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Leonor Kremer

Name of the laboratory

CNB-CSIC Protein Tools Unit
CNB-CSIC Protein Tools Unit
Centro Nacional de Biotecnología (CNB)
Spanish National Research Council (CSIC)
Darwin 3, Campus Cantoblanco
28049 Madrid, Spain
(+34) 915 854 614

Members of the laboratory

Head of the Unit
Leonor Kremer (PhD) lkremer@cnb.csic.es
Technicians
Mónica García-Gallo (PhD) mggallo@cnb.csic.es
María Teresa Martín (PhD) mtmartin@cnb.csic.es
Ana María García Cabrero (PhD) amgarcia@cnb.csic.es
Mercedes Llorente (BSc) llorente@cnb.csic.es
Ph.D. Students
Beatriz Somovilla Crespo bsomovilla@cnb.csic.es
Isabel Corraliza Gorjón icorraliza@cnb.csic.es
Master Students
Lisa Kochsiek lisa.kochsiek@uni-bielefeld.de

Laboratory activity

The CNB-CSIC Protein Tools Unit focuses on the design, production, characterization and use of proteins as specific molecular tools. The Unit generates monoclonal antibodies (mAb), performs studies of the immune response, develops specific immunoassays and analyzes biomolecular interactions using a Biacore 3000 surface plasmon resonance biosensor. It works in a horizontal manner, participating in research projects or on contract for specific goals. The activities, individually or as part of a project, are based on proposals from research groups within the CNB and from private or public research organizations and/or companies.

One of our main goals is to generate mAb not available commercially. These mAb, made for research purposes, can also be used for commercial applications. We also help basic research groups with technology transfer from research.

The Unit generated mAb against more than 60 different antigens, for use in basic and pre-clinical research, drug discovery, and diagnostics. We produced mAb to chromatin-associated proteins (Dido, ScoHET), centrosomal proteins (CAP350), immunoglobulins (canine IgE), blood proteins (coagulation Factor V, CD5L), neurodegenerative disease-related proteins (TAU, beta amyloid peptides), mitochondrial proteins (SCAM3), kinases (P38, PI3K, DGK, GSK3, PKD, SADB), viral antigens (TEGV, SARS-E, gp120) and other human pathogens (Candida famata, Anisakis spp).

Research activities

Monoclonal antibody development and characterization

We have developed mAb for structure-function studies, antigen mapping, neutralization, quantitative assays, pharmacokinetics, and affinity determination of enzymes (alkaline phosphatase, RNAse), nucleases (CEL I), transcription factors (DREAM), hormones and their receptors (LH, FSH, TSH, hCG, hGH, hGH antagonist B2036, Pegvisomant, GHR), growth factors and their receptors (IGF-1, IGF-2, EGF, IGF-1R, EGFR), growth factor-binding proteins (IGFBP-1, IGFBP-3, GHBP), cytoskeletal proteins (4.1R, WIP), integral membrane proteins (MAL, BENE), blood proteins (albumin, immunoglobulins, Factor VIII), chemokines (CCL1, CCL11, CCL20), virus (Phi 29, torovirus) and tumor antigens (FBP). These mAb have been used by various international research groups and have been described in more than 200 scientific publications.

Production and characterization of antibodies to murine chemokine receptors using rats and knockout mice

We have generated a panel of poly- and monoclonal antibodies to chemokine receptors for studies of receptor-ligand interactions and for phenotyping mouse models of human inflammatory diseases. Some anti-mouse CCR4 and CCR8 mAb were produced by immunization of rats and knockout mice. Several of these mAb block ligand-receptor binding. One of them has been used by various research groups to inhibit CCR8 function in distinct mouse models of inflammatory disease and has been licensed to a company for worldwide commercialization.

Production and characterization of mAb to human chemokine receptors

We have generated a panel of mouse mAb to human chemokine receptors CCR6 and CCR9. One mAb against hCCR6 has been licensed for commercialization and two mAb specific for hCCR9 have been licensed as antitumor agents.

Techniques available

  • Strategy design and implementation for immunization, immunogen production and analysis of animal immune responses
  • Poly- and monoclonal antibody production in rabbit, mouse, rat and hamster
  • Immunization with plasmids and transfected cells
  • Antibody characterization by ELISA, RIA, flow cytometry, Western blot
  • Purification and labeling of enzymes and antibodies
  • Development of immunoassays for antigen detection and quantification in biological fluids
  • Characterization of biomolecular interactions in real time using a Biacore 3000 biosensor robot. Determination of kinetic and affinity constants.

