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WO1994026787A1 - Procede pour generer des bibliotheques d'anticorps de phages specifiques de types cellulaires - Google Patents

Procede pour generer des bibliotheques d'anticorps de phages specifiques de types cellulaires Download PDF

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Publication number
WO1994026787A1
WO1994026787A1 PCT/US1994/005124 US9405124W WO9426787A1 WO 1994026787 A1 WO1994026787 A1 WO 1994026787A1 US 9405124 W US9405124 W US 9405124W WO 9426787 A1 WO9426787 A1 WO 9426787A1
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Prior art keywords
target cells
cell
phage particles
phage
target
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PCT/US1994/005124
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English (en)
Inventor
William T. Tse
Irving L. Weissman
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The Board Of Trustees Of The Leland Stanford Junior University
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Application filed by The Board Of Trustees Of The Leland Stanford Junior University filed Critical The Board Of Trustees Of The Leland Stanford Junior University
Publication of WO1994026787A1 publication Critical patent/WO1994026787A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Definitions

  • the invention relates generally to the field of cell type specific phage antibody libraries, and relates specifically to the field of generating such libraries by direct affinity selection on the surface of target cells.
  • the phage antibody technology is a recently developed technique that allows the expression of monoclonal antibodies on the surface of filamentous phage particles (see, for example, Smith, Science 228, 1315-1317 (1985); Barbas et al., Proc. Natl. Acad. Sci. USA 88, 7978-7982 (1991); Clackson et al., Nature 352, 624-628 (1991)).
  • One advantage of this technology is the linkage of the specificity of a monoclonal antibody with the genetic information encoding the specificity in the same phage particle, thus allowing enrichment of specific phage antibodies by binding to purified antigens attached to a solid phase, followed by elution and amplification of the phage particles.
  • Such a method is described in Kang et al., Proc.Natl.Acad.Sci.USA 88, 4363-4366 (1991).
  • micropanning or affinity column techniques researchers have used this technology to generate antibodies reactive to well-characterized antigens that can be purified in large quantities, either as natural or recombinant products, including various hapterts, tetanus toxoid, HTV and RSV glycoproteins, HBV core and surface antigens, progesterone, thyroglobulin, TNF alpha, CEA, soluble CD4, IL2R p55 chain, lysozyme, bovine serum albumin, trypsin, anhydrotrypsin, and L6 tumor-associated antigen.
  • natural or recombinant products including various hapterts, tetanus toxoid, HTV and RSV glycoproteins, HBV core and surface antigens, progesterone, thyroglobulin, TNF alpha, CEA, soluble CD4, IL2R p55 chain, lysozyme, bovine serum albumin, trypsin, anhydro
  • the present invention involves methods for using the phage antibody technology to generate antibodies directed against previously unknown and unpurified antigens on the cell surface, e.g., as a way to discover new antigens on important cell types. Since a phage antibody may be amplified by growing the phage in bacteria, it is possible to recover a single phage particle specifically bound to an antigen in order to generate enough monoclonal antibodies for detailed characterization of the antigen. The high sensitivity of the method enables isolation of antibodies directed against an unpurified antigen in the midst of numerous background antigens.
  • target antigens on the surface of a phenotypically defined population of cells are used to separate phage antibodies reactive to the antigens from the background.
  • the cell membrane of the target cells can be considered a solid phase support for a set of cell type specific antigens, and the intact target cells can be used as a convenient physical device to separate phage antibodies bound on the surface of the target cells from the unbound phage antibodies.
  • the inventive method for generating antibodies against previously uncharacterized and unpurified antigens on the surface of target cells in a cell population includes incubating a combinatorial library of antibodies expressed on the surface of filamentous phage particles with a target cell population under conditions sufficient to bind a portion of the phage particles to the target cells.
  • the target cells and bound phage particles are then separated from the unbound phage particles, and the bound phage particles are recovered.
  • These phage particles are then amplified to create an enriched library.
  • Monoclonal antibodies specific to the target cell are then isolated from the enriched library for subsequent use.
  • the phage antibody library may be preadsorbed with an excess of non-target cells to immunologically remove antibodies directed against antigens present on non-target cells.
  • the adsorption step may be performed after the phage antibody selection and amplification, or prior to the initial incubating step.
