EP0733070A1 - Procede de generation d'anticorps specifiques - Google Patents

Procede de generation d'anticorps specifiques

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Publication number
EP0733070A1
EP0733070A1 EP95905871A EP95905871A EP0733070A1 EP 0733070 A1 EP0733070 A1 EP 0733070A1 EP 95905871 A EP95905871 A EP 95905871A EP 95905871 A EP95905871 A EP 95905871A EP 0733070 A1 EP0733070 A1 EP 0733070A1
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EP
European Patent Office
Prior art keywords
antibody
cell
library
antibodies
phage
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP95905871A
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German (de)
English (en)
Inventor
Gary Barsomian
Diane P. Copeland
Dana Hillhouse
Tracy Johnson
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Genzyme Corp
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Genzyme Corp
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Publication date
Application filed by Genzyme Corp filed Critical Genzyme Corp
Publication of EP0733070A1 publication Critical patent/EP0733070A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

  • antibodies are synthesized and secreted into bodily fluids by plasma cells, a type of terminally differentiated B-lymphocyte. Exposure of the animal to a foreign molecule (i.e. via immunization) generally produces multiple plasma cell clones resulting in a heterogeneous mixture of antibodies (polyclonal antibodies) in the blood and other fluids.
  • the blood of an immunized animal can be collected, clotted, and the clot removed to leave a sera containing the antibodies produced in response to immunization. This remaining liquid or serum, which contains the polyclonal antibodies, is referred to as antiserum.
  • antiserum contains many different types of antibodies that are specific for many different antigens. Even in hyperimmunized animals, seldom are more than one tenth of the circulating antibodies specific for the particular immunogen used to immunized the animal. The use of these mixed populations of antibodies, though useful in many situations, can create a variety of different problems in immunochemical techniques. For example, such antiserum will generally be inadequate for use in distinguishing between the immunogen and closely related molecules which share many common determinants with the immunogen.
  • Monoclonal antibodies are traditionally made by isolating a single antibody secreting cell (e.g. a lymphocyte) from an immunized animal, fusing the lymphocyte with a myeloma (or other immortal) cell to form a hybrid cell (called a "hybridoma"), and then culturing the selected hybridoma cell in vivo or in vitro to yield antibodies which are identical in structure and specificity.
  • a single antibody secreting cell e.g. a lymphocyte
  • myeloma or other immortal
  • the antibody-secreting cell line is immortal, the characteristics of the antibody are reproducible from batch to batch.
  • the usefulness of monoclonal antibodies stems from three characteristics - their specificity of binding, their homogeneity, and their ability to be produced in virtually unlimited quantities. While production of monoclonal antibodies has resulted in production of antibodies of greater specificity to a particular antigen then polyclonal methods, there are nevertheless a number of limitations associated with these techniques and antibodies produced thereby.
  • a key aspect in the isolation of monoclonal antibodies relates to how many antibody producing hybridoma cells with different specificities can be practically established and sampled in response to immunization with a particular antigen, compared to how many theoretically need to be sampled in order to obtain an antibody having specific characteristics. For example, the number of different antibody specificities expressed at any one time by lymphocytes of the murine immune system is thought to be approximately 10 7 and represents only a small proportion of the potential repertoire of specificities.
  • Immunization regimens can provide enrichment of B-cells producing the desired antibodies.
  • typical protocols for isolating antibody producing B-cells permit sampling of generally less than 500 antibody producing hybridoma cells per immunized animal.
  • traditional techniques for the production of monoclonal antibodies statistically favor generation of monoclonal antibodies to immunodominant molecules, making isolation of antibodies specific for a rare or less immunodominant epitope difficult.
  • This problem can be further exacerbated by the fact that in many instances pure antigen is not available as an immunogen, particularly in the case of cell surface antigens. Immunization with intact cells frequently results in production of antibodies against irrelevant epitopes, especially for xenotypic immunization.
  • Neonatal tolerization and chemical immunosuppression are most commonly used to reduce clonal expansion of B cells in response to "background” antigen signals, thereby enriching for a population of B cells responsive to the epitopes of interest.
  • the practical application of a subtractive immunization technique can be very difficult, as the efficiency of immunosuppression is often not acceptable, or as in the case of cyclophosphamide immunosuppression, generally results in only a few antibody-producing hybridoma cells per immunotolerized animal (e.g. less than 100), making it unlikely that monoclonal antibodies can be isolated which are specific to the immunorecessive epitopes.
  • the present invention provides a method for generating an antibody which is specific for an immunorecessive epitope, and nucleic acid encoding the antibody.
  • the subject method generally comprises the steps of generating a variegated display library of antibody variable regions, and selecting from the library those antibody variable regions which have a desired binding specificity for the immunorecessive epitope.
  • the antibody variable regions used to generate the display library are cloned from an immunotolerance-derived antibody repertoire.
  • the antibody variable regions of the display library are presented by a replicable genetic display package in an immunoreactive context which permits the antibody to bind to an antigen that is contacted with the display package.
  • affinity selection techniques can be utilized to enrich the population of display packages for those having antibody variable regions which have a desired binding specificity for the immunorecessive epitope.
  • the display library can be a phage display library.
  • the display library can be generated on a bacterial cell-surface or a spore.
  • the subject method can be used to isolate antibodies which are specific for such immunorecessive epitopes as, for example, cell-type specific markers, including fetal cell markers such as fetal nucleated red blood markers, cancer cell markers such as colon cancer markers or metastatic tumor cell markers, stem cell markers such as markers for precursor nerve cells or hematopoietic stem cells.
  • cell-type specific markers including fetal cell markers such as fetal nucleated red blood markers, cancer cell markers such as colon cancer markers or metastatic tumor cell markers, stem cell markers such as markers for precursor nerve cells or hematopoietic stem cells.
  • the subject method can be used to generate antibodies which can discriminate by binding between a variant form of a protein and other related forms of the protein.
  • the variant protein can differ by one or more amino acid residues from other related proteins in order to give rise to the immunorecessive epitope, as well as vary antigenically from the related protein by virtue of glycosylation or other post-translational modification.
  • the variation can arise naturally, as between different isoforms of a protein family, illustrated by the apolipoprotein E family, or can be generated by genetic aberration, as illustrated by the neoplastic transforming mutations of oncogenic proteins or tumor suppressor proteins such as p53.
  • a specific antibody to an immunorecessive epitope can be generated by affinity purification of a antibody phage display library derived from an immunotolerance-derived antibody repertoire.
  • suitable host cells are transformed with a library of replicable phage vectors encoding a library of phage particles displaying a fusion antibody /coat protein, where the fusion protein includes a phage coat protein portion and an antibody variable region portion.
  • the antibody variable region is obtained from the immunotolerance-derived antibody repertoire.
  • the transformed cells are cultured, the phage particles are formed, and the antibody fusion proteins are expressed. Any of resulting phage particles which have an antibody variable region portion which specifically binds to a an immunorecessive epitope can be separated from those which do not specifically bind the immunorecessive epitope.
  • the present invention further pertains to novel immunorecessive antibody libraries produced by the subject method.
  • an antibody display library can be isolated which is enriched for antibodies that specifically bind an immunorecessive epitope of interest.
  • the display library comprises a population of display packages expressing a variegated V-gene library which has been cloned from an immunotolerance-derived antibody repertoire, and which has been further enriched after expression by the display package via affinity separation with the immunorecessive epitope. It is also contemplated by the present invention that individual antibodies, and genes encoding these antibodies, can be isolated from the antibody libraries of the subject method.
  • individual display packages can be obtained, and the antibody gene contained therein subcloned into other appropriate expression vectors suitable for production of the antibody for the desired use.
  • Figures 1A and IB show variable region PCR primers for amplifying the variable regions of both heavy and light chains from murine antibody genes.
  • Figure 2 shows a schematic representation of an Fab' expression cassette.
  • Figure 3 is a semi-log graph depicting the binding of phage antibody pools (phab) enriched on the HEL cell line (number indicates the round of enrichment). The graph provides additional comparison of the enriched phab pools with the binding of other immunoglobulins (T3, Anti-M and Wilma) to the HEL cells.
  • Figure 4 illustrates the percentage of cells (either HEL cells or mature white cells) stained by individual phab isolates generated by the subject method.
  • Figure 5 A shows the results of sequential rounds of pre-adsorption and enrichment on fetal liver cells for phab binding.
  • the increase in the percentage of phage antibodies binding to fetal liver cells is indicative enrichment for fetal cell binding phage antibodies.
  • the phage antibody library was derived using a V-gene library from an immunotolerized host animal.
  • Figure 5B compares the results of the immunotolerized experiment in Figure 5A with the results of sequential rounds of panning using phage antibody libraries derived immunized, but not tolerized, host animals.
  • Figure 6 show variable region PCR primers for amplifying the variable regions of both heavy and light chains from human antibody genes.
  • Figure 7 details the sequences for CDR3 regions of both heavy and light chains for individual phab isolates enriched on fetal cells.
  • Figures 8A and 8B illustrate the general features of the FB3-2 and H3-3 antibodies, respectively, including the framework regions (double underline; FRs), complementarity determining regions (CDRs), and constant regions (italics; IgGl CHI or kappa constant).
  • the present invention makes available a powerful directed approach for isolating specific antibodies which are extremely difficult or impossible to obtain by current methodologies, and thereby overcomes the deficiencies discussed above.
  • One aspect of the present invention is the synthesis of a method that combines immunotolerization and variegated display libraries to yield a dramatic and surprising synergism in the efficient isolation of antibodies having a desired binding affinity for an immunorecessive target epitope. Utilizing immunotolerance techniques such as subtractive immunization, a subset of lymphocytes producing antibodies against an immunorecessive target epitope are enriched in an immunized animal.
  • V-genes antibody variable region genes
  • the subject method selects genes encoding antibodies specific for the target epitope by (i) displaying the antibodies encoded by each variable region gene on the outer surface of a replicable genetic display package to create an antibody display library, and (ii) using affinity selection techniques to enrich the population of display packages for those containing V-genes encoding antibodies which have a desired binding specificity for the target epitope.
  • antibodies isolated by the subject method can have binding affinities greater than 10 8 M _1 , e.g., in the range of lO ⁇ **1 to lO ⁇ M **1 .
  • the specificity of these antibodies can be several fold, if not orders of magnitude, better than combinatorial and hybridoma generated antibodies, particularly with respect to antibodies for cell surface epitopes.
  • the subject method can provide antibodies which have no substantial background binding to other related cells, e.g., specificities greater than 10 fold binding to the target cells over background binding to the related cells.
  • antibodies can be generated which do not substantially cross-react with other epitopes, preferably having specificities greater than 20 fold over background, more preferably 50, 75 or 100 fold over background, and even more preferably more than 125 fold over background.
  • the term "antibody” in its various grammatical forms is art-recognized and includes immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
  • the simplest naturally occurring antibody comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • the light chains exist in two distinct forms called kappa (K) and lambda ( ⁇ ).
  • K kappa
  • ⁇ lambda
  • Each chain has a constant region (C) and a variable region (V).
  • Each chain is organized into a series of domains.
  • the light chains have two domains, corresponding to the C region and the other to the V region.
  • the heavy chains have four domains, one corresponding to the V region and three domains (1,2 and 3) in the C region.
  • the naturally occurring antibody has two arms (each arm being an Fab region), each of which comprises a V L and a VJJ region associated with each other. It is this pair of V regions (V L and V H ) that differ from one antibody to another (owing to amino acid sequence variations).
  • the variable domains for each of the heavy and light chains have the same general structure, including four framework regions (FRs), whose sequences are relatively conserved, connected by three hypervariable or complementarity determining regions (CDRs).
  • variable region of each chain can typically be represented by the general formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDRs for a particular variable region are held in close proximity to one and other by the framework regions, and with the CDRs from the other chain and which together are responsible for recognizing the antigen and providing an antigen binding site (ABS).
  • ABS antigen binding site
  • binding antigens can be performed by fragments of a naturally-occurring antibody, and as set out above, these antigen-binding fragments are also intended to be designated by the term "antibody".
  • binding fragments encompassed within the term antibody include (i) the Fab fragment consisting of the V L , V H , CL and C H 1 domains; (ii) the Fd fragment consisting of the V ⁇ and CHI domains; (iii) the Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) the dAb fragment (Ward et al., (1989) Nature 341 :544-546 ) which consists of a VJJ domain; (v) isolated CDR regions; and (vi) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • antibody variable region is likewise recognized in the art, and includes those portions of an antibody which can assemble to form an antigen binding site.
  • an antibody variable region can comprise each of the framework regions (FR1-
  • CDR1-CDR3 complementary determining regions
  • a desired binding specificity for an immunorecessive epitope refers to the ability of individual antibodies to specifically immunoreact with distinct antigens.
  • the desired binding specificity will typically be determined from the reference point of the ability of the antibody to differentially bind, and therefore distinguish between, two different antigens -particularly where the two antigens have unique epitopes which are present along with many common epitopes.
  • a desired binding affinity for an immunorecessive epitope can refer to the ability of an antibody to distinguish between related cells, such as between adult and fetal cells, or between normal and transformed cells.
  • the desired binding affinity can refer to the ability of the antibody to differentially bind a mutant form of a protein versus the wild-type protein, or alternatively, to discriminate in binding between different isoforms of a protein.
  • An antibody which binds specifically to an immunorecessive epitope is referred to as a "specific antibody”.
  • the term "relative specificity” refers to the ratio of specific immunoreactivity to background immunoreactivity (e.g., binding to non- target antigens). For instance, relative specificity for fetal cells can be expressed as the ratio of the percent binding to fetal cells to the percent binding to maternal cells.
  • Antibodies which have no substantial background binding to a non-target antigen, such as a maternal cell have large relative specificities (e.g., in excess of 10 fold over background binding).
  • an antibody binds to only a particular portion of the macromolecule, referred to herein as the "determinant" or "epitope".
  • the total number of antibodies produced by a population of antibody-producing cells in a particular animal is referred to a the "antibody repertoire”.
  • the extraordinary diversity of the antibody repertoire is a result of variability in the structures of the antigen binding sites amongst the individual antibodies which make up the repertoire.
  • immunogens refers to the exposure of an animal (that is capable of producing antibodies) to a foreign antigen so as to induce active immunity, which includes the production of antibodies to the foreign antigen.
  • Molecules that generate an immune response are called immunogens.
  • immunorecessive epitope which is also substituted from time to time with the terms "rare epitope” or "target epitope”, is intended to refer to epitopes that, in the context that it ordinarily occurs or can be isolated as an immunogen, are typically not efficient for use in generating an antibody response by immunization, at least so far as polyclonal and monoclonal antibody production is concerned. Such immunorecessive epitopes will generally be less abundant and/or less antigenic than other epitopes commonly associated with them in the immunogen.
  • Immunorecessive epitopes may be associated with, for example, cell surface antigens that are unique to a particular cell phenotype. In many instances, this cell surface antigen is not in and of itself available as an immunogen because no purified form of the antigen has been obtained.
  • an immunogen containing the immunorecessive epitope will also include many background epitopes which can act to decrease the overall percentage of B-lymphocytes activated by the immunorecessive epitope in the total B-lymphocyte population.
  • the immunogen can comprise the whole cell on which the immunorecessive epitope is expressed.
