre- re-targetingtargeting antibody antibody fragment. fragment. DespiteDespite theirtheir impressiveimpressive therapeutictherapeutic potential, much much less less is is known known about about the the developability developability propertiesproperties of of multispecifics multispecifics withwith respectrespect toto conventional antibodies (IgGs), which which is is a a major major limitation limitation toto their their development development andand broadbroad adoptionadoption asas therapeutics.therapeutics. Further Further exacerba exacerbatingting the the knowledge knowledge gap gap isis the the sheer sheer number number ofof multispecificmultispecific antibodyantibody formatsformats that have have been been developed, developed, each each with with its its own own specificspecific advantagesadvantages andand liabilitiesliabilities asas therapeuticstherapeutics [16–18]. [16–18]. Recent Recent advances advances in in the the ability ability to to screen screen antibodiesantibodies forfor developabilitydevelopability parametersparameters earlier in the discovery discovery process process are are likely likely to to streamline streamline the the developmentdevelopment ofof antibodyantibody therapeuticstherapeutics but, to date, no such general general approach approach exists exists for for multispecific multispecific antibodies. Because many multispecific antibodies are composed of recombined antibody fragments, antibodies. Because many multispecific antibodies are composed of recombined antibody fragments, and stabilization of the individual fragments is a common component of multispecific design and stabilization of the individual fragments is a common component of multispecific design strategies, strategies, this review will focus on the latest methods for optimizing the drug-like properties of this review will focus on the latest methods for optimizing the drug-like properties of antibody antibody fragments in addition to strategies for optimizing multispecific antibodies. Here we divide fragments in addition to strategies for optimizing multispecific antibodies. Here we divide the the critical antibody properties into three broad categories, namely: (i) physical and chemical stability, critical antibody properties into three
Load more

Recommended publications
  • Affibody Molecules for Epidermal Growth Factor Receptor Targeting
    Journal of Nuclear Medicine, published on January 21, 2009 as doi:10.2967/jnumed.108.055525 Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry Vladimir Tolmachev1-3, Mikaela Friedman4, Mattias Sandstrom¨ 5, Tove L.J. Eriksson2, Daniel Rosik2, Monika Hodik1, Stefan Sta˚hl4, Fredrik Y. Frejd1,2, and Anna Orlova1,2 1Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; 2Affibody AB, Bromma, Sweden; 3Department of Medical Sciences, Nuclear Medicine, Uppsala University, Uppsala, Sweden; 4Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden; and 5Section of Hospital Physics, Department of Oncology, Uppsala University Hospital, Uppsala, Sweden Noninvasive detection of epidermal growth factor receptor (EGFR) expression in malignant tumors by radionuclide molecu- he epidermal growth factor receptor (EGFR; other lar imaging may provide diagnostic information influencing pa- T designations are HER1 and ErbB-1) is a transmembrane tient management. The aim of this study was to evaluate a tyrosine kinase receptor that regulates cell proliferation, novel EGFR-targeting protein, the ZEGFR:1907 Affibody molecule, for radionuclide imaging of EGFR expression, to determine a motility, and suppression of apoptosis (1). Overexpression suitable tracer format (dimer or monomer) and optimal label. of EGFR is documented in several malignant tumors, such Methods: An EGFR-specific Affibody molecule, ZEGFR:1907, as carcinomas of the breast, urinary bladder, and lung, and 111 and its dimeric form, (ZEGFR:1907)2, were labeled with In using is associated with poor prognosis (2). A high level of EGFR 125 benzyl-diethylenetriaminepentaacetic acid and with I using expression could provide malignant cells with an advantage p-iodobenzoate.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,877,688 B2 Vasquez Et Al
    US008877688B2 (12) United States Patent (10) Patent No.: US 8,877,688 B2 Vasquez et al. (45) Date of Patent: *Nov. 4, 2014 (54) RATIONALLY DESIGNED, SYNTHETIC (56) References Cited ANTIBODY LIBRARIES AND USES THEREFOR U.S. PATENT DOCUMENTS 4.946,778 A 8, 1990 Ladner et al. (75) Inventors: Maximiliano Vasquez, Palo Alto, CA 5,118,605 A 6/1992 Urdea (US); Michael Feldhaus, Grantham, NH 5,223,409 A 6/1993 Ladner et al. 5,283,173 A 2f1994 Fields et al. (US); Tillman U. Gerngross, Hanover, 5,380,833. A 1, 1995 Urdea NH (US); K. Dane Wittrup, Chestnut 5,525.490 A 6/1996 Erickson et al. Hill, MA (US) 5,530,101 A 6/1996 Queen et al. 5,565,332 A 10/1996 Hoogenboom et al. (73) Assignee: Adimab, LLC, Lebanon, NH (US) 5,571,698 A 11/1996 Ladner et al. 5,618,920 A 4/1997 Robinson et al. (*) Notice: Subject to any disclaimer, the term of this 5,658,727 A 8, 1997 Barbas et al. patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 747 days. FOREIGN PATENT DOCUMENTS This patent is Subject to a terminal dis claimer. DE 19624562 A1 1, 1998 WO WO-88.01649 A1 3, 1988 (21) Appl. No.: 12/404,059 (Continued) OTHER PUBLICATIONS (22) Filed: Mar 13, 2009 Rader et al. (Jul. 21, 1998) Proceedings of the National Academy of (65) Prior Publication Data Sciences USA vol. 95 pp. 8910 to 8915.* US 201O/OO5638.6 A1 Mar. 4, 2010 (Continued) Primary Examiner — Heather Calamita Related U.S.