  • the removal of these antibodies can be done by subtraction hybridization of DNA from a phage antibody library enriched with the target cells to DNA from a library enriched with non-target cells.
  • the phenotypically defined target cell population may be isolated prior to incubating the target cells with the phage antibody library, or the target cells may be a subpopulation within a heterogeneous cell population and be isolated after incubating with the phage antibody library, thus copurifying the phage antibodies bound to the cell surface antigens.
  • FIGURE 1 is a flow chart illustrating an embodiment of the present inventive method.
  • FIGURE 2 is a flow chart illustrating another embodiment of the present inventive method.
  • FIGURES 3a - 3d are histograms showing indirect immunofluorescence patterns of KGla cells and mouse thymocytes stained with the unenriched and enriched antibody libraries.
  • the abscissa indicates relative fluorescence intensity, and the ordinate indicates relative cell number.
  • the process of the present invention includes a method to generate antibodies against cell surface antigens by direct affinity selection of phage antibodies on the surface of target cells.
  • a phage antibody library is constructed by standard methods and an aliquot of the library is used for incubation with the target cells.
  • Phage antibodies that are specific to and have a high affinity for antigens on the target cells will tightly bind to the cells and can be separated from the rest of the phage antibody library by washing and spinning down the cells.
  • the bound phage particles can then be eluted from the cell surface, recovered, and amplified to yield an enriched phage antibody library.
  • the present method involves a first step of incubating (100) a standard phage antibody library, such as a combinatorial library of antibodies expressed on the surface of filamentous phage particles, with a target cell population.
  • a standard phage antibody library such as a combinatorial library of antibodies expressed on the surface of filamentous phage particles
  • the cells are incubated under conditions sufficient to bind a portion of the phage particles to the target cells.
  • the target cell population is an isolated cell population.
  • the process of the present invention preferably utilizes a standard combinatorial phage antibody library of 10 ⁇ recombinants, which may be prepared using standard protocols, such as that described in Barbas et al., Methods 2, 119-124 (1991).
  • the cell population may be isolated according to any preselected criteria.
  • the target cells and the phage particles bound thereto are separated (102) from the unbound phage particles.
  • Phage antibodies that are specific to and have a high affinity for antigens on the target cells will tightly bind to the cells and can be separated from the rest of the phage antibody library by centrifugating the cells through a calf serum cushion.
  • Other standard separation methods known by those skilled in the relevant art may be used, examples including but not limited to, cell washing and centrifugation, filtration of the unbound phages through a membrane, and immobilization or capture of the cells on a solid phase.
  • the separated phage particles, or phage antibodies, are then recovered (104).
  • the bound phage particles may be eluted, for example using an acidic buffer, from the cell surface using standard methods.
  • the resulting recovered phage antibodies are amplified (106) to yield an enriched phage antibody library. Repeating this enrichment step several times generates a phage antibody library with high specificity for surface antigens on the target cells. Each round of enrichment increases the representation of antibodies with higher affinity for the target antigens but decreases the diversity of the library. Thus, the amount of enrichment will depend on the particular requirements of the user.
  • monoclonal antibodies specific for the target cells are isolated (108) from the library.
  • phage antibodies reactive to antigens on the target cell population are obtained.
  • the monoclonal antibodies are isolated (108) using methods known and available to those skilled in the art.
  • direct affinity selection of the phage antibodies on the cell surface is used as described substantially above.
  • a population of isolated target cells are incubated with the phage antibody library.
  • the target cells may be in the form of a cell line, or other substantially isolated population of target cells.
  • the target cell population is a subpopulation from a heterogeneous cell population before any isolation.
  • the target cell population may subsequently be separated (110) from other cells in the heterogeneous population based on phenotypic distinctions, or other appropriate distinctions, thus copurifying the phage particles bound to the cell surface (112).
  • the target cells are phenotypically defined target cell subpopulations.
  • This allows generation of antibodies against rare but phenotypically defined cell subpopulations that are too few in cell number to allow characterization by conventional methods.
  • the phage antibody library is first allowed to bind to the total and unpurified cell population.
  • the target subpopulation is then physically separated from the rest of the cells by means of its defining phenotypic characteristics.