  • the immunorecessive epitope can be a cell-type specific marker, such as a cancer cell marker, a fetal cell marker, or a stem cell marker.
  • an immunorecessive epitope can comprise an epitope unique to a variant form of a protein, such as a variant which differs by only one or two amino acid residues from a related protein.
  • the immunorecessive epitope can be a determinant of a mutant p53 which does not arise on the wild-type p53, or an epitope which unique to a particular isoform of human apolipoprotein E, such as ApoE4.
  • Tolerization refers to the process of diminishing an animal's immunological responsiveness to a potentially antigenic substance present in that animal, and the antigenic substance to which tolerance is created is refered to as a "toleragen". Tolerance results from the interaction of toleragen with antigen receptors on lymphocytes under conditions in which the lymphocytes, instead of becoming activated, are killed or rendered unresponsive. Tolerance to particular antigens, or more exactly, to particular epitopes of an antigen, can be induced by a number of means, including neonatal tolerization or chemically-induced tolerization, and can be the result of induced clonal deletion or clonal anergy. The route of administration of an antigen can also effect the ability of the antigen to act as either an immunogen or as a toleragen.
  • immunotolerizing means relates to a process whereby the antibody response to an immunorecessive epitope is unmasked by the deletion of an antibody response to the background epitopes. For instance, as a first step in the immunotolerizing means, an animal is exposed to a toleragen comprising the immunodominant epitopes. The toleragen, however, lacks the immunorecessive epitopes. After tolerance to these background epitopes has been induced, an immunogen which includes the immunorecessive epitopes, is administered to the animal.
  • the immunotolerizing means can be used to "enrich” for cells producing antibodies specific for an immunorecessive epitope.
  • background epitopes is further defined as those epitopes that are common between the immunogen and the toleragen, while the term “immunorecessive epitopes” is further understood to refer to epitopes unique to the immunogen (relative to the toleragen).
  • the immunogen and the toleragen will typically be closely related, as for example, in the instance of phenotypically related cells, or mutant or different isoforms of a protein.
  • immunotolerance-derived antibody repertoire refers to the population of antibody-producing cells, and their antibodies, generated by an immunotolerization which is intended to enrich for antibodies for an immunorecessive epitope.
  • variable V-gene library refers to a mixture of recombinant nucleic acid molecules encoding at least the antibody variable regions of one or both of the heavy and light chains of the immunotolerance-derived antibody repertoire.
  • a population of display packages into which the variegated V-gene library has been cloned and expressed on the surface thereof is likewise said to be a “variegated antibody display library” or "antibody display library”.
  • the language "replicable genetic display package” or "display package” describes a biological particle which has genetic information providing the particle with the ability to replicate.
  • the package can display a fusion protein including an antibody derived from the variegated V-gene library.
  • the antibody portion of the fusion protein is presented by the display package in an immunoreactive context which permits the antibody to bind to an antigen that is contacted with the display package.
  • the display package will generally be derived from a system that allows the sampling of very large variegated V-gene libraries, as well as easy isolation of the recombinant V-genes from purified display packages.
  • the display package can be, for example, derived from vegetative bacterial cells, bacterial spores, and bacterial viruses (especially DNA viruses).
  • a variegated mixture of display packages encoding at least a portion of the V-gene library is also referred to as an "antibody display library”.
  • differential binding means refer to the separation of members of the antibody display library based on the differing abilities of antibodies on the surface of each of the display packages of the library to bind to the target epitope.
  • the differential binding of an immunorecessive epitope by antibodies of the display can be used in the affinity separation of antibodies which specifically bind the immunorecessive epitope from antibodies which do not.
  • the same molecule or cell that was used as an immunogen in the immunotolerizing step can also be used in an affinity enrichment step to retrieve display packages expressing antibodies which specifically bind it.
  • the affinity selection protocol will also include a pre- enrichment step wherein display packages capable of specifically binding the background epitopes are removed.
  • affinity selection means include affinity chromatography, immunoprecipitation, fluorescence activated cell sorting, agglutination, and plaque lifts.
  • affinity chromatography includes bio-panning techniques using either purified, immobilized antigen as well as whole cells.
  • the display package is a phage particle which comprises an antibody fusion coat protein that includes the amino acid sequence of an antibody variable region from the variegated V-gene library.
  • a library of replicable phage vectors, especially phagemids (as defined herein), encoding a library of antibody fusion coat proteins is generated and used to transform suitable host cells.
  • Phage particles formed from the chimeric protein can be separated by affinity selection based on the ability of the antibody associated with a particular phage particle to specifically bind a target epitope.
  • each individual phage particle of the library includes a copy of the corresponding phagemid encoding the antibody fusion coat protein displayed on the surface of that package.
  • phage particles Purification of phage particles based on the ability of an antibody displayed on an individual particle to bind a particular epitope therefore also provides for isolation of the V-gene encoding that antibody.
  • exemplary phage for generating the present variegated antibody libraries include Ml 3, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, ⁇ , T4, T7, P2, P4, ⁇ X-174, MS2 and 2.
  • fusion protein and "chimeric protein” are art-recognized terms which are used interchangeably herein, and include contiguous polypeptides comprising a first polypeptide covalently linked via an amide bond to one or more amino acid sequences which define polypeptide domains that are foreign to and not substantially homologous with any domain of the first polypeptide.
  • One polypeptide from which the fusion protein is constructed comprises a recombinant antibody derived from the cloned V-gene library.
  • a second polypeptide portion of the fusion protein is typically derived from an outer surface protein or display anchor protein which directs the "display package" (as hereafter defined) to associate the antibody with its outer surface.
  • this anchor protein can be derived from a surface protein native to the genetic package, such as a viral coat protein.
  • the fusion protein comprises a viral coat protein and an antibody it will be referred to as an "antibody fusion coat protein".
  • the fusion protein may further comprise a signal sequence, which is a short length of amino acid sequence at the amino terminal end of the fusion protein, that directs at least a portion of the fusion protein to be secreted from the cytosol of a cell and localized on the extracellular side of the cell membrane.
  • Gene constructs encoding fusion proteins are likewise referred to a "chimeric genes" or "fusion genes”.
  • chimeric antibody is used to describe a protein including at least the antigen binding portion of an immunoglobulin molecule attached by peptide linkage to at least a part of another protein.
  • a chimeric antibody can be, for example, an interspecies chimera, having a variable region derived from a first species (e.g. a rodent) and a constant region derived from a second species (e.g. a human), or alternatively, having CDRs derived from a first species and FRs and a constant region from a second species.
  • vector refers to a DNA molecule, capable of replication in a host cell, into which a gene can be inserted to construct a recombinant DNA molecule.
  • the use of phage vectors rather than the phage genome itself provides greater flexibility to vary the ratio of chimeric antibody/coat protein to wild-type coat protein, as well as supplement the phage genes with additional genes encoding other variable regions, such as may be useful in the two chain antibody constructs described below.
  • helper phage describes a phage which is used to infect cells containing a defective phage genome or phage vector and which functions to complement the defect.
  • the defect can be one which results from removal or inactivation of phage genomic sequence required for production of phage particles.
  • helper phage are M13K07, and M13K07 gene III no. 3.
  • isolated as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
  • an isolated nucleic acid encoding one of the subject antibodies preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the antibodies gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequence.
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • the subject invention sets forth a method for rapid and efficient isolation of cell-type specific antibodies.
  • antibodies that specifically bind epitopes unique to fetal cells or, alternatively, epitopes unique to cancer cells can be generated by the subject method.
  • the subject method can be employed to generate antibodies to variant forms of a protein, and which can be used, for example, to detect a mutation of a protein or to differentiate amongst various isoforms of a protein.
  • the present invention can provide antibodies useful for purification, diagnostic, and therapeutic applications.
  • the invention concerns novel immunorecessive antibody libraries produced by the subject method, as well as individual antibodies isolated therefrom.
  • an antibody display library can be isolated which is enriched for antibodies that specifically bind an immunorecessive epitope of interest.
  • the display library comprises a population of display packages expressing a variegated V-gene library which has been cloned from an immunotolerance-derived antibody repertoire, and which has been further enriched after expression by the display package by affinity separation with the immunorecessive epitope.
  • antibody display libraries can be generated which are enriched for specific antibodies to cell surface markers, such as fetal cell of tumor cell markers, as well as variant forms of proteins.
  • the specificity of the antibodies enriched for in the subject library can be defined in terms of the particular immunogen/toleragen sets used. For example, where the specific antibody is desired for distinguishing between various cells of common or similar origin or phenotype, the cell to which a specific antibody is desired is used as the immunogen, while a related cell(s) from which it is to be distinguished is employed as the toleragen. Cell-type specific markers for the cell of interest are represented in the immunorecessive epitopes.
  • the toleragen can include maternal erythroid cells and the immunogen can be fetal erythroid cells.
  • the toleragen can comprise normal colon cells and the immunogen can be selected from a colon carcinoma cell line.
  • Other exemplary immunogen/toleragen sets useful for generating the subject antibody libraries, as well as individual antibodies therefrom, are provided in the following description and others will be apparent to those skilled in the art.
  • the subject libraries can be generated so as to be enriched for specific antibodies able to distinguish by binding between a variant form of a protein and other related forms of the protein.
  • the variant protein can differ by one or more amino acid residues from other related proteins in order to give rise to the immunorecessive epitope, as well as vary antigenically from the toleragen by virtue of glycosylation or other post-translational modification.
  • the variation can arise naturally, as between different isoforms of a protein family, illustrated by the apolipoprotein E family, or can be generated by genetic aberration, as illustrated by the neoplastic transforming mutations of oncogenic proteins or tumor suppressor proteins such as p53.
  • individual antibodies, and genes encoding these antibodies can be isolated from the antibody libraries of the subject method. For instance, after affinity enrichment of the antibody display library for antibodies which specifically bind the immunorecessive epitope, individual display packages can be obtained, and the antibody gene contained therein subcloned into other appropriate expression vectors suitable for production of the antibody for the desired use.
  • Immunotolerization can be employed in the present invention to generate an antibody repertoire, for use in subsequent V-gene cloning steps, in which the antibody response to an immunorecessive epitope(s) has been unmasked. Immunotolerization can be carried out in either in vivo or in vitro immunization systems. For instance, immunotolerization can be employed in the present invention to enrich the pool of activated B-lymphocytes in an immunized animal for cells producing antibodies directed to immunorecessive epitopes of interest. In a typical immunotolerization procedure of the subject method, an immunogen is introduced to the immune system of an animal some time after exposure to a toleragen.
  • the effect of the toleragen is to reduce or abrogate altogether any immunological response upon re-exposure of the animal to determinants of the toleragen.
  • the determinants composing the toleragen are generally a portion of those antigenic determinants comprising the immunogen (i.e. the background epitopes)
  • the reduced antibody response to the background epitopes upon challenge with the immunogen can act to unmask the antibody response to the immunorecessive epitopes of the immunogen.
  • unmasked it is meant that the population of antibody-producing cells directed to the immunorecessive epitopes effectively becomes a greater percentage of the overall population of antibody-producing cells in the animal (see Williams et al. (1992) Biotechniques 12:842-847).
  • immunotolerizing means includes subtractive immunization for enriching a pool of B-cells for clones producing antibodies specific for rare epitopes.
  • subtractive immunization is a two-step procedure. Step one is a suppression step in which a state of tolerance is induced in the immune system of a host animal to a specific set of molecules, the tolerogen. Step two is an immunizing step in which another set of molecules, the immunogen, is introduced to the immune system.
  • the molecules comprising the tolerogen are generally a subset of those comprising the immunogen. Ideally, the only molecules to which the immune system will generate the antibodies after exposure to the immunogen are those molecules present in the immunogen but not present in the tolerogen.
  • neonatal tolerization is utilized to generate an enriched pool of B-cells.
  • Neonatal tolerization utilizes the self-tolerization process of the developing immune system. For each species, a discrete developmental period exists during which the immune system classifies all molecules present in the body as self, resulting in an induced state of immunological tolerance to those molecules (Billingham et al. (1953) Nature 172:603-606; Golumbeski et al. (1986) Anal Biochem 154:373-381; Hasek et al.
  • mice or other host animals
  • the immune system should be immunologically responsive only to those molecules in the immunogen, but not in the tolerogen.
  • chemical immunosuppression is the immunotolerizing means employed to generate an enriched B-cell population for subsequent cloning of variable region genes (V-genes).
  • V-genes variable region genes
  • chemical immunosuppression via the cytotoxic drug cyclophosaphamide is technique useful for subtractive immunization
  • the tolerogen the tolerogen
  • cyclophosphamide the tolerogen
  • the immune system should be immunologically responsive only to those epitopes of the immunogen that are not found in the tolerogen.
  • subtractive immunization protocols are also available for use in the subject method, and include, for example, the use of interleukin-targeted toxins.
  • interleukin-targeted toxins include, for example, the use of interleukin-targeted toxins.
  • IL-2- toxin fusion proteins Kelley et al. (1988) PNAS 85:3980-3984) and IL-4-toxin fusion proteins (Lakkis et al. (1991) Eur J Immunol 21 :2253-2258) can be used to selectively induce tolerance to the epitopes of a toleragen.
  • the antibody repertoire of the resulting B-cell pool is cloned.
  • Methods are generally known, and can be applied in the subject method, for directly obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR.
  • mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11: 152-156).
  • a similar strategy can also been used to amplify human heavy and light chain variable regions
  • RNA is isolated from mature B cells of, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS
  • First-strand cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and ⁇ light chains, as well as primers for the signal sequence.
  • variable region PCR primers such as those shown in Figures 1A and IB (for mouse) or Figure 6 (for human)
  • the variable regions of both heavy and light chains are amplified, each alone or in combination, and ligated into appropriate vectors for further manipulation in generating the display packages.
  • Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.
  • the V-gene library cloned from the immunotolerance-derived antibody repertoire can be expressed by a population of display packages to form an antibody display library.
  • the display package on which the variegated antibody library is manifest it will be appreciated from the discussion provided herein that the display package will often preferably be able to be (i) genetically altered to encode at least a variable region of an antibody, (ii) maintained and amplified in culture, (iii) manipulated to display the antibody gene product in a manner permitting the antibody to interact with a target epitope during an affinity separation step, and (iv) affinity separated while retaining the antibody gene such that the sequence of the antibody gene can be obtained.
  • the display remains viable after affinity separation.
  • the display package comprises a system that allows the sampling of very large variegated antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages.
  • the most attractive candidates for this type of screening are prokaryotic organisms and viruses, as they can be amplified quickly, they are relatively easy to manipulate, and large number of clones can be created.
  • Preferred display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses).
  • the present invention also contemplates the use of eukaryotic cells (other than cells which naturally produce antibodies, i.e. B-cells), including yeast and their spores, as potential display packages.
  • kits for generating phage display libraries e.g. the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612
  • methods and reagents particularly amenable for use in generating the variegated antibody display library of the present invention can be found in, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication WO 92/20791 ; the Markland et al.
  • the display means of the package will comprise at least two components.
  • the first component is a secretion signal which directs the recombinant antibody to be localized on the extracellular side of the cell membrane (of the host cell when the display package is a phage). This secretion signal is characteristically cleaved off by a signal peptidase to yield a processed, "mature" antibody.