    [Show full text]
  • Phage Display Libraries for Antibody Therapeutic Discovery and Development
    antibodies Review Phage Display Libraries for Antibody Therapeutic Discovery and Development Juan C. Almagro 1,2,* , Martha Pedraza-Escalona 3, Hugo Iván Arrieta 3 and Sonia Mayra Pérez-Tapia 3 1 GlobalBio, Inc., 320, Cambridge, MA 02138, USA 2 UDIBI, ENCB, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Casco de Santo Tomas, Delegación Miguel Hidalgo, Ciudad de Mexico 11340, Mexico 3 CONACyT-UDIBI, ENCB, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Casco de Santo Tomas, Delegación Miguel Hidalgo, Ciudad de Mexico 11340, Mexico * Correspondence: [email protected] Received: 24 June 2019; Accepted: 15 August 2019; Published: 23 August 2019 Abstract: Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naïve, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.
    [Show full text]
  • The Generation of Synthetic Antibody Reagents for Clostridium Difficile Toxins
    The Generation of Synthetic Antibody Reagents for Clostridium difficile Toxins by Sylvia Cien Man Wong A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Sylvia Wong 2013 The Generation of Synthetic Antibody Reagents for Clostridium difficile Toxins Sylvia Cien Man Wong Master of Science Graduate Department of Molecular Genetics University of Toronto 2013 Abstract The symptoms of C. difficile infection are primarily caused by two toxins, toxin A and toxin B. Some strains produce a third known toxin, C. difficile transferase (CDT) toxin; however, its role in virulence remains unclear. I aimed to develop synthetic antibodies using phage display technology to block toxin entry by binding to the receptor-binding domain (RBD) of the toxins. I first described the generation of anti-toxin A and anti-toxin B Fabs. I presented Fab A3, which bound to the full-length toxin, but did not functionally inhibit toxin entry. In chapter 2, I described the generation of novel anti-CDTb antibodies. I further demonstrated that five of the anti-CDTb antibodies could functionally inhibit CDTb binding in an ELISA-based assay and on cultured cells. These antibodies can be used as tools to understand the toxins’ role in human disease and potentially be used as therapeutics. ii Acknowledgments I am very thankful for having the chance to work with many great scientists in Dr. Jason Moffat’s and Dr. Sachdev Sidhu’s labs. These people made the lab a very motivating, supportive, and intellectually stimulating environment, where I was able to learn and expand my critical thinking skills.
    [Show full text]
  • Wo 2008/048970 A2
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date (10) International Publication Number 24 April 2008 (24.04.2008) PCT WO 2008/048970 A2 (51) International Patent Classification: (72) Inventors; and A61K 39/395 (2006 01) (75) Inventors/Applicants (for US only): JOHNSTON, Stephen, Albert [US/US], 8606 S Dorsey Lane, Tempe, (21) International Application Number: AZ 85284 (US) WOODBURY, Neal [US/US], 1001 S PCT/US2007/081536 McAllister Avenue, Tempe, AZ 85287 (US) CHAPUT, John, C. [US/US], 3069 E Dry Creed Road, Phoenix, AZ (22) International Filing Date: 16 October 2007 (16 10 2007) 85048 (US) DIEHNELT, Chris, W. [US/US], 20941 N Leona Boulevard, Maπ copa, AZ 85239 (US) YAN, Hao (25) Filing Language: English [CN/US], 1300 N Brentwood Place, Chandler, AZ 85224 (26) Publication Language: English (US) (74) Agent: LIEBESCHUETZ, Joe, Townsend and Townsend (30) Priority Data: and Crew LLP , 8th floor, Two Embarcadero Center, San 60/852,040 16 October 2006 (16 10 2006) US Francisco, CA 941 11-3834 (US) 60/975,442 26 September 2007 (26 09 2007) US (81) Designated States (unless otherwise indicated, for every (71) Applicant (for all designated States except US): THE kind of national protection available): AE, AG, AL, AM, ARIZONA BOARD OF REGENTS, A BODY COR¬ AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, PORATE OF THE STATE OF ARIZONA acting for CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, and on behalf
    [Show full text]
  • Computational Design of Peptide Ligands Based on Antibody-Antigen Interface Properties
    Computational design of peptide ligands based on antibody-antigen interface properties By Benjamin Thomas VIART Thesis Advisor Prof. Dra. Liza Figueiredo Felicori Vilela Thesis Co-advisor Dr. Franck Molina Universidade Federal de Minas Gerais Instituto de Ciências Biológicas Programa de Pós-Graduação em Bioinformática Avenida Presidente Antônio Carlos, 6627 Pampulha 31270-901 Belo Horizonte - MG To my parents, ii Acknowledgments • Firstly, I would like to express my sincere gratitude to my advisor Dr. Prof. Liza Figueiredo Felicori Vilela for the continuous support of my Ph.D study, for her patience, motivation and knowledge. Her advices and guidance helped me better understand the scientific gait, the rules of research, the importance