  • the phage particles specifically bound to the surface antigens of the target cells are separated (112) out together with the target cell subpopulation.
  • phage antibodies reactive to antigens on the target subpopulation of a heterogeneous population may be selectively enriched by copurification of the target cells with their specific phage antibodies, exploiting the phenotypic characteristics of the target cells. Standard methods of cell separation known to those skilled in the art may be used.
  • the method to be used for the copurification (112) step may be any of the type known and available to those skilled in the art.
  • Exemplary methods applicable to target cells in suspension include fluorescence-activated cell sorting (FACS), immunological panning, magnetic bead separation, and affinity chromatography.
  • FACS fluorescence-activated cell sorting
  • the specific copurification method will depend on availability of materials, the type of target cell subpopulation, and other factors determinable by those skilled in the art.
  • the cell population used in conjunction with the present method may include whole tissue sections, such as frozen tissue sections, in which the target cell population is identifiable. Exemplary methods for identifying and isolating target cell subpopulations may include use of immunohistochemical markers, physical location of cell populations, or other methods known and used by those skilled in the art.
  • tissue sections the tissue section is incubated with the phage antibody library under conditions sufficient to bind phage particles to the target cells. The target cells and the bound phage particles are then microdissected from the tissue section, the phage particles are recovered and amplified, and monoclonal antibodies are isolated in accordance with the method described above.
  • the preferred embodiment of the invention makes use of the phenotypic characteristics of the target cells to identify phage antibodies of interest by means of their binding to the target cells.
  • phage antibodies are selected if they are shown to have an identifiable and preselected function, as compared with other phage antibodies without such function.
  • phage antibodies may be assayed, for example, for the functional consequences of a phage antibody-target cell binding.
  • One assay may be to measure intracellular calcium concentration.
  • Another effective assay may be used to detect cell activation, cell proliferation, or cell killing, or the blocking of these activities.
  • Yet another assay may be to analyze the expression of preselected genes.
  • Other functional methods of identifying phage antibodies known and available to those skilled in the art may be used.
  • the resulting phage antibody library may contain antibodies reactive to antigens unique to the target cells and antibodies reactive to antigens present on both the target cells and non-target cells. It may be desirable to selectively remove the antibodies reactive to antigens not unique to the target cells. This may be accomplished by preadsorbing the phage antibody library with an excess of non-target cells to immunologically remove phage antibodies directed against antigens present on non-target cells. This additional step may be performed either prior to incubating the phage antibody library with the cell population, or prior to isolating monoclonal antibodies from the enriched library.
  • the goal of selectively enriching phage antibodies directed against antigens present only on target cells and not on non-target cells can also be achieved by DNA subtraction hybridization.
  • two cell type specific phage antibody libraries are generated using the present inventive method, one for the target cells and one for the non-target cells.
  • DNA encoding antibodies directed against antigens present on both cell types are then removed from the first library by hybridizing DNA from the first library to an excess of DNA from the second library and subsequently removing the annealed hybrids.
  • the resulting subtracted phage antibody library will then contain mostly antibodies directed against antigens unique to the target cells. Standard methods of DNA subtraction hybridization known to those skilled in the relevant art, or variations of these methods, may be used in this step.
  • KGla cell line is an available human myeloblastoid cell line, and is characterized in several publications, including Koeffler et al., Blood 56, 265-273 (1980).
  • spleen cells are isolated from the animals, lymphocytes are separated on a Ficoll-PaqueTM gradient, commercially available from Pharmacia LKB Biotechnology, Inc., Piscataway, New Jersey, and total RNA is prepared using standard, known techniques.
  • a combinatorial phage antibody library of 10 ⁇ recombinants is prepared using standard protocols as described in Barbas et al., Methods 2, 119-124 (1991), and as described in further detail below.
  • KGla cells are incubated with phage particles from the combinatorial library in binding buffer on ice for 30 minutes, and then spun through a calf serum cushion three times. Phage particles bound to the surface of the cells are eluted in an acid buffer (such as 0.1 M HC1, adjusted with glycine to pH 2.2; and BSA lmg/ml), recovered, and used to infect XLl-Blue bacteria, available from Stratagene Cloning Systems, La Jolla, California. The phagemids in the infected bacteria are expanded in culture and rescued with helper phage VCSM13 to yield an enriched phage antibody library. The library is then used in another round of enrichment.