  • the second component is a display anchor protein which directs the display package to associate the antibody with its outer surface. As described below, this anchor protein can be derived from a surface or coat protein native to the genetic package.
  • the means for arraying the variegated antibody library comprises a derivative of a spore or phage coat protein amenable for use as a fusion protein.
  • the antibody component of the display will comprise, at a minimum, one of either the V H or V L regions cloned from B cells isolated in the subtractive immunization step. It will be appreciated, however, that the V H regions and/or the V L regions may contain, in addition to the variable portion of the antibodies, all or a portion of the constant regions.
  • the display library will include variable regions of both heavy and light chains in order to generate at least an Fv fragment.
  • the minimal antibody display as comprising the use of cloned V- ⁇ regions to construct the fusion protein with the display anchor protein. However, it should be readily understood that similar embodiments are possible in which the role of the V L and V H chains are reversed in the construction of the display library.
  • the V H portion of the antibody display is derived from isolated cells of the subtractive immunization step, but the V L chain is either absent or is a "fixed" V L (i.e. the same V L chain for every antibody of the display).
  • the V L chain can be contributed from a gene construct other than the construct encoding the V H chain, or from the host cell itself (i.e. a light chain producing myeloma cell), or added exogenously to the packages so as to recombine with Vp j chains already displayed on their surface.
  • the V L chain is derived from a variegated V L library also cloned from the same population of B cells from which the V H gene is cloned, in which case a preferred embodiment places the VL gene in the same construct as the V H gene such that both may be readily recovered together.
  • the cDNA encoding the light chain may be cloned directly into an appropriate site of the vector containing the heavy chain-coat protein library; or, alternatively, the light chain may be cloned as a separate library in a different plasmid vector, amplified, and subsequently the fragments cloned into the vector library encoding the heavy chain.
  • the V L chain is cloned so that it is expressed with a signal peptide leader sequence that will direct its secretion into the periplasm of the host cell. For example, several leader sequences have been shown to direct the secretion of antibody sequences in E.
  • coli such as OmpA (Hsiung et al. Bio/Technology (1986) 4:991-995), and (Better et al. Science 240:1041-1043), phoA (Skerra and Pluckthun, Science (1988) 240:1038).
  • the cloning site for the VL chain sequences in the phagemid should be placed so that it does not substantially interfere with normal phage function.
  • One such locus is the intergenic region as described by Zinder and Boeke, (1982) Gene 19:1-10.
  • the V L sequence is preferably expressed at an equal or higher-level than the H L -cpIII product (described below) to maintain a sufficiently high V L concentration in the periplasm and provide efficient assembly (association) of V L with V H chains.
  • a phagemid can be constructed to encode, as separate genes, both a V H /coat fusion protein and a V L chain. Under the appropriate induction, both chains are expressed and allowed to assemble in the periplasmic space of the host cell, the assembled antibody being linked to the phage particle by virtue of the V ⁇ chain being a portion of a coat protein fusion construct.
  • coli such as strain MCI 061
  • libraries may be constructed in fd-tet Bl of up to about 3 x 10 8 members or more.
  • Increasing DNA input and making modifications to the cloning protocol within the ability of the skilled artisan may produce increases of greater than about 10- fold in the recovery of transformants, providing libraries of up to 10 10 or more recombinants.
  • the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome.
  • a flexible linker As generally described in McCafferty et al., Nature (1990) 348:552-554, complete V H and V L domains of an antibody, joined by a flexible (Gly 4 -Ser) 3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity.
  • an important criteria for the present selection method can be that it is able to discriminate between antibodies of different affinity for a particular antigen, and preferentially enrich for the antibodies of highest affinity.
  • manipulating the display package to be rendered effectively monovalent can allow affinity enrichment to be carried out for generally higher binding affinities (i.e. binding constants in the range of 10 6 to 10 10 M **1 ) as compared to the broader range of affinities isolable using a multivalent display package.
  • the natural i.e.
  • the library of display packages will comprise no more than 5 to 10% polyvalent displays, and more preferably no more than 2% of the display will be polyvalent , and most preferably, no more than 1% polyvalent display packages in the population.
  • the source of the wild-type anchor protein can be, for example, provided by a copy of the wild-type gene present on the same construct as the antibody fusion protein, or provided by a separate construct altogether.
  • polyvalent displays can be generated to isolate a broader range of binding affinities. Such antibodies can be useful, for example, in purification protocols where avidity can be desirable.
  • Bacteriophage are attractive prokaryotic-related organisms for use in the subject method. Bacteriophage are excellent candidates for providing a display system of the variegated antibody library as there is little or no enzymatic activity associated with intact mature phage, and because their genes are inactive outside a bacterial host, rendering the mature phage particles metabolically inert. In general, the phage surface is a relatively simple structure. Phage can be grown easily in large numbers, they are amenable to the practical handling involved in many potential mass screening programs, and they carry genetic information for their own synthesis within a small, simple package.
  • choosing the appropriate phage to be employed in the subject method will generally depend most on whether (i) the genome of the phage allows introduction of the antibody gene either by tolerating additional genetic material or by having replaceable genetic material; (ii) the virion is capable of packaging the genome after accepting the insertion or substitution of genetic material; and (iii) the display of the antibody on the phage surface does not disrupt virion structure sufficiently to interfere with phage propagation.
  • phage One concern presented with the use of phage is that the mo ⁇ hogenetic pathway of the phage determines the environment in which the antibody will have opportunity to fold. Periplasmically assembled phage are preferred as the displayed antibodies will generally contain essential disulfides, and such antibodies may not fold correctly within a cell. However, in certain embodiments in which the display package forms intracellularly (e.g., where ⁇ phage are used), it has been demonstrated that the antibody may assume proper folding after the phage is released from the cell.
  • the preferred display means is a protein that is present on the phage surface (e.g. a coat protein).
  • Filamentous phage can be described by a helical lattice; isometric phage, by an icosahedral lattice.
  • Each monomer of each major coat protein sits on a lattice point and makes defined interactions with each of its neighbors. Proteins that fit into the lattice by making some, but not all, of the normal lattice contacts are likely to destabilize the virion by aborting formation of the virion as well as by leaving gaps in the virion so that the nucleic acid is not protected.
  • the antibody library is expressed and allowed to assemble in the bacterial cytoplasm, such as when the ⁇ phage is employed.
  • the induction of the protein(s) may be delayed until some replication of the phage genome, synthesis of some of the phage structural-proteins, and assembly of some phage particles has occurred.
  • the assembled protein chains then interact with the phage particles via the binding of the anchor protein on the outer surface of the phage particle.
  • the cells are lysed and the phage bearing the library-encoded receptor protein (that corresponds to the specific library sequences carried in the DNA of that phage) are released and isolated from the bacterial debris.
  • phage harvested from the bacterial debris are affinity purified.
  • the antigen or determinant can be used to retrieve phage displaying the desired antibody.
  • the phage so obtained may then be amplified by infecting into host cells. Additional rounds of affinity enrichment followed by amplification may be employed until the desired level of enrichment is reached.
  • the enriched antibody-phage can also be screened with additional detection-techniques such as expression plaque (or colony) lift (see, e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeled antigen is used as a probe.
  • additional detection-techniques such as expression plaque (or colony) lift (see, e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeled antigen is used as a probe.
  • the phage obtained from the screening protocol are infected into cells, propagated, and the phage DNA isolated and sequenced, and/or recloned into a vector intended for gene expression in prokaryotes or eukaryotes to obtain larger amounts of the particular antibody selected.
  • the antibody is also transported to an extra-cytoplasmic compartment of the host cell, such as the bacterial periplasm, but as a fusion protein with a viral coat protein.
  • the desired protein or one of its polypeptide chains if it is a multichain antibody
  • the viral coat protein which is processed and transported to the cell inner membrane.
  • Other chains if present, are expressed with a secretion leader and thus are also transported to the periplasm or other intraceUular by extra- cytoplasmic location.
  • the chains e.g.
  • Filamentous bacteriophages which include Ml 3, fl, fd, Ifl, Ike, Xf, Pfl, and Pf3, are a group of related viruses that infect bacteria. They are termed filamentous because they are long, thin particles comprised of an elongated capsule that envelopes the deoxyribonucleic acid (DNA) that forms the bacteriophage genome.
  • the F pili filamentous bacteriophage (Ff phage) infect only gram-negative bacteria by specifically adsorbing to the tip of F pili, and include fd, fl and Ml 3.
  • filamentous phage in general are attractive and Ml 3 in particular is especially attractive because: (i) the 3-D structure of the virion is known; (ii) the processing of the coat protein is well understood; (iii) the genome is expandable; (iv) the genome is small; (v) the sequence of the genome is known; (vi) the virion is physically resistant to shear, heat, cold, urea, guanidinium chloride, low pH, and high salt; (vii) the phage is a sequencing vector so that sequencing is especially easy; (viii) antibiotic-resistance genes have been cloned into the genome with predictable results (Hines et al.
  • Ml 3 is a plasmid and transformation system in itself, and an ideal sequencing vector. Ml 3 can be grown on Rec- strains of E. coli. The Ml 3 genome is expandable (Messing et al.
  • the mature capsule or Ff phage is comprised of a coat of five phage-encoded gene products: cpVIII, the major coat protein product of gene VIII that forms the bulk of the capsule; and four minor coat proteins, cpIII and cpIV at one end of the capsule and cpVII and cpIX at the other end of the capsule.
  • the length of the capsule is formed by 2500 to 3000 copies of cpVIII in an ordered helix array that forms the characteristic filament structure.
  • the gene Ill-encoded protein (cpIII) is typically present in 4 to 6 copies at one end of the capsule and serves as the receptor for binding of the phage to its bacterial host in the initial phase of infection.
  • the phage particle assembly involves extrusion of the viral genome through the host cell's membrane.
  • the major coat protein cpVIII and the minor coat protein cpIII are synthesized and transported to the host cell's membrane. Both cpVIII and cpIII are anchored in the host cell membrane prior to their inco ⁇ oration into the mature particle.
  • the viral genome is produced and coated with cpV protein.
  • cpV-coated genomic DNA is stripped of the cpV coat and simultaneously recoated with the mature coat proteins.
  • Both cpIII and cpVIII proteins include two domains that provide signals for assembly of the mature phage particle.
  • the first domain is a secretion signal that directs the newly synthesized protein to the host cell membrane.
  • the secretion signal is located at the amino terminus of the polypeptide and targets the polypeptide at least to the cell membrane.
  • the second domain is a membrane anchor domain that provides signals for association with the host cell membrane and for association with the phage particle during assembly.
  • This second signal for both cpVIII and cpIII comprises at least a hydrophobic region for spanning the membrane.
  • the 50 amino acid mature gene VIII coat protein (cpVIII) is synthesized as a 73 amino acid precoat (Ito et al. (1979) PNAS 76:1199-1203).
  • cpVIII has been extensively studied as a model membrane protein because it can integrate into lipid bilayers such as the cell membrane in an asymmetric orientation with the acidic amino terminus toward the outside and the basic carboxy terminus toward the inside of the membrane.
  • the first 23 amino acids constitute a typical signal-sequence which causes the nascent polypeptide to be inserted into the inner cell membrane.
  • SP-I signal peptidase
  • the sequence of gene VIII is known, and the amino acid sequence can be encoded on a synthetic gene.
  • Mature gene VIII protein makes up the sheath around the circular ssDNA.
  • the gene VIII protein can be a suitable anchor protein because its location and orientation in the virion are known (Banner et al. (1981) Nature 289:814-816).
  • the antibody is attached to the amino terminus of the mature Ml 3 coat protein to generate the phage display library.
  • manipulation of the concentration of both the wild-type cpVIII and Ab/cpVIII fusion in an infected cell can be utilized to decrease the avidity of the display and thereby enhance the detection of high affinity antibodies directed to the target epitope(s).
  • Another vehicle for displaying the antibody is by expressing it as a domain of a chimeric gene containing part or all of gene III.
  • expressing the V-gene as a fusion protein with gpIII can be a preferred embodiment, as manipulation of the ratio of wild-type gpIII to chimeric gpIII during formation of the phage particles can be readily controlled.
  • This gene encodes one of the minor coat proteins of Ml 3.
  • Genes VI, VII, and IX also encode minor coat proteins. Each of these minor proteins is present in about 5 copies per virion and is related to mo ⁇ hogenesis or infection. In contrast, the major coat protein is present in more than 2500 copies per virion.
  • the gene VI, VII, and IX also encode minor coat proteins.
  • IX proteins are present at the ends of the virion; these three proteins are not post- translationally processed (Rasched et al. (1986) Ann Rev. Microbiol. 41:507-541).
  • the single-stranded circular phage DNA associates with about five copies of the gene III protein and is then extruded through the patch of membrane-associated coat protein in such a way that the DNA is encased in a helical sheath of protein (Webster et al. in The
  • the successful cloning strategy utilizing a phage coat protein will provide: (1) expression of an antibody chain fused to the N- terminus of a coat protein (e.g., cpIII) and transport to the inner membrane of the host where the hydrophobic domain in the C-terminal region of the coat protein anchors the fusion protein in the membrane, with the N-terminus containing the antibody chain protruding into the periplasmic space and available for interaction with a second or subsequent chain (e.g., V L to form an Fv or Fab fragment) which is thus attached to the coat protein; and (2) adequate expression of a second or subsequent polypeptide chain if present (e.g., VL) and transport of this chain to the soluble compartment of the periplasm.
  • a coat protein e.g., cpIII
  • Pf3 is a well known filamentous phage that infects Pseudomonas aerugenosa cells that harbor an IncP-I plasmid. The entire genome has been sequenced ((Luiten et al. (1985) J Virol. 56:268-276) and the genetic signals involved in replication and assembly are known (Luiten et al. (1987) DNA 6:129-137).
  • the major coat protein of PF3 is unusual in having no signal peptide to direct its secretion. The sequence has charged residues ASP-7, ARG-37, LYS-40, and PHE44 which is consistent with the amino terminus being exposed.
  • a tripartite gene can be constructed which comprises a signal sequence known to cause secretion in P. aerugenosa, fused in-frame to a gene fragment encoding the antibody sequence, which is fused in-frame to DNA encoding the mature Pf3 coat protein.
  • DNA encoding a flexible linker of one to 10 amino acids is introduced between the antibody gene fragment and the Pf3 coat-protein gene.
  • This tripartite gene is introduced into Pf3 so that it does not interfere with expression of any Pf3 genes.
  • the bacteriophage ⁇ X174 is a very small icosahedral virus which has been thoroughly studied by genetics, biochemistry, and electron microscopy (see The Single Stranded DNA Phages (eds. Den hardt et al. (NY:CSHL Press, 1978)).
  • Three gene products of ⁇ X174 are present on the outside of the mature virion: F (capsid), G (major spike protein, 60 copies per virion), and H (minor spike protein, 12 copies per virion).
  • the G protein comprises 175 amino acids, while H comprises 328 amino acids.
  • the F protein interacts with the single-stranded DNA of the virus.
  • the proteins F, G, and H are translated from a single mRNA in the viral infected cells.
  • ⁇ X174 is not typically used as a cloning vector due to the fact that it can accept very little additional DNA.