Publications (2019-present)

  • Kremer L, Garcia-Sanz JA. Editorial: Is the Recent Burst of Therapeutic Anti-tumor Antibodies the Tip of an Iceberg? Front Immunol. 2018; 9: 442.
  • Somovilla-Crespo B, Martín Monzón MT, Vela M, Corraliza-Gorjón I, Santamaria S, Garcia-Sanz JA, Kremer L. 92R Monoclonal Antibody Inhibits Human CCR9(+) Leukemia Cells Growth in NSG Mice Xenografts. Front Immunol. 2018; 9: 77.
  • Cuesta-Mateos C, Alcaraz-Serna A, Somovilla-Crespo B, Muñoz-Calleja C. Monoclonal Antibody Therapies for Hematological Malignancies: Not Just Lineage-Specific Targets. Front Immunol. 2018; 8: 1936.
  • Corraliza-Gorjón I, Somovilla-Crespo B, Santamaria S, Garcia-Sanz JA, Kremer L. New Strategies Using Antibody Combinations to Increase Cancer Treatment Effectiveness. Front Immunol. 2017; 8: 1804.
  • Pose-Utrilla J, García-Guerra L, Del Puerto A, Martín A, Jurado-Arjona J, De León-Reyes NS, Gamir-Morralla A, Sebastián-Serrano Á, García-Gallo M, Kremer L, Fielitz J, Ireson C, Pérez-Álvarez MJ, Ferrer I, Hernández F, Ávila J, Lasa M, Campanero MR, Iglesias T. Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1. Nat Commun. 2017; 8: 2275.
  • Santamaria S, Delgado M, Kremer L, Garcia-Sanz JA. Will a mAb-Based Immunotherapy Directed against Cancer Stem Cells Be Feasible? Front Immunol. 2017; 8: 1509.
  • Roncador G, Engel P, Maestre L, Anderson AP, Cordell JL, Cragg MS, Šerbec VC, Jones M, Lisnic VJ, Kremer L, Li D, Koch-Nolte F, Pascual N, Rodríguez-Barbosa JI, Torensma R, Turley H, Pulford K, Banham AH. The European antibody network's practical guide to finding and validating suitable antibodies for research. MAbs. 2016; 8: 27-36.
  • Bartolini F, Andres-Delgado L, Qu X, Nik S, Ramalingam N, Kremer L, Alonso MA, Gundersen GG. An mDia1-INF2 formin activation cascade facilitated by IQGAP1 regulates stable microtubules in migrating cells. Mol Biol Cell. 2016; 27: 1797-808.
  • Ruiz MF, Alvarez M, Eirín-López JM, Sarno F, Kremer L, Barbero JL, Sánchez L. An Unusual Role for doublesex in Sex Determination in the Dipteran Sciara. Genetics. 2015, 200: 1181-99.
  • Vela M, Aris M, Llorente M, Garcia-Sanz JA, Kremer L. Chemokine receptor-specific antibodies in cancer immunotherapy: achievements and challenges. Front Immunol 2015; 6: 2.
  • González-Magaldi M, Martín-Acebes MA, Kremer L, Sobrino F. Membrane topology and cellular dynamics of foot-and-mouth disease virus 3A protein. PLoS One. 2014, 9: e106685.
  • Barbarroja-Escudero J, Prieto-Martin A, Monserrat-Sanz J, Reyes-Martin E, Diaz-Martin D, Antolin-Amerigo D, Rodriguez-Rodriguez M, Canseco-Gonzalez F, Kremer L, Martinez-A C, Alvarez-Mon M. Abnormal chemokine receptor profile on circulating T lymphocytes from nonallergic asthma patients. Int Arch Allergy Immunol. 2014, 164: 228-36.
  • Goñi GM, Epifano C, Boskovic J, Camacho-Artacho M, Zhou J, Bronowska A, Martín MT, Eck MJ, Kremer L, Gräter F, Gervasio FL, Perez-Moreno M, Lietha D. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes. Proc Natl Acad Sci U S A. 2014, 111: E3177-86.
  • Chamorro S, Vela M, Franco-Villanueva A, Carramolino L, Gutiérrez J, Gómez L, Lozano M, Salvador B, García-Gallo M, Martínez-A C, Kremer L. Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts. MAbs. 2014; 6: 1000-12.
  • Aranda JF, Reglero-Real N, Marcos-Ramiro B, Ruiz-Sáenz A, Fernández-Martín L, Bernabé-Rubio M, Kremer L, Ridley AJ, Correas I, Alonso MA, Millán J. MYADM controls endothelial barrier function through ERM-dependent regulation of ICAM-1 expression. Mol Biol Cell. 2013; 24: 483-94.
  • Sánchez-Martín D, Martínez-Torrecuadrada J, Teesalu T, Sugahara KN, Alvarez-Cienfuegos A, Ximénez-Embún P, Fernández-Periáñez R, Teresa Martín M, Molina-Privado I, Ruppen-Cañás I, Blanco-Toribio A, Cañamero M, Cuesta AM, Compte M, Kremer L, Bellas C, Alonso-Camino V, Guijarro-Muñoz I, Sanz L, Ruoslahti E, Alvarez-Vallina L. Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies. Proc Natl Acad Sci U S A. 2013, 110: 13791-6.
  • Somovilla-Crespo B, Alfonso-Pérez M, Cuesta-Mateos C, Carballo-de Dios C, Beltrán AE, Terrón F, Pérez-Villar JJ, Gamallo-Amat C, Pérez-Chacón G, Fernández-Ruiz E, Zapata JM, Muñoz-Calleja C. Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma. J Hematol Oncol. 2013; 6: 89.