  • an acid buffer such as 0.1 M HC1, adjusted with glycine to pH 2.2; and BSA lmg/ml
  • Table 1 lists the number of phage particles recovered after each round of enrichment, showing a steady increase in the number of recovered phage particles after each round.
  • a negative control experiment (results not shown) using mouse thymocytes as the target cells for binding with the phage shows no increase in the number of phage particles recovered.
  • FIGURES 3(a) through 3(d) are histograms showing fluorescence patterns of KGla cells and mouse thymocytes stained with the unenriched (a,c) or enriched (b,d) antibody libraries. The abscissa indicates relative fluorescence intensity and the ordinate indicates relative cell number.
  • Fab fragments are produced separately from ten individual clones picked at random from the library. FACS analysis of these samples (results not given) shows that the Fab fragments from all of the ten clones strongly react with KGla cells and with variable patterns, indicating that all of the ten monoclonal antibodies are specific for KGla and probably recognize at least several different surface antigens on the cells.
  • the entire enriched antibody library most likely contains a large number of monoclonal antibodies recognizing many different surface antigens on KGla cells.
  • KGla cells which bear the human CD34 antigen on their surface, are stained with CD34-specific antibodies according to Civin et al., T.Immunol 133, 157-165 (1984), and spiked at a ratio of 1 to 10 or 1 to 100 into mouse thymocytes, which are CD34 antigen negative.
  • the cell mixtures are incubated with an unenriched phage antibody library and sorted for CD34 antigen positive (CD34+) cells by FACS. Phage particles bound on the surface of the sorted cells are recovered by acid elution and used to infect XLl-Blue bacteria.
  • An enriched phage antibody library is then produce by helper phage rescue of the phagemids in the infected bacteria and used in another round of enrichment.
  • Table 2 the number of phage particles recovered from the sorted CD34+ cells increases dramatically after three rounds of selection, suggesting effective enrichment of specific phage antibody by the copurification method described in detail above.
  • Fab fragments are produced from the enriched library and used for staining KGla and mouse thymocytes. FACS analysis using these Fab fragments show a strong reaction with the KGla cells but no reaction with the mouse thymocytes (results not shown). Fab fragments from the unenriched library give signals similar to the background. This result demonstrates the feasibility of producing specific monoclonal antibodies directed against a rare population of cells using the combined strategies of cell surface selection and target cell copurification.

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Abstract

L'invention concerne un procédé pour générer des anticorps monoclonaux dirigés contre des antigènes non caractérisés précédemment et non purifiés, sur la surface de cellules cibles dans une population de cellules. Le procédé consiste à incuber (100) une bibliothèque combinatoire d'anticorps exprimés sur la surface de particules filamenteuses de phages avec une population de cellules cibles dans des conditions permettant de fixer une portion des particules de phages aux cellules cibles. Les cellules cibles et les particules de phages sont alors séparées (102) des particules de phages non liées et les particules de phages liées sont récupérées (104). Ces particules de phages sont alors amplifiées (106) pour créér une bibliothèque enrichie. Les anticorps monoclonaux spécifiques des cellules cibles sont alors isolés (108) de la bibliothèque enrichie, pour une utilisation ultérieure.