  • mutations in the viral G gene encoding the G protein
  • a copy of the wild-type G gene carried on a plasmid that is expressed in the same host cell (Chambers et al. (1982) Nuc Acid Res 10:6465-6473).
  • one or more stop codons are introduced into the G gene so that no G protein is produced from the viral genome.
  • the variegated antibody gene library can then be fused with the nucleic acid sequence of the H gene.
  • the second plasmid can further include one or more copies of the wild-type H protein gene so that a mix of H and Ab/H proteins will be predominated by the wild-type H upon inco ⁇ oration into phage particles.
  • Phage such as ⁇ or T4 have much larger genomes than do Ml 3 or ⁇ X174, and have more complicated 3-D capsid structures than M13 or ⁇ PX174, with more coat proteins to choose from.
  • bacteriophage ⁇ and derivatives thereof are examples of suitable vectors.
  • the intraceUular mo ⁇ hogenesis of phage ⁇ can potentially prevent protein domains that ordinarily contain disulfide bonds from folding correctly.
  • variegated libraries expressing a population of functional antibodies, including both heavy and light chain variable regions have been generated in ⁇ phage. (Huse et al.
  • library DNA When used for expression of antibody sequences, such as V H , V L , Fv (variable region fragment) or Fab, library DNA may be readily inserted into a ⁇ vector.
  • variegated antibody libraries have been constructed by modification of ⁇ ZAP II (Short et al. (1988) Nuc Acid Res 16:7583) comprising inserting both cloned heavy and light chain variable regions into the multiple cloning site of a ⁇ ZAP II vector (Huse et al. supra.).
  • a pair of ⁇ vectors may be designed to be asymmetric with respect to restriction sites that flank the cloning and expression sequences. This asymmetry allows efficient recombination of libraries coding for separate chains of the active protein.
  • a library expressing antibody light chain variable regions may be combined with one expressing antibody heavy chain variable regions (VJJ), thereby constructing combinatorial antibody or Fab expression libraries.
  • V L antibody light chain variable regions
  • VJJ antibody heavy chain variable regions
  • one ⁇ vector is designed to serve as a cloning vector for antibody light chain sequences
  • another ⁇ vector is designed to serve as a cloning vector for antibody heavy chain sequences in the initial steps of library construction.
  • a combinatorial library is constructed from the two ⁇ libraries by crossing them at an appropriate restriction site. DNA is first purified from each library, and the right and left arms of each respective ⁇ vector cleaved so as to leave the antibody chain sequences intact.
  • one strategy for displaying antibodies on bacterial cells comprises generating a fusion protein by inserting the antibody into cell surface exposed portions of an integral outer membrane protein (Fuchs et al. (1991) Bio/Technology 9:1370-1372).
  • any well-characterized bacterial strain will typically be suitable, provided the bacteria may be grown in culture, engineered to display the antibody library on its surface, and is compatible with the particular affinity selection process practiced in the subject method.
  • the preferred display systems include Salmonella typhirnurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli.
  • Salmonella typhirnurium Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli.
  • Many bacterial cell surface proteins useful in the present invention have been characterized, and works on the localization of these proteins and the methods of determining their structure include Benz et al. (1988) Ann Rev Microbiol 42: 359-3
  • LamB protein of E coli is a well understood surface protein that can be used to generate a variegated library of antibodies on the surface of a bacterial cell (see, for example, Ronco et al.
  • LamB of E. coli is a porin for maltose and maltodextrin transport, and serves as the receptor for adso ⁇ tion of bacteriophages ⁇ and K10. LamB is transported to the outer membrane if a functional N-terminal signal sequence is present (Benson et al. (1984) PNAS 81:3830-3834). As with other cell surface proteins, LamB is synthesized with a typical signal-sequence which is subsequently removed.
  • the variegated antibody gene library can be cloned into the LamB gene such that the resulting library of fusion proteins comprise a portion of LamB sufficient to anchor the protein to the cell membrane with the antibody fragment oriented on the extracellular side of the membrane.
  • Secretion of the extracellular portion of the fusion protein can be facilitated by inclusion of the LamB signal sequence, or other suitable signal sequence, as the N-terminus of the protein.
  • the E. coli LamB has also been expressed in functional form in S. typhimurium (Harkki et al. (1987) Mol Gen Genet 209:607-611), V. cholerae (Harkki et al. (1986) Microb Pathol 1 :283-288), and K. pneumonia (Wehmeier et al. (1989) Mol Gen Genet 215:529-536), so that one could display a population of antibodies in any of these species as a fusion to E. coli LamB. Moreover, K. pneumonia expresses a maltoporin similar to LamB which could also be used. In P. aeruginosa, the Dl protein (a homologue of LamB) can be used (Trias et al.
  • Bacterial spores also have desirable properties as display package candidates in the subject method. For example, spores are much more resistant than vegetative bacterial cells or phage to chemical and physical agents, and hence permit the use of a great variety of affinity selection conditions. Also, Bacillus spores neither actively metabolize nor alter the proteins on their surface. However, spores have the disadvantage that the molecular mech ⁇ anisms that trigger sporulation are less well worked out than is the formation of Ml 3 or the export of protein to the outer membrane of E.
  • Bacteria of the genus Bacillus form endospores that are extremely resistant to damage by heat, radiation, desiccation, and toxic chemicals (reviewed by Losick et al. (1986) Ann Rev Genet 20:625-669). This phenomenon is attributed to extensive intermolecular cross- linking of the coat proteins.
  • Bacillus spores can be the preferred display package. Endospores from the genus Bacillus are more stable than are, for example, exospores from Streptomyces.
  • Bacillus subtilis forms spores in 4 to 6 hours, whereas Streptomyces species may require days or weeks to sporulate.
  • genetic knowledge and manipulation is much more developed for B. subtilis than for other spore-forming bacteria. Viable spores that differ only slightly from wild-type are produced in B. subtilis even if any one of four coat proteins is missing (Donovan et al. (1987) J Mol Biol 196:1-10).
  • plasmid DNA is commonly included in spores, and plasmid encoded proteins have been observed on the surface of Bacillus spores (Debro et al. (1986) J Bacteriol 165:258-268).
  • the variegated antibody display is subjected to affinity enrichment in order to select for antibodies which bind preselected antigens.
  • affinity separation or “affinity enrichment” includes, but is not limited to (1) affinity chromatography utilizing immobilizing antigens, (2) immunoprecipitation using soluble antigens, (3) fluorescence activated cell sorting, (4) agglutination, and (5) plaque lifts.
  • the library of display packages are ultimately separated based on the ability of the associated antibody to bind an epitope on the antigen of interest. See, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No.
  • the display library will be pre-enriched for antibodies specific for the rare epitope by first contacting the display library with a source of the background epitope, such as the toleragen, in order to further remove antibodies which bind the background epitopes. Subsequently, the display package is contacted with the target antigen and antibodies of the display which are able to specifically bind the antigen are isolated.
  • a source of the background epitope such as the toleragen
  • the target antigen is immobilized on an insoluble carrier, such as sepharose or polyacrylamide beads, or, alternatively, the wells of a microtitre plate.
  • an insoluble carrier such as sepharose or polyacrylamide beads, or, alternatively, the wells of a microtitre plate.
  • the cells on which the antigen is displayed may serve as the insoluble matrix carrier.
  • the population of display packages is applied to the affinity matrix under conditions compatible with the binding of the antibody to a target antigen. The population is then fractionated by washing with a solute that does not greatly effect specific binding of antibodies to the target antigen, but which substantially disrupts any non-specific binding of the display package to the antigen or matrix.
  • a certain degree of control can be exerted over the binding characteristics of the antibodies recovered from the display library by adjusting the conditions of the binding incubation and subsequent washing.
  • the temperature, pH, ionic strength, divalent cation concentration, and the volume and duration of the washing can select for antibodies within a particular range of affinity and specificity. Selection based on slow dissociation rate, which is usually predictive of high affinity, is a very practical route. This may be done either by continued incubation in the presence of a saturating amount of free hapten (if available), or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated antibody-display package is prevented, and with increasing time, antibody-display packages of higher and higher affinity are recovered.
  • antibodies with special characteristics may be used in affinity purification of various proteins when gentle conditions for removing the protein from the antibody are required.
  • Specific examples are antibodies which depend on Ca ++ for binding activity and which released their haptens in the presence of EGTA. (see, Hopp et al. (1988) Biotechnology 6:1204-1210).
  • Such antibodies may be identified in the recombinant antibody library by a double screening technique isolating first those that bind hapten in the presence of Ca ++ , and by subsequently identifying those in this group that fail to bind in the presence of EGTA.
  • specifically bound display packages can be eluted by either specific deso ⁇ tion (using excess antigen) or non-specific deso ⁇ tion (using pH, polarity reducing agents, or chaotropic agents).
  • the elution protocol does not kill the organism used as the display package such that the enriched population of display packages can be further amplified by reproduction.
  • the list of potential eluants includes salts (such as those in which one of the counter ions is Na + , NH4 + , Rb + , SO4 2 -, H2PO4-, citrate, K + , Li + , Cs + , HSO4-, CO3 2 -, Ca 2+ , Sr 2+ , Cl " , PO4 2 -, HCO3-, Mg 2 + , Ba2 + , Br, HPO 4 2* ⁇ or acetate), acid, heat, and, when available, soluble forms of the target antigen (or analogs thereof).
  • salts such as those in which one of the counter ions is Na + , NH4 + , Rb + , SO4 2 -, H2PO4-, citrate, K + , Li + , Cs + , HSO4-, CO3 2 -, Ca 2+ , Sr 2+ , Cl " , PO4 2 -, HCO3-, Mg 2 +
  • buffer components especially eluates
  • Neutral solutes such as ethanol, acetone, ether, or urea, are examples of other agents useful for eluting the bound display packages.
  • affinity enriched display packages are iteratively amplified and subjected to further rounds of affinity separation until enrichment of the desired binding activity is detected.
  • the specifically bound display packages, especially bacterial cells need not be eluted per se, but rather, the matrix bound display packages can be used directly to inoculate a suitable growth media for amplification.
  • the fusion protein generated with the coat protein can interfere substantially with the subsequent amplification of eluted phage particles, particularly in embodiments wherein the cpIII protein is used as the display anchor.
  • the cpIII protein is used as the display anchor.
  • some antibody constructs because of their size and/or sequence, may cause severe defects in the infectivity of their carrier phage. This causes a loss of phage from the population during reinfection and amplification following each cycle of panning.
  • the antibody can be derived on the surface of the display package so as to be susceptible to proteolytic cleavage which severs the covalent linkage of at least the antigen binding sites of the displayed antibody from the remaining package.
  • such a strategy can be used to obtain infectious phage by treatment with an enzyme which cleaves between the antibody portion and cpIII portion of a tail fiber fusion protein (e.g. such as the use of an enterokinase cleavage recognition sequence).
  • DNA prepared from the eluted phage can be transformed into host cells by electroporation or well known chemical means.
  • the cells are cultivated for a period of time sufficient for marker expression, and selection is applied as typically done for DNA transformation.
  • the colonies are amplified, and phage harvested for a subsequent round(s) of panning.
  • the nucleic acid encoding the V-genes for each of the purified display packages can be recloned in a suitable eukaryotic or prokaryotic expression vector and transfected into an appropriate host for production of large amounts of protein.
  • the isolated V-gene lacks a portion of a constant region and it is desirable that the missing portion be provided, simple molecular cloning techniques can be used to add back the missing portions.
  • the binding affinity of the antibody can be confirmed by well known immunoassay techniques with the target epitope (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)).
  • Antibody Compositions, and Immunoassay Kits Another aspect of the present invention concerns chimeric antibodies, e.g., altered antibodies in which at least the antigen binding portion of an immunoglobulin isolated by the method described above is cloned into another protein, preferably another antibody.
  • chimeric antibodies contemplated by the present invention
  • further manipulation of the subject antibodies can be used to complete the portion of the constant region isolated from the V-gene library, as well as to facilitate "class switching" whereby all or a portion of the constant region of the antibody isolated from the V-gene library is replaced with a different constant region, e.g., with the constant region(s) from a different IgG, such as IgGl, IgG2 or IgG3, or the constant region(s) from one of IgE, IgA, IgD or IgM.
  • single chain antibodies and other recombinant fragments can be generated from the cloned genes.
  • humanized antibody is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, the remaining immunoglobulin-derived portions of the molecule, as necessary to substantially reduce the immunogenicity of the molecule in human subjects, being derived from a human immunoglobulin.
  • the antigen binding site may include, for example, either complete variable domains fused to constant domains, or only the CDRs grafted to the appropriate framework regions in human variable domains.
  • Such antibodies are the equivalents of the recombinant antibodies described above, but may be less immunogenic when administered to humans, and therefore more likely to be tolerated upon injected in a patient.
  • any of the H3-3, FB3-2 or F4-7 antibodies described in the Examples below can be prepared to include human constant regions for each of the heavy and light chains of these mouse-derived genes.
  • the portion of the antibody gene encoding the murine constant region can be substituted with a gene encoding a human constant region (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
  • the subject antibodies can also be "humanized” by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
  • General reviews of "humanized” chimeric antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207; and by Oi et al. (1986) BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain. Sources of such nucleic acids are well known to those skilled in the art. The cD ⁇ A encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
  • Suitable "humanized” antibodies can be alternatively produced by CDR replacement (see U.S. Patent 5,225,539 to Winter; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060).
  • the D ⁇ A sequence encoding the chimeric variable domain may be prepared by oligonucleotide synthesis. This requires that at least the framework region sequence of the first antibody and at least the CDRs sequences of the subject antibody are known or can be readily determined. Determining these sequences, the synthesis of the D ⁇ A from oligonucleotides and the preparation of suitable vectors each involve the use of known techniques which can readily be carried out by a person skilled in the art in light of the teaching given herein.
  • the D ⁇ A sequence encoding the altered variable domain may be prepared by primer directed oligonucleotide site-directed mutagenesis.
  • This technique in essence involves hybridizing an oligonucleotide coding for a desired mutation with a single strand of D ⁇ A containing the mutation and using the single strand as a template for extension of the oligonucleotide to produce a strand containing the mutation.
  • This technique in various forms, is described by: Zoller et al. (1982) Nuc Acids Res 10:6487-6500; ⁇ orriset al. (1983) NMc Acids Res 11:5103-5112; Zoller et al. (1984) DNA 3:479-488; and Kramer et al. (1982)
  • oligonucleotides used for site-directed mutagenesis may be prepared by oligonucleotide synthesis or may be isolated from DNA coding for the variable domain of the subject antibody by use of suitable restriction enzymes. Such long oligonucleotides will generally be at least 30 residues long and may be up to or over 80 residues in length.
  • PCR techniques for generating fusion proteins can be used to generate the chimeric antibody.
  • PCR amplification of gene fragments, both CDR and FR regions can be carried out using anchor primers which give rise to complementary overhangs between two consecutive CDR and FR fragments which can subsequently be annealed to generate a chimeric V-gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • the antigen binding sites of the subject antibodies can also be used to generate a fusion protein which includes protein sequences from non-immunoglobulin molecules.