  • González-Magaldi M, Postigo R, de la Torre BG, Vieira YA, Rodríguez-Pulido M, López-Viñas E, Gómez-Puertas P, Andreu D, Kremer L, Rosas MF, Sobrino F. Mutations that hamper dimerization of foot-and-mouth disease virus 3A protein are detrimental for infectivity. J Virol. 2012; 86: 11013-23.
  • Cortés I, Sánchez-Ruíz J, Zuluaga S, Calvanese V, Marqués M, Hernández C, Rivera T, Kremer L, González-García A, Carrera AC. p85β phosphoinositide 3-kinase subunit regulates tumor progression. Proc Natl Acad Sci U S A. 2012; 109: 11318-23.
  • Ruiz-Sáenz A, Kremer L, Alonso MA, Millán J, Correas I. Protein 4.1R regulates cell migration and IQGAP1 recruitment to the leading edge. J Cell Sci. 2011; 124: 2529-38.
  • Aranda JF, Reglero-Real N, Kremer L, Marcos-Ramiro B, Ruiz-Sáenz A, Calvo M, Enrich C, Correas I, Millán J, Alonso MA. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration. Mol Biol Cell. 2011, 22:1252-62.
  • López-Santalla M, Salvador-Bernáldez M, González-Alvaro I, Castañeda S, Ortiz AM, García García MI, Kremer L, Roncal F, Mulero J, Martínez-A C, Salvador JM. Tyr323-dependent p38 activation is associated with rheumatoid arthritis and correlates with disease activity. Arthritis Rheum. 2011 Mar 30. doi: 10.1002/art.30375.
  • Nogales A, Galán C, Márquez-Jurado S, García-Gallo M, Kremer L, Enjuanes L, Almazán F. Immunogenic characterization and epitope mapping of transmisible gastroenteritis virus RNA dependent RNA polymerase. J Virol Methods. 2011, 175:7-13.
  • Nieto-Torres JL, De diego ML, Alvarez E, Jiménez-Guardeño JM, Regla-Nava JA, Llorente M, Kremer L, Shuo S, Enjuanes L. Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein. Virology. 2011, 415:69-82.
  • Del Valle I, Elvira G, Garcia-Benzaquen L, Armesilla-Diaz A, Kremer L, Garcia-Sanz JA, Martinez S, Silva A. Characterization of novel monoclonal antibodies able to identify neurogenic niches and arrest neurosphere proliferation and differentiation. Neuroscience. 2010; 169: 1473-85.
  • Hoenicka J, Quiñones-Lombraña A, España-Serrano L, Alvira-Botero X, Kremer L, Pérez-González R, Rodríguez-Jiménez R, Jiménez-Arriero MA, Ponce G, Palomo T. The ANKK1 gene associated with addictions is expressed in astroglial cells and upregulated by apomorphine. Biol Psychiatry. 2010, 67:3-11.
  • Galán-Díez M, Arana DM, Serrano-Gómez D, Kremer L, Casasnovas JM, Ortega M, Cuesta-Domínguez A, Corbí AL, Pla J, Fernández-Ruiz E. Candida albicans beta-glucan exposure is controlled by the fungal CEK1-mediated mitogen-activated protein kinase pathway that modulates immune responses triggered through dectin-1. Infect Immun. 2010, 78:1426-36.
  • Koduru S, Kumar L, Massaad MJ, Ramesh N, Le Bras S, Ozcan E, Oyoshi MK, Kaku M, Fujiwara Y, Kremer L, King S, Fuhlbrigge R, Rodig S, Sage P, Carman C, Alcalde P, Luscinskas FW, Geha RS. Cdc42 interacting protein 4 (CIP4) is essential for integrin-dependent T-cell trafficking. Proc Natl Acad Sci U S A. 2010, 107:16252-6.
  • Pérez de la Lastra JM, Kremer L, de la Fuente J. Recent advances in the development of immunoadhesins for immune therapy and as anti-infective agents. Recent Pat Antiinfect Drug Discov 2009 Nov 1 [Epub ahead of print]
  • Prieto I, Kouznetsova A, Fütterer A, Trachana V, Leonardo E, Alonso Guerrero A, Cano Gamero M, Pacios-Bras C, Leh H, Buckle M, Garcia-Gallo M, Kremer L, Serrano A, Roncal F, Albar JP, Barbero JL, Martínez-A C, van Wely KH. Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis. Chromosoma. 2009, 118:617-632
  • Cuesta AM, Sánchez-Martín D, Sanz L, Bonet J, Compte M, Kremer L, Blanco FJ, Oliva B, Alvarez-Vallina L. In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences. PLoS One 2009, 4:e5381
  • Greciano PG, Ruiz MF, Kremer L, Goday C. Two new chromodomain-containing proteins that associate with heterochromatin in Sciara coprophila chromosomes. Chromosoma 2009, 118:361-376
  • Gascón S, García-Gallo M, Renart J, Díaz-Guerra M. Endoplasmic reticulum-associated degradation of the NR1 but not the NR2 subunits of the N-methyl-D-aspartate receptor induced by inhibition of the N-glycosylation in cortical neurons. J Neurosci Res 2007, 85:1713-1723