PCT/US1994/005124 1993-05-07 1994-05-04 Procede pour generer des bibliotheques d'anticorps de phages specifiques de types cellulaires WO1994026787A1 (fr)

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022972A2 (fr) * 1995-12-15 1997-06-26 Pharmacia & Upjohn Company Procede pour la detection simultanee d'une cible therapeutique pour un etat pathologique et de sa molecule de type anticorps neutralisant
US5721342A (en) * 1989-11-13 1998-02-24 British Technology Group Limited Control of milk secretion
WO1998034110A1 (fr) * 1997-01-31 1998-08-06 Active Biotech Ab Identification in situ de structures cibles p.ex. procede de selection in vivo destine a une banque de bacteriophages
WO1998039469A1 (fr) * 1997-03-04 1998-09-11 Bio-Technology General Corp. Isolement de ligands peptidiques a specificite tissulaire et utilisation de ces derniers pour cibler des substances pharmaceutiques sur des organes
WO1998049286A2 (fr) * 1997-05-01 1998-11-05 Board Of Regents, The University Of Texas System Evolution orientee d'enzymes et d'anticorps
WO1999041402A2 (fr) * 1998-02-11 1999-08-19 Maxygen, Inc. Ciblage de vecteurs de vaccins genetiques
WO1999056129A1 (fr) * 1998-04-24 1999-11-04 The Regents Of The University Of California Procedes de selection d'anticorps d'internalisation
WO2000014215A1 (fr) * 1998-09-07 2000-03-16 Eberhard-Karls-Universität Tübingen Procede de selection de peptides pour le transport cible de medicaments et de marqueurs et peptides decouverts a l'aide dudit procede
US6057098A (en) * 1997-04-04 2000-05-02 Biosite Diagnostics, Inc. Polyvalent display libraries
US6190856B1 (en) * 1996-05-22 2001-02-20 The Johns Hopkins University Methods of detection utilizing modified bacteriophage
WO2001025492A1 (fr) * 1999-10-02 2001-04-12 Biosite Diagnostics Inc. Anticorps humains
US6265150B1 (en) 1995-06-07 2001-07-24 Becton Dickinson & Company Phage antibodies
US6277583B1 (en) * 1996-02-07 2001-08-21 Conjuchem, Inc. Affinity labeling libraries and applications thereof
US6680209B1 (en) 1999-12-06 2004-01-20 Biosite, Incorporated Human antibodies as diagnostic reagents
US6924359B1 (en) 1999-07-01 2005-08-02 Yale University Neovascular-targeted immunoconjugates
US7045283B2 (en) 2000-10-18 2006-05-16 The Regents Of The University Of California Methods of high-throughput screening for internalizing antibodies
US7132510B2 (en) 2000-12-29 2006-11-07 Bio-Technology General (Israel) Ltd. Specific human antibodies for selective cancer therapy
US7135287B1 (en) 1999-10-02 2006-11-14 Biosite, Inc. Human antibodies
US7244826B1 (en) 1998-04-24 2007-07-17 The Regents Of The University Of California Internalizing ERB2 antibodies
US7288249B2 (en) 1999-03-01 2007-10-30 Genentech, Inc. Antibodies for cancer therapy and diagnosis
US7390619B1 (en) 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0460607A2 (fr) * 1990-06-05 1991-12-11 Bristol-Myers Squibb Company Nouvel anticorps monoclonal contre un antigène nouveau associé aux tumeurs humaines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0460607A2 (fr) * 1990-06-05 1991-12-11 Bristol-Myers Squibb Company Nouvel anticorps monoclonal contre un antigène nouveau associé aux tumeurs humaines

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HARLOW et al., "Antibodies a Laboratory Manual", Published 1988, by COLD SPRING HARBOR LABORATORY (N.Y.), pages 496-497. *
J. IMMUNOLOGY, Vol. 135, No. 4, issued October 1985, LEDBETTER et al., "Antibodies to TP67 and TP44 Augment and Sustain Prolifferative Response of Activated T Cells", pages 2331-2336. *
NATURE, Vol. 308, issued 08 March 1984, HENDRICK et al., "Isolation of CDNA Clones Encoding T Cell-Specific Membrane-Associated Proteins", pages 149-153. *

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721342A (en) * 1989-11-13 1998-02-24 British Technology Group Limited Control of milk secretion
US6858384B2 (en) * 1995-06-07 2005-02-22 Crucell Holland B.V. Phage antibodies
US6265150B1 (en) 1995-06-07 2001-07-24 Becton Dickinson & Company Phage antibodies