  • such chimeric antibodies can include: proteins domains which render the protein cytotoxic or cytostatic, such as the addition of Pseudomonas exotoxin or Diphtheria toxin domains (see, for example, Jung et al. (1994) Proteins 19:35-47; Seetharam et al. (1991) J Biol Chem 266:17376-17381; and Nichols et al. (1993) J Biol Chem 268:5302-5308); DNA- binding polypeptides for facilitating DNA transport (see, for example, U.S.
  • catalytic domains which provide an enzymatic activity associated with the immunoglobulin, such as a phosphatase or peroxidase activity
  • purification polypeptides to simplify purification of the antibody, such as a glutathione-S-transferase polypeptide for purification of the antibody with a glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al.
  • the present invention also makes available isolated forms of the subject antibodies which are isolated from, or otherwise substantially free of other cellular and extracellular proteins, especially antigenic proteins, or other extracellular factors, with which the antibodies normally bind.
  • substantially free of other cellular or extracellular proteins also referred to herein as "contaminating proteins”
  • substantially pure or purified preparations are defined as encompassing preparations of the subject antibodies having less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein.
  • Functional forms of the subject antibodies can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
  • purified it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins.
  • purified as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8%o by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • pure as used herein preferably has the same numerical limits as “purified” immediately above. "Isolated” and “purified” do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g.
  • compositions of the subject antibodies may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and may depend on such as factors as intended route of administration, age and body weight of patient.
  • biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation.
  • the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the antibody, e.g., its specificity and/or affinity, its use in the pharmaceutical preparation of the invention is contemplated.
  • Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations".
  • such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the antibody in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids.
  • pharmaceutically acceptable vehicles or diluents include, although not exclusively, solutions or freeze-dried powders of the antibody in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids.
  • excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH.
  • Similar solutions may also be used for the pharmaceutical compositions of the antibodies in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH).
  • Still another aspect of the present invention concerns assay kits that can be used for detecting an immunorecessive epitope(s) in a sample, for example.
  • the assay kits generally provide an antibody for the immunorecessive epitope, derivatized with a label group that can be ultimately detected, as for example, by spectrophotometric techniques (including FACS) or radiographic techniques.
  • the label can be any one of a number of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • the label group can be a functional group selected from the group consisting of horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, luciferase, urease, fluorescein and analogs thereof, rhodamine and analogs thereof, allophycocyanin, R-phycoerythrin, erythrosin, europiam, luminol, luciferin, coumarin analogs, 125 I, 131 I, 3 H, 35 S, 1 C and 2 P.
  • Assay kits provided according to the invention may include a selection of several different types of the subject antibodies.
  • the antibodies may be in solution or in lyophilized form.
  • the antibodies may come pre-attached to a solid support, or they may be applied to the surface of the solid support when the kit is used.
  • the labeling means may come pre-associated with the antibody, or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use.
  • Many types of detectable labels are available and could make up one or more components of a kit.
  • Various detectable labels are known in the art, and it is generally recognized that a suitable label group is one which emits a detectable signal.
  • label groups can be used, depending on the type of immunoassay conducted.
  • Useful labels include those which are fluorescent, radioactive, phosphorescent, chemiluminescent, bioluminescent, and free radical.
  • the label groups may include polypeptides (e.g., enzymes or proteins), polymers, polysaccharides, receptors, cofactors, and enzyme inhibitors.
  • Kits of the invention may also include additional reagent.
  • the additional reagent can include blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like.
  • the solid phase surface may be in the form of microtiter plates, microspheres, or the like, composed of polyvinyl chloride, polystyrene, or the like materials suitable for immobilizing proteins.
  • the subject method of the present invention can be applied advantageously to the production of antibodies useful in purification, diagnostic, and therapeutic applications.
  • the antibody libraries which can be generated by the subject method provide a greater population of high affinity antibodies to the immunorecessive epitope of interest, as well as establish a broader pool of display packages comprising antibodies specific for the immunorecessive epitope.
  • the more effective access of the antibody repertoire provided by the display libraries of the present invention allows more efficient enrichment to occur by, for example, affinity selection means.
  • immunorecessive epitopes can be defined in terms of the toleragen and immunogen used in the subtractive immunization step, and are therefore unique to the immunogen with respect to the toleragen.
  • the desired antibody is to distinguish between various cells of common or similar origin or phenotype
  • the cell to be specifically bound by an antibody of the present invention is used as an immunogen, while the related cells from which it is to be distinguished are employed as the toleragen.
  • Table 1 provides exemplary systems of immunogen/toleragen sets which can be employed in the subject method to isolate antibodies which specifically bindepitopes unique to the immunogen.
  • the choice of toleragen and immunogen can provide antibodies specific to, for example, tumor cell markers, fetal cell markers, and stem cell markers.
  • the subject method can be used to generate antibodies which can discriminate between a variant form of a protein and other related forms of the protein by employing an immunogen comprising a variant protein, such as a mutant form of a protein or a particular isoform of a family of proteins, and a toleragen comprising the wild-type protein or alternate isoforms of the variant protein.
  • an immunogen comprising a variant protein, such as a mutant form of a protein or a particular isoform of a family of proteins
  • a toleragen comprising the wild-type protein or alternate isoforms of the variant protein.
  • the difference in determinants i.e. the immmunorecessive epitopes
  • the variant protein and wild-type (or other isoforms) will typically consist of only a few differences in amino acid residues (i.e. less than 15%, but preferably on the order of only one to three residues difference).
  • immunogens and toleragens can be used in the present invention to derive antibodies which can specifically bind variant forms of oncoproteins or tumor suppressor proteins, as well as of hemoglobin, apolipoprotein E, LDL receptor, cardiac ⁇ -myosin, sodium or other ion channels, collagen, glucokinase, or transthyretin.
  • Table 1 Table 1
  • the subject method is employed to generate antibodies for a cell-type specific marker.
  • the present method can be employed to produce antibodies directed specifically to fetal cell- specific markers.
  • specific antibodies for markers of fetal nucleated red blood cells can be generated by the subject method employing maternal erythroid cells as a toleragen and fetal erythroid cells as an immunogen.
  • antibodies generated by the subject method can be used to separate fetal cells from maternal blood by, for instance, fluorescence- activated cell sorting (FACS).
  • FACS fluorescence- activated cell sorting
  • the isolated fetal cells such as fetal nucleated erythrocytes, represent a non-invasive source of fetal DNA for prenatal genetic screening and offer a powerful and safe alternative to more invasive procedures than, for example, amniocentesis or chronic villus sampling.
  • the present invention contemplates the generation of antibodies specific for a tumor cell-specific marker.
  • the subject method can be employed advantageously to generate antibodies which are able to differentiate between normal cells and their transformed counte ⁇ arts.
  • Such antibodies may be suitable for both diagnostic and therapeutic uses.
  • antibodies can be selected in the present assay which detect cell-specific markers found on neoplastic or hype ⁇ lastic cells.
  • Antibodies so obtained can be used to identify transformed cells and thereby used to diagnose cancers and tumors such as adenocarcinomas, papillomas, squamous and transitional cell carcinomas, anaplastic carcinomas, carcinoid tumors, mesotheliomas, hepatomas, melanomas, and germ cell tumors.
  • antibodies mays also be used to selectively destroy transformed cells, both in vivo and in vitro, such as through the discriminatory activation of complement at the cell surface of a transformed cell bound by the antibody, or by delivery of toxins, or by delivery of nucleic acid constructs for gene therapy.
  • antibodies specific for colon cancer markers can be generated in the present invention by suing normal colon cells as a toleragen and cells derived from a colon carcinoma as an immunogen.
  • the subject method can be engaged to produce antibodies that specifically inhibit metastasis of highly metastatic tumor cells.
  • Such antibodies designed to recognize unique epitopes on highly metastatic variants of tumor cells (i.e.
  • the immunotolerance-derived antibody repertoires used in the subject method can be generated using a differentiated nerve cell as a toleragen and an embryonic nerve cell, such as a neural crest cell or uncommitted progenitor cell, as an immunogen.
  • the immunogen can comprise a hematopoietic stem cell, and the toleragen can be a committed stem cell.
  • the subject method can be applied to the generation of antibodies which can discern between variant proteins.
  • Such antibodies can be used to distinguish various naturally occurring isoforms of a protein, as well as to detect mutations which may have arisen in a protein.
  • antibodies can be produced by the present invention which can be used in immunochemical assays for detecting cell transformations arising due to mutation of an oncogene or anti-oncogene.
  • the subject method can be used to generate antibodies which discriminate between wild-type ras and a mutant form of ras.
  • useful antibodies for detecting ras-induced transformation of a cell can be generated by the subject method using a Ser-17- Asn variant of ras as an immunogen, and wild-type ras as a toleragen..
  • diagnostically useful antibodies can be produced by the present invention which specifically bind and discriminate between wild-type and variant tumor suppressor proteins.
  • inactivating mutations of either the p53 or Rb tumor suppressors can lead to escape from cell senescence and lead to transformation.
  • the subject method can be used to generate antibodies specific for a variant p53, the ability to distinguish between the wild-type and mutant forms arising through recognition of a unique epitope created by mutation, such as Arg-273->Cys, Tyr-163- Asn, Val-157- Phe, or Cys-238->Phe.
  • Appropriate immunogen/toleragen sets would therefore include p53 mutants and wild-type p53.
  • the subject method can also be used to produce antibodies for detecting variant hemoglobin molecules, and which subsequently can be employed as diagnostic tools for detecting hemoglobinopathies, such as sickle cell anemia and ⁇ -thalassemia.
  • hemoglobinopathies such as sickle cell anemia and ⁇ -thalassemia.
  • a large number of such abnormalities, most resulting from single-point mutations, have been observed as abnormal hemoglobins of embryonic, fetal, neonatal, and adult disorders (see, for review, Huisman (1993) Baillieres Clin Haematol 6:1-30). Therefore, antibodies to unique epitopes of hemoglobin variants can be of great use in detecting and quantitating both normal and abnormal hemoglobin levels.
  • the immunogen is apolipoprotien E4 (ApoE4) and the toleragen comprises other ApoE isoforms
  • specific antibodies can be isolated by the subject method which can be used to measure ApoE4 levels in plasma or serum of a patient.
  • the presence of the ApoE4 variant has been linked to increased susceptibility to Alzheimer's disease (Strittmatter et al. (1993) PNAS 90:8098-8102) as well as significant impact on variation of cholesterol lipid and lipoprotein levels in individuals (Rail et al. (1992) J. Intern. Med. 231:653-659; and Weisgraber et al. (1990) J. Lipid Res. 31:1503-1511).
  • specific antibodies to other ApoE isoforms can be generated, including antibodies which can specifically bind ApoE2 or ApoE5.
  • LDL receptor variants which can be useful, for example, in predicting risk of diagnosing familial hypercholesterolemia; specific antibodies to cardiac ⁇ -myosin variants, which can be used to diagnose hypertrophic cardiomyopathy; specific antibodies to variant forms of sodium or ion channels, such as which arise in congenital hyperkalemic periodic paralysis; antibodies to collagen variants, such as Cys-579 collagen, which can be indicative of a predisposing factor in risk of familial osteoarthritis; specific antibodies to a variant of glucokinase, such as which arise in non-insulin-dependent diabetes mellitus; and antibodies specific for a mutant of transthyretin, such as which might arise in familial amyloidotic polyneuropathy.
  • the subject method has been applied advantageously to the development of antibodies for cell-surface markers of fetal cells and transformed cells.
  • practice of the subject method can yield a library of antibodies which are amenable to very rapid enrichment.
  • This invention represents the first instance that antibodies specific for unknown/unisolated cell-surface antigens have been generated using a combinatorial display library.
  • Figure 5A reveals the rapid enrichment of specific antibodies from the immunotolerized V-gene library.
  • Figure 5B demonstrates that phage libraries prepared by prior art techniques (non-tolerized #1 and #2) do not show significant enrichment from one round of panning to the next (compare tolerized to non-tolerized #1 and #2).
  • phage libraries prepared by prior art techniques do not show significant enrichment from one round of panning to the next (compare tolerized to non-tolerized #1 and #2).
  • antibodies that discriminate between fetal and maternal blood cells with only the same approximate performance as anti-CD71 antibodies were obtained.
  • the subject method provides a library containing a rich source of high affinity antibodies which permit detection of specific antibodies by, for example, panning on live cells, FACS assays or cell based ELISA.
  • a library containing a rich source of high affinity antibodies which permit detection of specific antibodies by, for example, panning on live cells, FACS assays or cell based ELISA.
  • individual antibody display packages were enriched 5000 to 3,600,000 fold in only a single round of selection.
  • DNA sequence analyses of particular isolates depict a remarkable history of affinity maturation of both heavy and light chains, suggesting an unexpectedly efficient access to the immunological repertoire.
  • the instant method enables selection of antibodies having both discriminating specificity and high binding affinity for an immunorecessive epitope.
  • comparison of antibodies isolated by the subject method with antibodies available through the use of prior art techniques reveals that the combinatorially-derived antibodies of the present invention tend to be orders of magnitude better with respect to each of specificity and affinity relative to antibodies available in the prior art.
  • the genes for three of the antibodies which demonstrate both desirable specificity and binding affinity have been sequenced. As described in Example 2, the F4-7 and H3-3 antibodies were originally isolated with a panning regimen including fetal liver cells.
  • H3-3 antibody recognized fetal blood cells of early gestational age (e.g., ⁇ 16 weeks), but also stained fetal cells of later gestational ages, albeit less well. This probably reflects the use of fetal liver, which consists predominantly of the earliest blood cell precursors, for both immunization and enrichment. However, it is demonstrated below that the population of antibodies enriched from the library could be biased to select antibodies specific for epitopes present on fetal blood cells of later gestational ages.
  • One of the isolates, FB3-2 was characterized and found to have an extraordinarily low background staining level on adult blood cells (e.g., less than 0.1%).
  • a guide to the nucleic acid and amino acid sequences for each of these clones is provided in Table 2, and the overall structure of the variable region for each of the heavy and light chains are provided in Figures 8 A and 8B.
  • the antibodies isolated by the present method are not apparently available by other prior art techniques and in fact displayed performance characteristics which greatly su ⁇ assed those obtained by previous methods.
  • the antibodies achieved by the subject method employing an identical immunotolerization step, but coupled instead with the use of hybridoma techniques, only a few antibodies which showed fetal cell selectivity were obtained.
  • the specificity for one of the best of these antibodies, "anti-Em” is shown in Table 3.
  • Fetal cell selective antibodies isolated by other groups using other hybridoma technologies were also compared.
  • anti-CD71 antibodies are believed to be among the best of the fetal cell specific antibodies.
  • antibodies generated by the instant method perform with superior qualities relative to each of the antibodies obtained by immunotolerance (anti-Em) and hybridoma (anti-CD71) techniques.
  • Anti-Em 5.0 ⁇ g fetal liver 50.0% 2.5 fold 5.0 ⁇ g maternal PBMC 20.0%
  • each of the anti-Em and anti-CD71 antibodies are considered to be of excellent specificity with respect to anti-fetal cell antibodies derived by methods in the prior art. Yet, as Table 3 illustrates, the level background binding to maternal peripheral blood mononuclear cells (PBMC) is many times higher for these antibodies relative to the background staining of maternal cells using the subject antibodies. Consequently, although the anti-Em, anti-CD71 antibodies and the like stain fetal cells very well, their background staining on maternal blood of greater than 5 percent provides substantial room for improvement of antibodies useful for retrieving a very small population of fetal blood cells from maternal blood samples. One estimate of fetal cell concentrations in maternal blood provides 1 fetal cell in
  • Another feature of the antibodies derived from the subject method which feature also apparently exceeds the antibodies of the prior art, pertains to the binding affinity of these antibodies for fetal cell-bound antigens.