Patents

  • Inventors: Chamorro S, Franco A, García Sanz JA, Kremer L, Martínez Alonso C, Vela Cuenca M, Carramolino L. “Antibodies against CCR9 and applications thereof”. EP13382469.8, PCT/EP2014/075578 (2014), CSIC.
  • Inventors: Kremer L, Carrasco Llamas L, Pisa García D. “Monoclonal antibody against the protein GAPDH from Candida famata”. 200930966 (2009), CSIC and Universidad Autónoma Foundation.
  • Inventors: Kremer L, Llorente M, Casasnovas JM, Fernández Ruiz E, Galán Diez M. “Antibody anti-dectin-1, hybridoma producer of this antibody and its applications”. P200801766 (2008), CSIC and Biomedical Research Foundation of the La Princesa Hospital.
  • Inventors: Bernard A, Garmendia C, Abad JL, Gonzalez MA, Llorente M, Martinez-A C, Serrano F. “Multifunctional genetic constructions having a high capacity to inhibit the expression of CCR5 on the cell surface”. WO2004013330 (2004), Genetrix SL and CSIC.
  • Inventors: Kremer L, Mellado JM, Rodríguez Frade JM, Martínez Alonso C. “Method for determining cytokine receptor activation by the use of an antibody”. WO9841872, CA2285036, AU6530498, US2003077663, PCT/SE98/00496 (1998 & 2003), Pharmacia SA and CSIC.
  • Inventors: Llorente M, Mendez E, Lopez E. “Method for extracting gluten contained in heat-processed and non-heat-processed foodstuffs, compatible with an enzyme-linked immunosorbent assay, composition and kits comprising said composition”. WO02092633 (2002), CSIC.
  • Inventors: Kremer L, Mellado JM, Rodríguez-Frade JM, Martínez Alonso C, Hansson YE. “Monoclonal antibodies binding human growth hormone (hGH)”. SE 9601231-5, W09736929, NZ331868, AU2525597, US5945296 (1997), Pharmacia AB.