DOCSLIB.ORG

Thesis + Long Intro

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Thesis + Long Intro Engineered discoidin domain from factor VIII binds αvβ3 integrin with antibody-like affinity Thesis by Gene Kym In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2014 (Defended April 23rd, 2014) ii © 2014 Gene Kym All Rights Reserved iii ACKNOWLEDGEMENTS I would like to thank my family for encouraging my studies and keeping me focused on long-term goals. I am thankful for the teachers who educated me, the mentors who trained me, my colleagues in student organizations who showed me the power of teamwork, and my friends for teaching me about the world outside the of academia. I am grateful to Steve, the Mayo Lab, and the individuals at Caltech who made my doctoral years a time of exploration, learning, and fun. iv ABSTRACT Alternative scaffolds are non-antibody proteins that can be engineered to bind new targets. They have found useful niches in the therapeutic space due to their smaller size and the ease with which they can be engineered to be bispecific. We sought a new scaffold that could be used for therapeutic ends and chose the C2 discoidin domain of factor VIII, which is well studied and of human origin. Using yeast surface display, we engineered the C2 domain to bind to αvβ3 integrin with a 16 nM affinity while retaining its thermal stability and monomeric nature. We obtained a crystal structure of the engineered domain at 2.1 Å resolution. We have christened this discoidin domain alternative scaffold the “discobody.” v TABLE OF CONTENTS Acknowledgements ........................................................................................................ iii Abstract ........................................................................................................................... iv Table of contents ............................................................................................................. v Figures and tables ........................................................................................................... vi Abbreviations ............................................................................................................... viii Chapter I: Introduction to alternative scaffolds .............................................................. 1 Chapter II: Discobody engineering and characterization ............................................. 25 Appendix A: Detailed discobody data .......................................................................... 61 Bibliography .................................................................................................................. 82 vi FIGURES AND TABLES Figure 1-1. Antibody domain schematic .............................................................. 19 Figure 1-2. Early antibody alternatives ................................................................ 21 Figure 1-3. Alternative scaffold structures ........................................................... 23 Figure 2-1. Topology diagrams of discobody and traditional scaffolds .............. 40 Figure 2-2. Flow cytometry plots of discobody loop grafts ................................. 41 Figure 2-3. FACS plots of sequential library sorts and Eng-Db sequence .......... 42 Figure 2-4. Binding characterization of Eng-Db .................................................. 43 Figure 2-5. Soluble Eng-Db competition binding ................................................ 43 Figure 2-6. Crystal structure of Eng-Db ............................................................... 44 Figure 2S-1. Potential linker orientations of bispecific Dbs .................................. 45 Figure 2S-2. On rate of Eng-Db using multiple antigen concentrations ................ 46 Figure 2S-3. Off rate of Eng-Db using single antigen concentration ..................... 48 Figure 2S-4. On rate of Eng-Db using single antigen concentration ..................... 50 Figure 2S-5. Equilibrium binding constant of Eng-Db .......................................... 52 Figure 2S-6. Melting temperature of Eng-Db ......................................................... 54 Figure 2S-7. Gel filtration elution profiles of Eng-Db ........................................... 54 Figure 2S-8. Displayed Eng-Db competition with Dbs ......................................... 55 Figure 2S-9. Displayed fibronectin competition with Dbs ..................................... 56 Figure 2S-10. Eng-Db structure compared to RGD peptide .................................... 57 Table 2-1. Eng-Db characterization summary .................................................... 58 Table 2S-1. Crystal structure data collection and refinement statistics ................ 59 Figure A-1. Discoidin family structural information ............................................ 61 Figure A-2. Factor VIII and the C2 domain .......................................................... 62 Figure A-3. Hydrophobic feet and the 4-Ala mutation of the C2 domain ............ 62 Figure A-4. αvβ3 integrin and the RGD motif ...................................................... 63 Figure A-5. Yeast surface display schematics ....................................................... 