  • HEL human erythro-leukemic
  • the association constant (K ⁇ exceeded 10 9 .
  • monomeric H3-3 and FB3-2 Fab' fragments displayed association constants of 6x1 O ⁇ M **1 and 8xl0 10 M"- respectively.
  • Dimeric forms of the recombinant antibodies had even greater binding affinities, with K a s of 5xl0 12 M ** - for H3-3 and lxlO ⁇ M **1 for FB3-2 respectively.
  • the subject method makes available antibodies specific for immunorecessive epitopes, in which antibodies are characterized by association constants for the immunorecessive epitopes which are greater than 10 6 M" 1 , preferably greater than 10 8 M _1 , more preferably greater than about lO- ⁇ M" 1 , and even more preferably greater than 10 10 M"-, lO- 'M *-1 , or 10 12 M"-, e.g., K a in the range of 10- ⁇ M- 1 to lO ⁇ M" 1 .
  • the subject method accommodates the isolation of antibodies which have a low level of background staining.
  • the relative specificity of these antibodies can be several fold, if not orders of magnitude, better than combinatorial and hybridoma generated antibodies, particularly with respect to antibodies for cell surface epitopes.
  • the subject method can provide antibodies which have no substantial background binding to other related cells, e.g., relative specificities greater than 10 fold binding to the target cells over background binding to the related cells.
  • antibodies can be generated which do not substantially cross-react with other epitopes, preferably having specificities greater than 20 fold over background, more preferably 50, 75 or 100 fold over background, and even more preferably more than 125 fold over background.
  • anti-fetal cell antibodies generated by the instant method were tested by fluorescence-activated cell sorting ("FACS efficiency assay") and were each demonstrated to have relative specificities greater than 125 fold over background.
  • FACS efficiency assay fluorescence-activated cell sorting
  • the anti-CD71 and anti-Fe antibodies were found to have relative specificities of 7.7 and 2.5 fold over background, respectively.
  • specificity of fetal cell specific antibodies produced by the subject method can also be characterized in terms of a background staining of maternal cells relative to antibodies of the prior art, such as anti-CD71 antibodies.
  • the subject antibodies preferably stain two times less non-fetal cells relative to an anti-CD71 antibody, more preferably at least five times less, and even more preferably at least twenty times less than an anti-CD71 antibody.
  • Such comparisons can be made using standard immunoassays, such as the FACS efficiency assay of Example 4.
  • Exemplary anti-CD71 (e.g., anti-Transferrin receptor) antibodies include the 5E9 antibody (ATCC HB21), the L5.1 antibody (ATCC HB84) and the L01.1 antibody (Beckton Dickinson Catalog No. 347510).
  • 5E9 antibody ATCC HB21
  • the L5.1 antibody ATCC HB84
  • L01.1 antibody Beckton Dickinson Catalog No. 347510
  • each antibody can be further engineered without departing from the pu ⁇ ose and intent of the present invention.
  • a chimeric FB3-2 antibody can be generated which includes the variable regions from the heavy chain (residues El -S 121, SEQ ID No. 51) and light chain (residues Dl-Kl 11, SEQ ID No. 53).
  • chimeric F4-7 antibodies can be provided which include the heavy chain (residues E1-S121, SEQ ID No. 55) and light chain (residues Dl-Kl 11, SEQ ID No. 57) variable regions from the F4-7 antibody described below.
  • chimeric H3- 3 antibodies are also contemplated, as for example antibodies including the variable regions from the heavy chain (residues El -SI 15, SEQ ID No. 59) and light chain (residues Dl-Kl 11, SEQ ID No. 61) of the H3-3 antibody.
  • chimeric antibodies can be generated including heavy and light chain variable regions, each represented by the general formula: FR(1)-CDR(1)-FR(2)- CDR(2)-FR(3)-CDR(3)-FR(4), wherein CDR(l), CDR(2) and CDR(3) represent complementarity determining regions from the subject antibody, and FR(1), FR(2), FR(3) and FR(4) are framework regions from a second antibody.
  • chimeric FB3-2 antibodies can be generated which include a heavy chain in which CDR(l) is SYWLE, CDR(2) is EILFGSGSAHYNEKFKG and CDR(3) is GDYGNYGDYFDY, and a light chain in which CDR(l) is RASQSVSTSRYSYMH, CDR(2) is FASNLES and CDR(3) is HSWEIPYT.
  • a chimeric F4-7 antibody can be made including a heavy chain in which CDR(l) is SSWLE, CDR(2) is EILFGSGSAHYNEKFKG and CDR(3) is GDYGNYGDYFDY, and a light chain in which CDR(l) is RVRQSVSTSSHSYMH, CDR(2) is YASNLES and CDR(3) is HSWEIPYT.
  • chimeric H3-3 antibodies can be provided, which antibodies include a heavy chain having a CDR(l) of DYYMY, a CDR(2) of TISDDGTYTYYADSVKG and a CDR(3) of DPLYGS, and a light chain in which CDR(l) is RSSQSLVHSNGNTYLH, CDR(2) is KVSNRFS and CDR(3) is SQSTHVLT.
  • the associated framework regions can be derived from an unrelated antibody, preferably a human antibody.
  • the present invention further pertains to methods of producing the subject recombinant antibodies.
  • a host cell transfected with nucleic acid vectors directing expression of nucleotide sequences encoding an antibody (or fragment) can be cultured under appropriate conditions to allow expression of the antibody to occur, and if required, assembly of a heavy/light chain dimer.
  • the antibody may be secreted and isolated from a mixture of cells and medium containing the recombinant antibody.
  • a cell culture includes host cells, media and other by-products. Suitable media for cell culture are well known in the art.
  • the recombinant antibody peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying antibodies, including protein-A:sepharose and ion-exchange chromatography, gel filtration chromatography, ultrafiltration and electrophoresis.
  • the recombinant antibody is a fusion protein containing a domain which facilitates its purification, such as a GST fusion protein or a poly(His) fusion protein.
  • This invention also pertains to a host cell transfected to express a recombinant form of the subject antibody.
  • the host cell may be any prokaryotic or eukaryotic cell, and the choice can be based at least in part on the desirability of such post-translation modifications as glycosylation.
  • a nucleotide sequence derived from the cloning of an anti-fetal cell or anti-oncogenic cell antibody by the subject method, encoding all or a selected portion of the variable region can be used to produce a recombinant form of an antibody via microbial or eukaryotic cellular processes.
  • the cell line which is transformed to produce the recombinant antibody is an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line.
  • the cell line may also include a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Ban * virus.
  • the immortalised cell line is a myeloma cell line or a derivative thereof.
  • the recombinant antibody gene can be produced by ligating nucleic acid encoding the subject antibody protein, or the heavy and light chains thereof, into vectors suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
  • Expression vectors for production of recombinant forms of the subject antibody include plasmids and other vectors.
  • suitable vectors for the expression of an antibody include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, inco ⁇ orated by reference herein).
  • These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
  • an antibody is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequences of the variable regions for each of the heavy and light chain genes of the H3-3 or FB3-2 antibodies.
  • the preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAJ/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papillomavirus
  • pHEBo Epstein-Barr virus
  • the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
  • suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
  • the subject method has been applied advantageously to the development of antibodies for cell-surface markers of fetal and transformed cells.
  • the present invention can yield a remarkable library of antibodies which are amenable to very rapid enrichment.
  • individual antibody display packages were enriched 5000 to 3,600,000 fold in only a single round of selection.
  • DNA sequence analyses of particular isolates gave a remarkable history of affinity maturation of both heavy and light chains, suggesting an unexpectedly efficient access to the immunological repertoire.
  • DNA modifying enzymes were obtained from New England Biolabs (Beverly, MA) and used under conditions recommended by the suppliers.
  • Taq polymerase was obtained from Perkin Elmer (Norwalk, CT).
  • a set of DNA fragments (1 Kb ladder) obtained from Life Technologies (Gaithersburg, MD) was used as a standard for molecular weight of DNA fragments by agarose gel electrophoresis.
  • DNA primers were custom synthesized by Genosys, Inc. (The Woodlands, TX) or Cruachem, Inc. (Sterling, VA).
  • Deoxyadenosine 5'[ ⁇ -( 35 S)thio]triphosphate was purchased from New England Nuclear (Boston, MA).
  • Polyclonal biotinylated anti-M13 antibody was obtained from 5 prime-3 prime (Boulder, CO). Streptavidin-Alkaline phosphatase and Polyclonal goat anti-mouse kappa-alkaline phosphatase were from Fisher Biotech (Pittsburgh, PA).
  • Bacterial strains and culture E. coli strains XL-1, SolR, and LE392 were obtained from Stratagene (LaJolla, CA).
  • Lambda phage resistant XL-1 was isolated by standard methods and is described in this work.
  • E. coli was grown to stationary phase at 30 or 37°C with shaking in Erlenmeyer flasks filled to one-tenth their nominal capacity with LB, SOB, 2X YT, NZY medium (Sambrook, 1989) or TB medium:0.1 M KH2PO4 buffer, buffer, pH 7.5 containing 12 g bacto-tryptone, 24 g yeast extract, and 5.04 g glycerol per liter (phosphate buffer was autoclaved separately).
  • agar Difco, Detroit MI
  • Glucose supplement was to 0.5% Carbenicillin, chloramphenicol, and kanamycin were added when necessary to 50, 30, and 50 ug/ml, respectively.
  • E. coli cloning vector lambda SurfZapTM and helper phages ExAssistTM and VCS
  • Ml 3 were obtained from Stratagene.
  • PBMC peripheral blood mononuclear cells
  • Fetal blood mononuclear cells were prepared by standard Ficoll-Hypaque gradient techniques.
  • Fetal blood mononuclear cells were prepared from fetal liver obtained from abortuses at 12-20 weeks gestation, at which age the liver is the principal hematopoietic organ. Cells were freed from surrounding connective tissue by passage through sterile microscreens in the presence of sterile Ca-Mg-free PBS.
  • the resulting cell suspension was diluted up to 20 ml in PBS and the blood mononuclear cell fraction obtained by standard Ficoll-Hypaque gradient centrifugation. After recovery from the Ficoll interface, both adult and fetal cells were washed twice in sterile Ca-Mg-free PBS the resuspended in the PBS at 2xl0 7 cells per ml.
  • mice at 6 weeks of age were injected intra-peritoneally ("I.P.") with lxlO 7 adult PBMC in 500 ul PBS.
  • the adult PBMC injection was followed 10 minutes later by I.P. injection of cyclophosphamide at 100 mg/kg.
  • the cyclophosphamide was repeated at 24 and 48 hours. After an additional 14 days, the tolerization was repeated.
  • mice were immunized with fetal mononuclear blood cells by I.P. injection of lxl0 7 fetal cells in 500 ul PBS. After an additional 2 weeks, the mice were once again tolerized with adult PBMC as described for the first round of tolerization. Finally, three weeks later, the mice were again immunized with fetal blood mononuclear cells by I.P. injection of lxl 0 7 fetal cells in 500 ul PBS. The fetal cell immunization was repeated in 24 and 48 hours. After an additional 24 hours, the mice were sacrificed. The spleens were harvested and immediately frozen in liquid nitrogen.
  • RNA was isolated from spleens or from Hybridoma cell lines using standard methods (Chomczynski, U.S. Patent No. 4,843,155). RNA preparations were stored in RNAase free water (Sambrook, J. et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)) at -70°C until use. A Superscript pre amplification kit from Life Technologies was used to prepare first strand cDNA as recommended by the supplier. Isolation of DNA
  • Isolation of plasmid DNA from E. coli for DNA sequence or restriction analyses was by alkaline lysis (Birnboim and Doly, 1979). Bulk preparation of plasmid DNA was carrier out using nucleobond column chromatography as described by the manufacturer Macherey- Nagel (Duren, Germany). All cultures for isolation of plasmid DNA from E. coli clones containing antibody clones were grown overnight with shaking at 37°C in 2xYT medium containing 0.5% glucose and 50 ug/ml carbenicillin.
  • a set of degenerate primers shown in Figures 1A and IB, was designed to minimize bias toward limited sets of PCR products from the repertoire of antibody coding regions encoded in spleenic mRNA, as well as to amplify >90% of the mouse kappa chain and heavy chain Fab encoding sequences. Amplifications of kappa chain or heavy chain coding sequences were accomplished using 5 separate primer pairs for each.
  • the primers also contained restriction enzyme site to allow the ligation of the light and heavy chain PCR products into a bacterial Fab expression cassette suitable for insertion in the Surf-Zap vector (Stratagene). PCR reactions were carried out in an Automated BioSystems temp-cycler (Essex, MA) using the following protocol.
  • RNA was converted to cDNA using a Superscript first strand synthesis kit (BRL).
  • BBL Superscript first strand synthesis kit
  • each PCR product from five separate reactions were combined to generate a kappa chain and separate heavy chain product pool.
  • the pools were then purified by first removing protein and debris with a PVDF spin filter (Millipore) followed by removal of low molecular weight components using a 30,000 MW cut off spin filter as recommended by the supplier (Millipore).
  • Approximately 5 ug of each product pool was digested in 300 ul of Sfil buffer with 50 units of Sfil for 2 h at 50°C. Enzyme and small end fragments generated by Sfil digestion were removed with the spin column procedure described above.
  • Sfil digested light chain products were ligated to Sfil digested heavy chain products (approximately 2 ug each) in a 50 ul volume overnight at 4°C.
  • the ligation mixture was then purified with spin columns as above and digested with 50 units each of Notl and Spel restriction enzymes in 100 ul.
  • the digestion products were resolved by agarose gel electrophoreses and the 1.4 kb kappa chain heavy chain encoding dimer was purified using Gene Clean II (Promega) as recommended by the supplier.
  • Gene Clean II Promega
  • PCR products were purified as described above. Approximately 2 ug of each product was treated separately at 37°C for 1 h in a 50 ul volume containing 5 units T4 polymerase, 5 mM dTTP. Products were purified as for PCR products, and approximately 500 ng of each product was ligated at room temperature for 3 h in a 25 ul volume of ligation buffer (Promega) containing 2 units of DNA ligase.
  • Fab encoding dimer from either method were amplified under standard conditions using a 5' kappa chain primer and 3' heavy chain primer shown in Figure 2 except that annealing was at 55°C for 1 min., and the extension time was extended to 4 min. at 72°C. Generally 12-25 cycles under these conditions yielded approximately 1-2 ug of 1.4 kb kappa- heavy chain dimer.