of the details. I would like to thank her as well for making feel welcome in Brazil, helping me with the transition to this new culture that is now part of me as the French culture is part of her. • Secondly, I would like to deeply thank my co-advisor Dr Franck Molina, without whom I would never have done this PhD. I am forever grateful to him for believing in me and helping me achieve this goal. • From the department, I would like to thanks Prof. Jader, Prof. Vasco, Prof. Miguel, Prof. Lucas, Prof. Gloria as well as Sheila. • This thesis is dedicated to my parents, Frédéric and Françoise Viart, who helped me throughout my life to become a good person. I thank them for their unconditional love and support. • I would like to thank all my family, especially my sisters, Sophie and Lisa, for their smiles and grimaces; my grand-parents, Bob, Nanou and Mémé for their support and love and also my uncle, Bruno, for all his help when I was in first year of medical school.
    [Show full text]
  • Nanobodies and Recombinant Binders in Cell Biology
    JCB: Review Nanobodies and recombinant binders in cell biology Jonas Helma,1 M. Cristina Cardoso,2 Serge Muyldermans,3 and Heinrich Leonhardt1 1Department of Biology II, Ludwig Maximilians University Munich and Center for Integrated Protein Science Munich, 82152 Planegg-Martinsried, Germany 2Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany 3Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1050 Brussels, Belgium Antibodies are key reagents to investigate cellular pro- exclusive to small and stable binding molecules and cannot cesses. The development of recombinant antibodies and be performed easily with full-length antibodies, as a result of crucial inter- and intramolecular disulphide bridges that do not binders derived from natural protein scaffolds has ex- form in the cytoplasm. Thus, researchers have found a plethora panded traditional applications, such as immunofluores- of new applications in which binders have been combined with cence, binding arrays, and immunoprecipitation. In enzymatic or structural functionalities in living systems. addition, their small size and high stability in ectopic en- The development of in vitro screening techniques has been a decisive step for the rise and generation of recombinant vironments have enabled their use in all areas of cell re- binding reagents. These methods include classic phage display Downloaded from search, including structural biology, advanced microscopy, but also bacterial and yeast display as well as ribosomal and and intracellular expression. Understanding these novel mRNA display. With such in vitro display techniques at hand, directed evolution strategies and genetic manipulation of binder reagents as genetic modules that can be integrated into sequences allow targeted engineering of key features, such as cellular pathways opens up a broad experimental spec- specificity, valence, affinity, and stability, enable derivatization trum to monitor and manipulate cellular processes.
    [Show full text]
  • Article in Press
    G Model MIMM-4561; No. of Pages 12 ARTICLE IN PRESS Molecular Immunology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Molecular Immunology j ournal homepage: www.elsevier.com/locate/molimm Review Alternative molecular formats and therapeutic applications for ଝ bispecific antibodies ∗ Christoph Spiess, Qianting Zhai, Paul J. Carter Department of Antibody Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA a r t i c l e i n f o a b s t r a c t Article history: Bispecific antibodies are on the cusp of coming of age as therapeutics more than half a century after they Received 28 November 2014 ® were first described. Two bispecific antibodies, catumaxomab (Removab , anti-EpCAM × anti-CD3) and Received in revised form ® blinatumomab (Blincyto , anti-CD19 × anti-CD3) are approved for therapy, and >30 additional bispecific 30 December 2014 antibodies are currently in clinical development. Many of these investigational bispecific antibody drugs Accepted 2 January 2015 are designed to retarget T cells to kill tumor cells, whereas most others are intended to interact with two Available online xxx different disease mediators such as cell surface receptors, soluble ligands and other proteins. The modular architecture of antibodies has been exploited to create more than 60 different bispecific antibody formats. Keywords: These formats vary in many ways including their molecular weight, number of antigen-binding sites, Bispecific antibodies spatial relationship between different binding sites, valency for each antigen, ability to support secondary Antibody engineering Antibody therapeutics immune functions and pharmacokinetic half-life. These diverse formats provide great opportunity to tailor the design of bispecific antibodies to match the proposed mechanisms of action and the intended clinical application.