63 Figure A-6. Yeast display expression of 4-Ala by flow cytometry ..................... 64 Figure A-7. Soluble expression of 4-Ala ............................................................... 64 Figure A-8. AgRP binding control by flow cytometry ......................................... 65 Figure A-9. AgRP equilibrium titration by flow cytometry ................................. 66 vii Figure A-10. AgRP equilibrium titration curve fit .................................................. 66 Figure A-11. Measurements for loop grafting ......................................................... 67 Figure A-12. Close look at loop graft measurements .............................................. 67 Figure A-13. SGRGDNDLV loop graft equilibrium titration ................................ 68 Figure A-14. SGRGDNDLV loop graft curve fit ................................................... 68 Figure A-15. AVTGRGDSPASS loop graft equilibrium titration ......................... 69 Figure A-16. AVTGRGDSPASS loop graft curve fit ............................................. 69 Figure A-17. Normalized binding curves for control and loop grafts .................... 70 Figure A-18. Library construction schematics ........................................................ 71 Figure A-19. FACS library population lift per sorting round ................................. 71 Figure A-20. Sequences from sort round I-III ......................................................... 72 Figure A-21. Converged sequences from sort round V .......................................... 72 Figure A-22. RPRGDIE equilibrium titration by flow cytometry .......................... 73 Figure A-23. RPRGDIE curve fit ............................................................................ 73 Figure A-24. ACRGDTC equilibrium titration by flow cytometry ........................ 74 Figure A-25. ACRGDTC curve fit .......................................................................... 74 Figure A-26. Db constructs for bacterial expression ............................................... 75 Figure A-27. Db bacterial expression gel ................................................................ 75 Figure A-28. TEV cleavage of Eng-Db ................................................................... 76 Figure A-29. Db constructs for yeast secretion expression ..................................... 76 Figure A-30. Db yeast secretion expression gel filtration and gel .......................... 77 Figure A-31. Db yeast display levels compared to traditional scaffolds ............... 77 Figure A-32. Db yeast display levels quantified ..................................................... 78 Figure A-33. Db soluble protein thermal melts ....................................................... 78 Figure A-34. Crystals collected for X-ray diffraction ............................................. 79 Figure A-35. Diffraction during pre-data collection ............................................... 79 Table A-1. Sequences of Db constructs ............................................................... 80 viii ABBREVIATIONS Da Daltons Db Discobody ECM Extracellular matrix E. coli Escherichia coli FACS Fluorescence-activated cell sorting fVIII Factor VIII IgG Immunoglobulin G Kd dissociation constant kDa kilodalton koff dissociation rate kon association rate LB lysogeny broth, also known as Luria-Bertani medium mAb Monoclonal antibody µM micromolar mM millimolar nM nanomolar PCR polymerase chain reaction PS phosphatidylserine S. cerevisea Saccharomyces cerevisea 1 Chapter 1 INTRODUCTION TO ALTERNATIVE SCAFFOLDS Monoclonal antibodies (mAbs) can be engineered to bind specific molecular targets with high affinity, and were heralded as being “magic bullets” to target cancer. Despite their increasing commercial success, mAbs have had limited therapeutic value for many diseases [1-4]. A richer understanding of how mAbs engage with and utilize the human immune system is beginning to change how mAbs are engineered [5]. Additionally, antibody engineering innovations [6] have lead to novel mAb formats, including bispecifics [7-9], small domains [10-12], and fusions to cytotoxic payloads [13] or nanoparticles [14]. Small domain alternative scaffolds such as fibronectin or DARPins are driving many of these later generation technologies [11,12,15]. While alternative
Load more

Recommended publications
  • Wo2015188839a2
    Downloaded from orbit.dtu.dk on: Oct 08, 2021 General detection and isolation of specific cells by binding of labeled molecules Pedersen, Henrik; Jakobsen, Søren; Hadrup, Sine Reker; Bentzen, Amalie Kai; Johansen, Kristoffer Haurum Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Pedersen, H., Jakobsen, S., Hadrup, S. R., Bentzen, A. K., & Johansen, K. H. (2015). General detection and isolation of specific cells by binding of labeled molecules. (Patent No. WO2015188839). General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/188839
    [Show full text]
  • Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage
    Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen B.S. Biomedical Engineering University of Minnesota – Twin Cities, 2012 Submitted to the Department of Biological Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering at the Massachusetts Institute of Technology February 2019 © 2019 Massachusetts Institute of Technology. All rights reserved. Signature of Author: ____________________________________________________________ Griffin Clausen Department of Biological Engineering January 14, 2019 Certified by: ___________________________________________________________________ Angela Belcher James Mason Crafts Professor of Biological Engineering and Materials Science Thesis Supervisor Accepted by: ___________________________________________________________________ Forest White Professor of Biological Engineering Graduate Program Committee Chair This doctoral thesis has been examined by the following committee: Amy Keating Thesis Committee Chair Professor of Biology and Biological Engineering Massachusetts Institute of Technology Angela Belcher Thesis Supervisor James Mason Crafts Professor of Biological Engineering and Materials Science Massachusetts Institute of Technology Paul Blainey Core Member, Broad Institute Associate Professor of Biological Engineering Massachusetts Institute of Technology 2 Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen Submitted
    [Show full text]
  • Strategies and Challenges for the Next Generation of Therapeutic Antibodies
    FOCUS ON THERAPEUTIC ANTIBODIES PERSPECTIVES ‘validated targets’, either because prior anti- TIMELINE bodies have clearly shown proof of activity in humans (first-generation approved anti- Strategies and challenges for the bodies on the market for clinically validated targets) or because a vast literature exists next generation of therapeutic on the importance of these targets for the disease mechanism in both in vitro and in vivo pharmacological models (experi- antibodies mental validation; although this does not necessarily equate to clinical validation). Alain Beck, Thierry Wurch, Christian Bailly and Nathalie Corvaia Basically, the strategy consists of develop- ing new generations of antibodies specific Abstract | Antibodies and related products are the fastest growing class of for the same antigens but targeting other therapeutic agents. By analysing the regulatory approvals of IgG-based epitopes and/or triggering different mecha- biotherapeutic agents in the past 10 years, we can gain insights into the successful nisms of action (second- or third-generation strategies used by pharmaceutical companies so far to bring innovative drugs to antibodies, as discussed below) or even the market. Many challenges will have to be faced in the next decade to bring specific for the same epitopes but with only one improved property (‘me better’ antibod- more efficient and affordable antibody-based drugs to the clinic. Here, we ies). This validated approach has a high discuss strategies to select the best therapeutic antigen targets, to optimize the probability of success, but there are many structure of IgG antibodies and to design related or new structures with groups working on this class of target pro- additional functions.
    [Show full text]
  • WO 2014/152006 A2 25 September 2014 (25.09.2014) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/152006 A2 25 September 2014 (25.09.2014) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, A61K 39/395 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/US20 14/026804 KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (22) International Filing Date: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 13 March 2014 (13.03.2014) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (25) Filing Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (26) Publication Language: English ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 61/791,953 15 March 2013 (15.03.2013) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (71) Applicant: INTRINSIC LIFESCIENCES, LLC UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, [US/US]; 505 Coast Boulevard South, Suite 408, La Jolla, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, California 92037 (US).
    [Show full text]
  • WO 2018/144999 Al 09 August 2018 (09.08.2018) W ! P O PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/144999 Al 09 August 2018 (09.08.2018) W ! P O PCT (51) International Patent Classification: Lennart; c/o Orionis Biosciences NV, Rijvisschestraat 120, A61K 38/00 (2006.01) C07K 14/555 (2006.01) Zwijnaarde, B-9052 (BE). TAVERNIER, Jan; c/o Orionis A61K 38/21 (2006.01) C12N 15/09 (2006.01) Biosciences NV, Rijvisschestraat 120, Zwijnaarde, B-9052 C07K 14/52 (2006.01) (BE). (21) International Application Number: (74) Agent: ALTIERI, Stephen, L. et al; Morgan, Lewis & PCT/US2018/016857 Bockius LLP, 1111 Pennsylvania Avenue, NW, Washing ton, D.C. 20004 (US). (22) International Filing Date: 05 February 2018 (05.02.2018) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (25) Filing Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (26) Publication Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (30) Priority Data: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 62/454,992 06 February 2017 (06.02.2017) US KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (71) Applicants: ORIONIS BIOSCIENCES, INC. [US/US]; MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 275 Grove Street, Newton, MA 02466 (US).