  • This product was purified using spin columns as described above and then digested in a200 ul volume containing 75 units each of Not I and Spe I restriction enzymes. Digestion products were purified as described above except that a 100,000 MW spin column (Amicon) was used to more efficiently remove primers from the digestion products. Purified 1.4 kb dimers were stored at 4°C until use. Construction of variegated Fab clone banks
  • Ligation of Not I-Spe I digested 1.4 kb Fab encoding fragments was as follows: 0.2 ug of digested products was ligated to 2 ug of lambda surf-zap arms in 10 ul of Promega ligation buffer containing 3 units of T4 ligase overnight at 4°C. Aliquots of the ligation mixture were then packaged into lambda heads using a Giga-pack Gold packaging kit as recommended by the supplier (Stratagene). Packaging reactions were titered on E. coli L ⁇ 392 and pooled to yield a primary library. This primary library was then amplified in E. coli LE392 using conventional methods. Generally 5xl0 9 E.
  • coli XL1 cells were infected in 10 ml of 10 mM MgSO 4 with 107 invitro packaged SURF-ZAP primary clones for 10 min. at 37°C.
  • the infected cells were added to 100 ml of NZY top agarose at 50°C.
  • the mixture was immediately plated onto two 20x20 cm plates containing NZY agar, allowed to solidify, and then incubated for 8-16 h.
  • the amplified library was harvested by rocking with an overlay of 25 ml of SM buffer of 2 h.
  • Phagemid clone bank was rescued from the primary lambda SURF-ZAP library by super infection with Ml 3 exassist helper phage essentially as recommended by Stratagen.
  • 10- - E. coli XL1 cells were infected with 10 10 lambda clones from our amplified surf-zap library and 10 12 Exassist M13 phage. After growth for 3.5 h in LB or TB medium, the cells were removed by centrifugation. The exassist rescued library was treated for 70°C for 20 min. and then stored at 4°C.
  • Phage antibodies were generated by infection of E. coli SOLR 1 : 1 with rescued phagemid to generate a population of carbenicillin resistant antibody clone containing cells representing a 10-100 fold excess over the primary library size. Transduced cells were grown to early log phase in TB medium containing carbenicillin, and then infected with a ten fold excess of VCS M13 helper phage to cells. After 1 h at 37°C, kanamycin was added and the culture was incubated at 30°C with shaking until early stationary phase. Cells were removed by centrifugation, and phage antibodies were recovered from the supernatant by harvested by centrifugation, dissolved in 1 ml of TE buffer and then PEG precipitated a second time. Phage antibodies were dissolved in 1 ml TE or PBS buffer and stored at 4°C.
  • Cell specific phage antibodies were isolated by enrichment on whole cells. Cells were prepared for enrichment by washing twice in blocking buffer (0.1% hydrolyzed casein, 3% BSA, in Hanks Buffered Salt Solution). For the first round of enrichment 10 1 1 phage antibodies in 200 ul of blocking buffer were added to 10 6 cells and incubated on ice for 1 h. Non-specific phage antibodies were then removed by washing 8 times with cold blocking buffer. Cells were harvested after each wash by centrifugation at 3500 ⁇ m in an Eppendorf micro centrifuge. Cell surface bound phage antibodies were then eluted in 500 ul of 0.2 M Glycine pH2.2 containing 3 M urea and 0.5% BSA.
  • phage antibodies were titered on XL1 cells, and then amplified by the following protocol. Eluted phage antibodies in 200 ul SM buffer were added to 5x10 9 XL1 plating cells in 1 ml of 10 mM MgSO 4 and incubated for 10 min. at room temperature. Infected cells were then used to inoculate 100 ml of TB broth in a 2 L flask and incubated at 30°C with shaking.
  • E. coli XL1 was infected with dilutions of phage antibody pools and plated on LB medium containing 0.5% glucose and 50 ug/ml carbenicllin.
  • 20 mm culture tubes containing 2 ml of 2xYT medium with 50 ug/ml carbenicllin were inoculated with isolated colonies and grown overnight at 30°C with shaking. The following morning 1 ml of culture was gently shaken at 37°C for 1 h and then infected with 10 1 1 M13 VCS phage.
  • Flow cytometric assay of phage antibody binding to whole cells A flow cytometry protocol was devised for the testing of phage antibody binding to surface markers on whole cells, lxl 0 6 adult or fetal mononuclear cells were dispensed into a 2 ml microtube and washed with blocking buffer as in the phage enrichment procedure. For initial assay, 2x10 10 phage were added to the washed cells and the volume brought to 100 ul with casein/BSA/HBSS. The phage were incubated with the cells for one hour at 4°C. The phage-cells were washed three times with 1 ml blocking buffer.
  • Biotinylated sheep anti- phage polyclonal antibody (5 Prime -2 Prime) was added to the phage-cells at 5-7.5 ul per sample, optimized for each lot of polyclonal antibody. Volume was once again brought up to 100 ul with blocking buffer. The anti-phage was incubated 90 minutes at 4°C. Excess anti- phage was removed by washing three times with blocking buffer. Streptavidin-FITC (Jackson Immunoresearch) was diluted 1:50 in Ca-Mg-free PBS and 250 ul added to each sample of phage-cells. After a 30 minute 4°C incubation, the phage-cells were washed twice with blocking buffer and fixed by adding 400 ul 0.5% formaldehyde.
  • Relative binding activity of each clone was determined by evaluation of two parameters: (1) scatter pattern vs. intensity of fluorescence, for determination of relative cell surface epitope number and uniformity of expression for each phage clone, with higher, more uniform, numbers being most desirable; (2) titration of phage and retention of fluorescent binding intensity - for determination of relative phage antibody affinities.
  • soluble antibody Fab
  • Fab soluble antibody
  • the anti-phage/streptavidin-FITC was replaced by a goat anti-IgG-FITC F(ab') polyclonal antibody (TAGO) that recognized the K chain of the Fab fragments.
  • TAGO goat anti-IgG-FITC F(ab') polyclonal antibody
  • 30 ul of the goat anti-IgG-FITC diluted 1 :10 in 2.5% normal human serum was used per sample. The dilution in human serum ensured that any cross-reactivity of the polyclonal with human blood cell antigens would be minimized.
  • Example 1 Enrichment of phage antibodies on cancer cells.
  • a combinatorial phage display library of IgGl and kappa chain derived Fabs containing 6xl0 7 primary clones was constructed from a mouse which had been tolerized with adult human blood and immunized with fetal liver cells.
  • cultures containing antibody clones or pools of clones were always in rich media (TB or 2xYT containing 1% glucose).
  • cultures used to produce phage antibodies were harvested as close to peak growth as possible since binding activity was found to fall beyond the start of stationary phase of growth.
  • the human erythro-leukemic cell line (HEL) carries onco/fetal cell surface markers also found on fetal liver cells. This characteristic and the ability to culture this cell made it a reliable source of cells to develop methods for enrichment of cell line specific antibodies from the above phage library. The binding of phage antibody pools enriched on this cell line
  • Example 2 Enrichment of phage antibodies on fetal cells. To maximize the chances of isolating fetal cell specific clones, the phage antibody library described in Example 1 was pre-absorbed on adult nucleated blood prior to each enrichment cycle on fetal liver cells in addition to enrichments without pre-adso ⁇ tion. The results of sequential rounds of pre-adso ⁇ tion and enrichment on fetal liver cells are shown in Figure 5A. The increase in the percentage of phage antibodies binding to fetal liver cells indicated enrichment for fetal cell binding phage antibodies.
  • Table 4 shows the distribution of different phage antibody types at different stages of enrichment on HEL or Fetal cells with or without preadso ⁇ tion on adult cells. It is likely that the three classes of phage antibodies recognize three different epitopes based upon the difference in their staining profiles on fetal liver and adult cells.
  • HEL cell surface binding isolates seen on a consistent cell source
  • pan-fetal specific antibodies emphasizes the power of the present approach, which yielded 13 different versions of three classes of pan-fetal specific antibodies.
  • Affinity of purified antibodies was determined by Scatchard analysis. Varying amounts of antibody in significant excess were incubated for 16 hours at 4°C with a constant number of HEL cells. After extensive washes, bound antibody was eluted from cells at pH 2, and quatitated in an ELISA. For Scatchard analysis, free antibody was assumed to be equivalent to the total added. The K a for each antibody was obtained from the negative slope of the line from the plot of bound versus bound/free antibody. All points were done in triplicate; the correlation coefficient for all reported slopes was greater than 90%.
  • hybridoma-derived antibodies such as anti-CD71 and anti-EM
  • reactivity of these antibodies with fetal and maternal cells was tested by analytical flow cytometry (FACS efficiency assay). Briefly, lxl 0 6 cells per sample were stained with indicated amounts of FITC-conjugated pure antibody. 10,000 cells were analyzed for each sample. The results, provided in Table 3 above, are reported as "% positive", indicating the percentage of cells that were found to stain above background fluorescence as established by an isotype-matched negative control antibody.
  • H3-3 antibody for fetal as opposed to adult hematopoietic cells was further demonstrated by FACS and subsequent fluorescent in situ hybridization (FISH) analysis of sorted cells.
  • FISH fluorescent in situ hybridization
  • MOLECULE TYPE Other nucleic acid
  • MOLECULE TYPE peptide
  • FRAGMENT TYPE internal
  • SEQUENCE DESCRIPTION SEQ ID NO:37;
  • MOLECULE TYPE protein
  • Gly Glu lie Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu Lys Phe 50 55 60
  • ATC CAT CCT GTG GAG GAG GAG GAT ACT GCA ACA TAT TAC TGT CAG CAC 348 lie His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gin His 80 85 90 AGT TGG GAG ATT CCG TAC ACG TTC GGA GGG GGG ACC AAG CTG GAA ATA 396 Ser Trp Glu lie Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie 95 100 105 110 AAA 399
  • MOLECULE TYPE protein
  • Lys Leu Leu lie Lys Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60
  • Trp lie Gly Glu lie Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu 50 55 60
  • Gly Glu lie Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu Lys Phe 50 55 60
  • ATC CAT CCT GTG GAG GAG GAG GAT ACT GCA ACA TAT TAC TGT CAG CAC 348 lie His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gin His 80 85 90
  • Lys Leu Leu lie Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn lie His 65 70 75 80
  • Glu lie Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg 100 105 110
  • GCC CCT GGA TCT GCT GCC CAA ACT AAC TCC ATG GTG ACC CTG GGA TGC 492 Ala Pro Gly Ser Ala Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys 130 135 140 CTG GTC AAG GGC TAT TTC CCT GAG CCA GTG ACA GTG ACC TGG AAC TCT 540 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser 145 150 155
  • MOLECULE TYPE protein
  • Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80
  • Asp Lys Lys lie Val Pro Arg Asp Cys 210 215
  • CAG TCT CCA AAG CTC CTG ATC TAC AAG GTT TCC AAC CGG TTT TCT GGG 252 o

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Abstract

L'invention concerne un procédé pour générer un anticorps spécifique d'un épitope immunorécessif et un acide nucléique codant l'anticorps. Ce procédé consiste généralement à générer une bibliothèque de visualisation diversifiée de zones variables d'anticorps et à sélectionner dans cette bibliothèque les zones qui présentent une spécificité de liaison requise pour l'épitope immunorécessif. Les zones variables d'anticorps utilisées pour générer la bibliothèque de visualisation sont clonées à partir d'un répertoire d'anticorps dérivés de l'immunotolérance.