WO1997022972A3 (fr) * 1995-12-15 1997-08-21 Upjohn Co Procede pour la detection simultanee d'une cible therapeutique pour un etat pathologique et de sa molecule de type anticorps neutralisant
WO1997022972A2 (fr) * 1995-12-15 1997-06-26 Pharmacia & Upjohn Company Procede pour la detection simultanee d'une cible therapeutique pour un etat pathologique et de sa molecule de type anticorps neutralisant
US6277583B1 (en) * 1996-02-07 2001-08-21 Conjuchem, Inc. Affinity labeling libraries and applications thereof
US6190856B1 (en) * 1996-05-22 2001-02-20 The Johns Hopkins University Methods of detection utilizing modified bacteriophage
WO1998034110A1 (fr) * 1997-01-31 1998-08-06 Active Biotech Ab Identification in situ de structures cibles p.ex. procede de selection in vivo destine a une banque de bacteriophages
US6727062B1 (en) 1997-01-31 2004-04-27 Active Biotech Ab Identification of target structures E.G. in vivo selection method for a phage library
WO1998039469A1 (fr) * 1997-03-04 1998-09-11 Bio-Technology General Corp. Isolement de ligands peptidiques a specificite tissulaire et utilisation de ces derniers pour cibler des substances pharmaceutiques sur des organes
US6986986B1 (en) 1997-04-04 2006-01-17 Joe Buechler Polyvalent display libraries
US6057098A (en) * 1997-04-04 2000-05-02 Biosite Diagnostics, Inc. Polyvalent display libraries
WO1998049286A3 (fr) * 1997-05-01 1999-03-25 Univ Texas Evolution orientee d'enzymes et d'anticorps
WO1998049286A2 (fr) * 1997-05-01 1998-11-05 Board Of Regents, The University Of Texas System Evolution orientee d'enzymes et d'anticorps
WO1999041402A3 (fr) * 1998-02-11 1999-11-11 Maxygen Inc Ciblage de vecteurs de vaccins genetiques
WO1999041402A2 (fr) * 1998-02-11 1999-08-19 Maxygen, Inc. Ciblage de vecteurs de vaccins genetiques
US7390619B1 (en) 1998-02-11 2008-06-24 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines
US9388244B2 (en) 1998-04-24 2016-07-12 The Regents Of The University Of California Internalizing ErbB2 antibodies
US8974792B2 (en) 1998-04-24 2015-03-10 The Regents Of The University Of California Internalizing erbB2 antibodies
WO1999056129A1 (fr) * 1998-04-24 1999-11-04 The Regents Of The University Of California Procedes de selection d'anticorps d'internalisation
US8173424B2 (en) 1998-04-24 2012-05-08 The Regents Of The University Of California Internalizing ErbB2 antibodies
US7892554B2 (en) 1998-04-24 2011-02-22 The Regents Of The University Of California Internalizing ErbB2 antibodies
US7244826B1 (en) 1998-04-24 2007-07-17 The Regents Of The University Of California Internalizing ERB2 antibodies
WO2000014215A1 (fr) * 1998-09-07 2000-03-16 Eberhard-Karls-Universität Tübingen Procede de selection de peptides pour le transport cible de medicaments et de marqueurs et peptides decouverts a l'aide dudit procede
US7288249B2 (en) 1999-03-01 2007-10-30 Genentech, Inc. Antibodies for cancer therapy and diagnosis
US7858092B2 (en) 1999-07-01 2010-12-28 Yale University Neovascular-targeted immunoconjugates
US6924359B1 (en) 1999-07-01 2005-08-02 Yale University Neovascular-targeted immunoconjugates
US8071104B2 (en) 1999-07-01 2011-12-06 Yale University Neovascular-targeted immunoconjugates
US8388974B2 (en) 1999-07-01 2013-03-05 Yale University Neovascular-targeted immunoconjugates
US7135287B1 (en) 1999-10-02 2006-11-14 Biosite, Inc. Human antibodies
US6794132B2 (en) 1999-10-02 2004-09-21 Biosite, Inc. Human antibodies
WO2001025492A1 (fr) * 1999-10-02 2001-04-12 Biosite Diagnostics Inc. Anticorps humains
US7189515B2 (en) 1999-12-06 2007-03-13 Biosite, Inc. Human antibodies as detection reagents
US6680209B1 (en) 1999-12-06 2004-01-20 Biosite, Incorporated Human antibodies as diagnostic reagents
US7045283B2 (en) 2000-10-18 2006-05-16 The Regents Of The University Of California Methods of high-throughput screening for internalizing antibodies
US7132510B2 (en) 2000-12-29 2006-11-07 Bio-Technology General (Israel) Ltd. Specific human antibodies for selective cancer therapy

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