    [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]
  • Generation of Novel Intracellular Binding Reagents Based on the Human Γb-Crystallin Scaffold
    Generation of novel intracellular binding reagents based on the human γB-crystallin scaffold Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I-Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg Institut für Biochemie und Biotechnologie von Ewa Mirecka geboren am 17. Dezember 1976 in Gdynia, Polen Table of contents Table of contents 1. INTRODUCTION.........................................................................................................1 1.1 Monoclonal antibodies as a biomolecular scaffold..........................................................1 1.2 Binding molecules derived from non-immunoglobulin scaffolds.....................................3 1.2.1 Alternative protein scaffolds – general considerations............................................................ 3 1.2.2 Application of alternative binding molecules ........................................................................... 6 1.3 Affilin – novel binding molecules based on the human γB-crystallin scaffold................... 6 1.3.1 Human γB-crystallin as a molecular scaffold........................................................................... 6 1.3.2 Generation of a human γB-crystallin library and selection of first-generation Affilin molecules ................................................................................................................................ 8 1.4 Selection of binding proteins by phage display ...................................................................
    [Show full text]
  • WO 2018/098356 Al 31 May 2018 (31.05.2018) W !P O PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/098356 Al 31 May 2018 (31.05.2018) W !P O PCT (51) International Patent Classification: co, California 94124 (US). DUBRIDGE, Robert B.; 825 A61K 39/395 (2006.01) C07K 16/28 (2006.01) Holly Road, Belmont, California 94002 (US). LEMON, A61P 35/00 (2006.01) C07K 16/46 (2006.01) Bryan D.; 2493 Dell Avenue, Mountain View, California 94043 (US). AUSTIN, Richard J.; 1169 Guerrero Street, (21) International Application Number: San Francisco, California 941 10 (US). PCT/US20 17/063 126 (74) Agent: LIN, Clark Y.; WILSON SONSINI GOODRICH (22) International Filing Date: & ROSATI, 650 Page Mill Road, Palo Alto, California 22 November 201 7 (22. 11.201 7) 94304 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Langi English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/426,069 23 November 2016 (23. 11.2016) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 62/426,077 23 November 2016 (23. 11.2016) US HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (71) Applicant: HARPOON THERAPEUTICS, INC. KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, [US/US]; 4000 Shoreline Court, Suite 250, South San Fran MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, cisco, California 94080 (US).
    [Show full text]
  • Anticalins Versus Antibodies: Made-To-Order Binding Proteins for Small Molecules Gregory a Weiss * and Henry B Lowman
    Minireview R177 Anticalins versus antibodies: made-to-order binding proteins for small molecules Gregory A Weiss * and Henry B Lowman Engineering proteins to bind small molecules presents a challenge as daunting as Department of Protein Engineering, Genentech, Inc., drug discovery, for both hinge upon our understanding of receptor^ligand 1 DNA Way, South San Francisco, CA 94080, USA molecular recognition. However, powerful techniques from combinatorial *Present address: Department of Chemistry, molecular biology can be used to rapidly select arti¢cial receptors. While University of California, Irvine, CA 92697, USA traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable Correspondence: HB Lowman receptor for small molecule binding. `Anticalins', engineered lipocalin variants, E-mail: [email protected] offer some advantages over traditional antibody technology and illuminate Keywords: Antibody; Anticalin; Small molecule features of molecular recognition between receptors and small molecule ligands. ligand Received: 19 June 2000 Accepted: 10 July 2000 Published: 1 August 2000 Chemistry & Biology 2000, 7:R177^R184 1074-5521 / 00 / $ ^ see front matter ß 2000 Published by Elsevier Science Ltd. PII: S 1 0 7 4 - 5 5 2 1 ( 0 0 ) 0 0 0 1 6 - 8 Introduction of randomly mutated proteins displayed on the surface of The challenge of discovering small molecule ligands for ¢lamentous bacteriophage, viruses capable of infecting protein receptors is well appreciated by, among others, only bacteria (reviewed in [4]). Diversity can be targeted the pharmaceutical industry, which spent the majority of to particular regions of a displayed protein through stan- its $24 billion pharmaceutical research and development dard molecular biology techniques [5], or can be distrib- budget in 1999 on small molecule drug discovery [1].
    [Show full text]
  • WO 2017/013231 Al 26 January 2017 (26.01.2017) P O P C T
    (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 2017/013231 Al 26 January 2017 (26.01.2017) P O P C T (51) International Patent Classification: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C07K 14/705 (2006.01) C07K 16/00 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (21) International Application Number: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, PCT/EP20 16/067468 MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (22) International Filing Date: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 2 1 July 20 16 (21 .07.2016) SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 62/194,882 2 1 July 2015 (21.07.2015) TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 62/364,414 20 July 2016 (20.07.2016) TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (71) Applicants : XIANG, Sue D.