    [Show full text]
  • EURL ECVAM Recommendation on Non-Animal-Derived Antibodies
    EURL ECVAM Recommendation on Non-Animal-Derived Antibodies EUR 30185 EN Joint Research Centre This publication is a Science for Policy report by the Joint Research Centre (JRC), the European Commission’s science and knowledge service. It aims to provide evidence-based scientific support to the European policymaking process. The scientific output expressed does not imply a policy position of the European Commission. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of this publication. For information on the methodology and quality underlying the data used in this publication for which the source is neither Eurostat nor other Commission services, users should contact the referenced source. EURL ECVAM Recommendations The aim of a EURL ECVAM Recommendation is to provide the views of the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) on the scientific validity of alternative test methods, to advise on possible applications and implications, and to suggest follow-up activities to promote alternative methods and address knowledge gaps. During the development of its Recommendation, EURL ECVAM typically mandates the EURL ECVAM Scientific Advisory Committee (ESAC) to carry out an independent scientific peer review which is communicated as an ESAC Opinion and Working Group report. In addition, EURL ECVAM consults with other Commission services, EURL ECVAM’s advisory body for Preliminary Assessment of Regulatory Relevance (PARERE), the EURL ECVAM Stakeholder Forum (ESTAF) and with partner organisations of the International Collaboration on Alternative Test Methods (ICATM). Contact information European Commission, Joint Research Centre (JRC), Chemical Safety and Alternative Methods Unit (F3) Address: via E.
    [Show full text]
  • Monobodies and Other Synthetic Binding Proteins for Expanding Protein Science
    Reviews Protein Science DOI 10.1002/pro.3148 Monobodies and Other Synthetic Binding Proteins for Expanding Protein Science Fern Sha,1,4 Gabriel Salzman,1 Ankit Gupta1,2 and Shohei Koide1,2,3* 1Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA 2Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY 10016, USA 3Department of Biochemistry and Molecular Pharmacology New York University School of Medicine, New York, NY 10016, USA 4Present address, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20148, USA *Correspondence to: Shohei Koide, Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY 10016, USA. E-mail: [email protected] Abstract Synthetic binding proteins are constructed using non-antibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function,
    [Show full text]
  • An Affibody Molecule Is Actively Transported Into the Cerebrospinal
    International Journal of Molecular Sciences Article An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor Sebastian W. Meister , Linnea C. Hjelm , Melanie Dannemeyer, Hanna Tegel, Hanna Lindberg, Stefan Ståhl and John Löfblom * Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Centre, SE-106 91 Stockholm, Sweden; [email protected] (S.W.M.); [email protected] (L.C.H.); [email protected] (M.D.); [email protected] (H.T.); [email protected] (H.L.); [email protected] (S.S.) * Correspondence: [email protected]; Tel.: +46-8-790-9659 Received: 6 March 2020; Accepted: 22 April 2020; Published: 23 April 2020 Abstract: The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule ZSYM73. Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.
    [Show full text]
  • WO 2019/068007 Al Figure 2
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/068007 Al 04 April 2019 (04.04.2019) W 1P O PCT (51) International Patent Classification: (72) Inventors; and C12N 15/10 (2006.01) C07K 16/28 (2006.01) (71) Applicants: GROSS, Gideon [EVIL]; IE-1-5 Address C12N 5/10 (2006.0 1) C12Q 1/6809 (20 18.0 1) M.P. Korazim, 1292200 Moshav Almagor (IL). GIBSON, C07K 14/705 (2006.01) A61P 35/00 (2006.01) Will [US/US]; c/o ImmPACT-Bio Ltd., 2 Ilian Ramon St., C07K 14/725 (2006.01) P.O. Box 4044, 7403635 Ness Ziona (TL). DAHARY, Dvir [EilL]; c/o ImmPACT-Bio Ltd., 2 Ilian Ramon St., P.O. (21) International Application Number: Box 4044, 7403635 Ness Ziona (IL). BEIMAN, Merav PCT/US2018/053583 [EilL]; c/o ImmPACT-Bio Ltd., 2 Ilian Ramon St., P.O. (22) International Filing Date: Box 4044, 7403635 Ness Ziona (E.). 28 September 2018 (28.09.2018) (74) Agent: MACDOUGALL, Christina, A. et al; Morgan, (25) Filing Language: English Lewis & Bockius LLP, One Market, Spear Tower, SanFran- cisco, CA 94105 (US). (26) Publication Language: English (81) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of national protection available): AE, AG, AL, AM, 62/564,454 28 September 2017 (28.09.2017) US AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, 62/649,429 28 March 2018 (28.03.2018) US CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (71) Applicant: IMMP ACT-BIO LTD.
    [Show full text]