    [Show full text]
  • Protein Scaffolds Proteingerüste Structures Protéiques
    (19) TZZ _ _T (11) EP 2 215 246 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/10 (2006.01) C12N 15/62 (2006.01) 07.01.2015 Bulletin 2015/02 C07K 14/78 (2006.01) C40B 40/10 (2006.01) (21) Application number: 08845766.8 (86) International application number: PCT/US2008/012398 (22) Date of filing: 31.10.2008 (87) International publication number: WO 2009/058379 (07.05.2009 Gazette 2009/19) (54) PROTEIN SCAFFOLDS PROTEINGERÜSTE STRUCTURES PROTÉIQUES (84) Designated Contracting States: US-B1- 6 482 410 US-B1- 6 818 418 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • KOIDE A ET AL: "The fibronectin type III domain RO SE SI SK TR as a scaffold for novel binding proteins", JOURNAL OF MOLECULAR BIOLOGY, LONDON, (30) Priority: 31.10.2007 US 984209 P GB, vol. 284, no. 4, 11 December 1998 (1998-12-11), pages 1141-1151, XP004455886, (43) Date of publication of application: ISSN: 0022-2836, DOI: DOI:10.1006/JMBI. 11.08.2010 Bulletin 2010/32 1998.2238 • BATORI V ET AL: "Exploring the potential of the (73) Proprietor: MedImmune, LLC monobody scaffold:effects of loop elongation on Gaithersburg, MD 20878 (US) the stability of a fibronectin type III domain", PROTEIN ENGINEERING, OXFORD UNIVERSITY (72) Inventors: PRESS, SURREY, GB, vol. 15, no. 12, 1 January • WU, Herren 2002 (2002-01-01), pages 1015-1020, Boyds,MD 20841 (US) XP002996047, ISSN: 0269-2139, DOI: DOI: • BACA, Manuel 10.1093/PROTEIN/15.12.1015 Gaithersburg,MD 20878 (US) • KOIDE AKIKO ET AL: "Monobodies: antibody •SWERS,Jeffrey mimics based on the scaffold of the fibronectin Rockville,MD 20852 (US) type III domain", METHODS IN MOLECULAR • CHACKO, Benoy BIOLOGY, HUMANA PRESS INC, NJ, US, vol.
    [Show full text]
  • Bispecific Immunomodulatory Antibodies for Cancer Immunotherapy
    Published OnlineFirst June 9, 2021; DOI: 10.1158/1078-0432.CCR-20-3770 CLINICAL CANCER RESEARCH | REVIEW Bispecific Immunomodulatory Antibodies for Cancer Immunotherapy A C Belen Blanco1,2, Carmen Domínguez-Alonso1,2, and Luis Alvarez-Vallina1,2 ABSTRACT ◥ The recent advances in the field of immuno-oncology have here referred to as bispecific immunomodulatory antibodies, dramatically changed the therapeutic strategy against advanced have the potential to improve clinical efficacy and safety profile malignancies. Bispecific antibody-based immunotherapies have and are envisioned as a second wave of cancer immunotherapies. gained momentum in preclinical and clinical investigations Currently, there are more than 50 bispecific antibodies under following the regulatory approval of the T cell–redirecting clinical development for a range of indications, with promising antibody blinatumomab. In this review, we focus on emerging signs of therapeutic activity. We also discuss two approaches for and novel mechanisms of action of bispecific antibodies inter- in vivo secretion, direct gene delivery, and infusion of ex vivo acting with immune cells with at least one of their arms to gene-modified cells, which may become instrumental for the regulate the activity of the immune system by redirecting and/or clinical application of next-generation bispecific immunomod- reactivating effector cells toward tumor cells. These molecules, ulatory antibodies. Introduction of antibodies, such as linear gene fusions, domain-swapping strat- egies, and self-associating peptides and protein domains (Fig. 1B). The past decade has witnessed a number of cancer immunotherapy Multiple technology platforms are available for the design of bsAbs, breakthroughs, all of which involve the modulation of T cell–mediated allowing fine-tuning of binding valence, stoichiometry, size, flexi- immunity.
    [Show full text]