EP95905871A 1993-12-08 1994-12-08 Procede de generation d'anticorps specifiques Withdrawn EP0733070A1 (fr)

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US16402293A 1993-12-08 1993-12-08
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US35040094A 1994-12-06 1994-12-06
US350400 1994-12-06
PCT/US1994/014106 WO1995015982A2 (fr) 1993-12-08 1994-12-08 Procede de generation d'anticorps specifiques

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101563366B (zh) * 2006-10-19 2012-10-03 健泰科生物技术公司 抗notch3激动性抗体及其在制备治疗notch3相关疾病的药物中的用途

Families Citing this family (484)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7429646B1 (en) 1995-06-05 2008-09-30 Human Genome Sciences, Inc. Antibodies to human tumor necrosis factor receptor-like 2
CN1036794C (zh) * 1995-05-30 1997-12-24 苏州医学院 抗人活化血小板单克隆抗体，其制备方法及应用
AU6353596A (en) * 1995-06-30 1997-02-05 Kobenhavns Universitet Recombinant antibodies from a phage display library, directed against a peptide-mhc complex
US7888466B2 (en) 1996-01-11 2011-02-15 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US5932449A (en) * 1996-02-01 1999-08-03 The United States Of America As Represented By The Secretary Of The Army Detection of botulinum toxin
JP2001512560A (ja) * 1996-10-08 2001-08-21 ユー―ビスイス ベスローテン フェンノートシャップ 標的に対し特異的な親和性を有するペプチドおよびタンパク質の選択のための方法および手段
JP2002506625A (ja) 1998-03-19 2002-03-05 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド サイトカインレセプター共通γ鎖様
JP2002537769A (ja) 1999-02-26 2002-11-12 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド ヒトエンドカインαおよび使用方法
EP1157041B1 (fr) 1999-03-01 2005-06-01 Genentech, Inc. Anticorps destines au traitement et au diagnostic du cancer
US7291714B1 (en) 1999-06-30 2007-11-06 Millennium Pharmaceuticals, Inc. Glycoprotein VI and uses thereof
US20040001826A1 (en) 1999-06-30 2004-01-01 Millennium Pharmaceuticals, Inc. Glycoprotein VI and uses thereof
CA2405557C (fr) 2000-04-12 2013-09-24 Human Genome Sciences, Inc. Proteines hybrides d'albumine
US20030031675A1 (en) 2000-06-06 2003-02-13 Mikesell Glen E. B7-related nucleic acids and polypeptides useful for immunomodulation
WO2001096528A2 (fr) 2000-06-15 2001-12-20 Human Genome Sciences, Inc. Facteur de necrose tumorale humain delta et epsilon
WO2002002641A1 (fr) 2000-06-16 2002-01-10 Human Genome Sciences, Inc. Anticorps se liant de maniere immunospecifique a un stimulateur de lymphocyte b
AU2001271502A1 (en) * 2000-06-26 2002-01-08 Gpc Biotech Ag Methods and compositions for isolating biologically active antibodies
US7381799B2 (en) * 2000-08-22 2008-06-03 Ergon Pharmaceuticals Llc Compositions and methods for inhibiting angiogenesis
GB0022978D0 (en) 2000-09-19 2000-11-01 Oxford Glycosciences Uk Ltd Detection of peptides
EP2412384A1 (fr) 2000-11-28 2012-02-01 MedImmune, LLC Procédés d'administration/dosage d'anticorps anti-rsv pour la prophylaxie et le traitement
DE60143544D1 (de) 2000-12-12 2011-01-05 Medimmune Llc Moleküle mit längeren halbwertszeiten, zusammensetzungen und deren verwendung
CN1564826A (zh) 2001-02-09 2005-01-12 人类基因组科学公司 人类g蛋白趋化因子受体(ccr5)hdgnr10
US8981061B2 (en) 2001-03-20 2015-03-17 Novo Nordisk A/S Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof
DK1385864T3 (da) 2001-04-13 2010-08-16 Human Genome Sciences Inc Anti-VEGF-2-antistoffer
WO2002097033A2 (fr) 2001-05-25 2002-12-05 Human Genome Sciences, Inc. Anticorps se liant de maniere immunospecifique a des recepteurs de trail
EP2261250B1 (fr) 2001-12-21 2015-07-01 Human Genome Sciences, Inc. Protéines de fusion d'albumine et GCSF
GB0207533D0 (en) 2002-04-02 2002-05-08 Oxford Glycosciences Uk Ltd Protein
AU2003226065B2 (en) 2002-04-12 2009-02-26 Ludwig Institute For Cancer Research, Ltd Recombinant anti-interleukin-9 antibodies
EP2070949B1 (fr) 2002-06-10 2013-01-16 Vaccinex, Inc. Anticorps anti-C35 et leur application pour le traitement du cancer
US7425618B2 (en) 2002-06-14 2008-09-16 Medimmune, Inc. Stabilized anti-respiratory syncytial virus (RSV) antibody formulations
US7425620B2 (en) 2002-08-14 2008-09-16 Scott Koenig FcγRIIB-specific antibodies and methods of use thereof
DE60336406D1 (de) 2002-10-16 2011-04-28 Purdue Pharma Lp Antikörper, die an zellassoziiertes ca 125/0722p binden, und verfahren zu deren anwendung
DE10303974A1 (de) 2003-01-31 2004-08-05 Abbott Gmbh & Co. Kg Amyloid-β(1-42)-Oligomere, Verfahren zu deren Herstellung und deren Verwendung
AU2004213053C1 (en) 2003-02-20 2009-07-16 Seagen Inc. Anti-CD70 antibody-drug conjugates and their use for the treatment of cancer and immune disorders
WO2004085648A2 (fr) 2003-03-19 2004-10-07 Biogen Idec Ma Inc. Proteine de liaison de recepteur de nogo
WO2004091510A2 (fr) 2003-04-11 2004-10-28 Medimmune, Inc. Anticorps il-9 recombinants et leurs utilisations
WO2005010040A1 (fr) 2003-07-15 2005-02-03 Barros Research Institute Antigene eimeria tenella utilise dans l'immunotherapie de la coccidiose
JP2007511738A (ja) 2003-08-08 2007-05-10 ジーンニュース インコーポレーテッド 変形性関節症のバイオマーカー及びその使用
JP4934426B2 (ja) 2003-08-18 2012-05-16 メディミューン，エルエルシー 抗体のヒト化
IL158287A0 (en) 2003-10-07 2004-05-12 Yeda Res & Dev Antibodies to nik, their preparation and use
SI1983000T1 (sl) 2003-11-21 2015-12-31 Ucb Biopharma Sprl Postopek za zdravljenje multiple skleroze z zaviranjem aktivnosti il-17
GB0329825D0 (en) 2003-12-23 2004-01-28 Celltech R&D Ltd Biological products
EP1729795B1 (fr) 2004-02-09 2016-02-03 Human Genome Sciences, Inc. Proteines hybrides d'albumine
WO2005097184A2 (fr) 2004-03-26 2005-10-20 Human Genome Sciences, Inc. Anticorps contre le recepteur nogo
CA2578670C (fr) 2004-04-15 2017-03-07 University Of Florida Research Foundation, Inc. Neuro-proteines utilisees comme biomarqueurs pour deceler une lesion du systeme nerveux et d'autres troubles neurologiques
CA2562764A1 (fr) 2004-04-23 2005-11-03 Richard Kroczek Methode de traitement d'etats induits par des lymphocytes t consistant en une depletion de cellules a icos positif in vivo
PT1776136E (pt) 2004-06-24 2012-12-05 Biogen Idec Inc Tratamento de estados que envolvem desmielinização
CA2576193A1 (fr) 2004-08-03 2006-02-16 Biogen Idec Ma Inc. Influence du taj sur les fonctions neuronales
CA2580921C (fr) 2004-09-21 2016-04-12 Medimmune, Inc. Anticorps dirige contre le virus respiratoire syncytial et procedes de production de vaccins associes
WO2006047417A2 (fr) 2004-10-21 2006-05-04 University Of Florida Research Foundation, Inc. Detection de biomarqueurs du recepteur cannabinoide et utilisations
EP2422811A2 (fr) 2004-10-27 2012-02-29 MedImmune, LLC Modulation d'une spécificité d'anticorps par adaptation sur mesure de son affinité a une antigène apparente
GB0426146D0 (en) 2004-11-29 2004-12-29 Bioxell Spa Therapeutic peptides and method
JP5265923B2 (ja) 2005-02-07 2013-08-14 ジーンニュース インコーポレーテッド 軽度の変形性関節症のバイオマーカーおよびその使用
WO2006089133A2 (fr) 2005-02-15 2006-08-24 Duke University Anticorps anti-cd19 et leur utilisation en oncologie
US8211430B2 (en) 2005-03-04 2012-07-03 Curedm Group Holdings, Llc Methods and pharmaceutical compositions for treating type 1 diabetes mellitus and other conditions
EP1869192B1 (fr) 2005-03-18 2016-01-20 MedImmune, LLC Rearrangement de l'infrastructure d'anticorps
GB0506912D0 (en) 2005-04-05 2005-05-11 Celltech R&D Ltd Biological products
US9889197B2 (en) 2005-04-15 2018-02-13 Macrogenics, Inc. Covalently-associated diabody complexes that possess charged coil domains and that are capable of enhanced binding to serum albumin
AU2006236439B2 (en) 2005-04-15 2012-05-03 Macrogenics, Inc. Covalent diabodies and uses thereof
AU2006236225C1 (en) 2005-04-19 2013-05-02 Seagen Inc. Humanized anti-CD70 binding agents and uses thereof
CA2607281C (fr) 2005-05-05 2023-10-03 Duke University Traitements des maladies auto-immunes par anticorps anti-cd19
US7393919B2 (en) 2005-05-25 2008-07-01 Cure Dm, Inc. Peptides, derivatives and analogs thereof, and methods of using same
US20070110757A1 (en) 2005-06-23 2007-05-17 Ziping Wei Antibody formulations having optimized aggregation and fragmentation profiles
CN103146708A (zh) 2005-06-30 2013-06-12 Abbvie公司 Il-12/p40结合蛋白
CN101495509B (zh) 2005-07-08 2015-04-22 比奥根艾迪克Ma公司 Sp35抗体及其用途
NZ565631A (en) 2005-08-03 2011-01-28 Adelaide Res & Innovation Pty Polysaccharide synthases
EP2500356A3 (fr) 2005-08-19 2012-10-24 Abbott Laboratories Immunoglobuline à double domaine variable et ses utilisations
EP2495257A3 (fr) 2005-08-19 2012-10-17 Abbott Laboratories Immunoglobuline à double domaine variable et ses utilisations
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
EP1926757B1 (fr) 2005-09-14 2012-02-22 UCB Pharma, S.A. Conjugué anticorps-polymère à structure en peigne
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
ATE518007T1 (de) 2005-10-21 2011-08-15 Genenews Inc Verfahren und vorrichtung zur korrelierung der ebenen von biomarker-produkten mit erkrankungen
JP2009517340A (ja) 2005-11-04 2009-04-30 バイオジェン・アイデック・エムエイ・インコーポレイテッド ドーパミン作動性ニューロンの神経突起成長および生存を促進するための方法
EA014900B1 (ru) 2005-11-07 2011-02-28 Зе Скрипс Ресеч Инститьют Композиции и способы контроля специфичности передачи сигналов, опосредуемой тканевым фактором
ES2409835T3 (es) 2005-11-28 2013-06-28 Zymogenetics, Inc. Antagonistas de IL-21
DK1976877T4 (en) 2005-11-30 2017-01-16 Abbvie Inc Monoclonal antibodies to amyloid beta protein and uses thereof
PL1954718T3 (pl) 2005-11-30 2015-04-30 Abbvie Inc Przeciwciała skierowane przeciwko A globulomerowi, ich reszty wiążące antygeny, odpowiednie hybrydomy, kwasy nukleinowe, wektory, komórki gospodarze, sposoby wytwarzania tych przeciwciał, kompozycje zawierające te przeciwciała, zastosowania tych przeciwciał i sposoby stosowania tych przeciwciał
WO2007064882A2 (fr) 2005-12-02 2007-06-07 Biogen Idec Ma Inc. Traitement d'etats lies a la demyelination
BRPI0619595B8 (pt) 2005-12-09 2022-12-13 Ucb Pharma Sa anticorpo neutralizante, sequência de dna isolada, vetor de clonagem ou expressão, célula hospedeira, processo para a produção de um anticorpo, composição farmacêutica, e, uso de um anticorpo neutralizante humanizado
WO2007089601A2 (fr) 2006-01-27 2007-08-09 Biogen Idec Ma Inc. Antagonistes des récepteurs nogo
EP2650306A1 (fr) 2006-03-06 2013-10-16 Aeres Biomedical Limited Anticorps humanisés anti CD22 et leur utilisation dans le traitement de l'oncologie, la transplantation et les maladies auto-immunes
GB0611116D0 (en) 2006-06-06 2006-07-19 Oxford Genome Sciences Uk Ltd Proteins
CA2655080A1 (fr) 2006-06-14 2007-12-21 Macrogenics, Inc. Procedes de traitement de troubles auto-immuns utilisant des anticorps monoclonaux a toxicite reduite
ES2599319T3 (es) 2006-06-26 2017-02-01 Macrogenics, Inc. Anticuerpos específicos de Fc RIIB y métodos de uso de éstos
US7572618B2 (en) 2006-06-30 2009-08-11 Bristol-Myers Squibb Company Polynucleotides encoding novel PCSK9 variants
MX2009000709A (es) 2006-07-18 2009-02-04 Sanofi Aventis Anticuerpo antagonista contra epha2 para el tratamiento de cancer.
US8058402B2 (en) 2006-08-28 2011-11-15 Kyowa Hakko Kirin Antagonistic human LIGHT-specific human monoclonal antibodies
US7915388B2 (en) 2006-09-08 2011-03-29 Abbott Laboratories Interleukin-13 binding proteins
WO2008048545A2 (fr) 2006-10-16 2008-04-24 Medimmune, Llc. Molécules ayant des demi-vies réduites, compositions et leurs utilisations
GB0620729D0 (en) 2006-10-18 2006-11-29 Ucb Sa Biological products
EP1914242A1 (fr) 2006-10-19 2008-04-23 Sanofi-Aventis Nouveau anticorps Anti-CD38 pour le traitement du cancer
AR063494A1 (es) * 2006-10-19 2009-01-28 Genentech Inc Anticuerpos agonistas anti notch3 y su uso en el tratamiento de las enfermedades relacionadas con el gen notch3
MX2009004881A (es) 2006-11-09 2009-05-21 Irm Llc Anticuerpos agonistas de trkb y usos de los mismos.
US8785400B2 (en) 2006-11-22 2014-07-22 Curedm Group Holdings, Llc Methods and compositions relating to islet cell neogenesis
US8455626B2 (en) 2006-11-30 2013-06-04 Abbott Laboratories Aβ conformer selective anti-aβ globulomer monoclonal antibodies
ES2523915T5 (es) 2006-12-01 2022-05-26 Seagen Inc Agentes de unión a la diana variantes y usos de los mismos
CN101678100A (zh) 2006-12-06 2010-03-24 米迪缪尼有限公司 治疗系统性红斑狼疮的方法
KR20150004933A (ko) 2006-12-18 2015-01-13 제넨테크, 인크. 항-ｎｏｔｃｈ3 길항제 항체 및 ｎｏｔｃｈ3-관련 질환의 예방 및 치료에 있어서 그의 용도
US8128926B2 (en) 2007-01-09 2012-03-06 Biogen Idec Ma Inc. Sp35 antibodies and uses thereof
ES2673153T3 (es) 2007-01-09 2018-06-20 Biogen Ma Inc. Anticuerpos Sp35 y usos de los mismos
US8685666B2 (en) 2007-02-16 2014-04-01 The Board Of Trustees Of Southern Illinois University ARL-1 specific antibodies and uses thereof
WO2008101184A2 (fr) 2007-02-16 2008-08-21 The Board Of Trustees Of Southern Illinois University Anticorps spécifiques d'arl-1
WO2008104803A2 (fr) 2007-02-26 2008-09-04 Oxford Genome Sciences (Uk) Limited Protéines
EP3118221B1 (fr) 2007-02-26 2019-08-21 Oxford BioTherapeutics Ltd Proteines
US20100311767A1 (en) 2007-02-27 2010-12-09 Abbott Gmbh & Co. Kg Method for the treatment of amyloidoses
JP5721951B2 (ja) 2007-03-22 2015-05-20 バイオジェン アイデック マサチューセッツ インコーポレイテッド 抗体、抗体誘導体、および抗体断片を含む、ｃｄ１５４に特異的に結合する結合タンパク質ならびにその使用
US20110130544A1 (en) 2007-03-30 2011-06-02 Medimmune, Llc Antibodies with decreased deamidation profiles
EP2164868B1 (fr) 2007-05-04 2015-03-25 Technophage, Investigação E Desenvolvimento Em Biotecnologia, SA Domaines variables transgéniques d'anticorps de lapin et leurs utilisations
US20080279851A1 (en) 2007-05-07 2008-11-13 Medlmmune, Llc Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease
SI3072525T1 (en) 2007-05-14 2018-06-29 Astrazeneca Ab Procedures for lowering the levels of basophiles
AU2008265984B2 (en) 2007-06-21 2014-07-17 Macrogenics, Inc. Covalent diabodies and uses thereof
KR20100080519A (ko) 2007-08-30 2010-07-08 큐어디엠 인코포레이티드 프로섬 펩타이드 및 이의 유사체의 조성물 및 이의 이용 방법
GB0717337D0 (en) 2007-09-06 2007-10-17 Ucb Pharma Sa Method of treatment
ES2667729T3 (es) 2007-09-26 2018-05-14 Ucb Biopharma Sprl Fusiones de anticuerpos con doble especificidad
AU2008324800B2 (en) 2007-11-05 2014-03-27 Astrazeneca Ab Methods of treating scleroderma
EP3115469B1 (fr) 2007-11-19 2020-04-29 Celera Corporation Marqueurs du cancer du poumon et utilisations de ceux-ci
JP5490714B2 (ja) 2007-11-28 2014-05-14 メディミューン，エルエルシー タンパク質製剤
GB0800277D0 (en) 2008-01-08 2008-02-13 Imagination Tech Ltd Video motion compensation
WO2009088105A1 (fr) 2008-01-11 2009-07-16 Gene Techno Science Co., Ltd. Anticorps humanisés anti-intégrine α9 et leurs utilisations
EP2244729B1 (fr) 2008-01-18 2016-11-02 MedImmune, LLC Anticorps modifiés par l'introduction de cysteine pour conjugaison spécifique
CA2713981C (fr) 2008-02-08 2016-11-01 Medimmune, Llc Anticorps anti-ifnar1 ayant une affinite reduite pour le ligand fc
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
WO2009118300A1 (fr) 2008-03-25 2009-10-01 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Traitement du cancer par régulation à la baisse de frizzled-4 et/ou frizzled-1
AU2009231991B2 (en) 2008-04-02 2014-09-25 Macrogenics, Inc. HER2/neu-specific antibodies and methods of using same
JP5785490B2 (ja) 2008-04-02 2015-09-30 マクロジェニクス，インコーポレーテッド Ｂｃｒ複合体特異的抗体およびその使用方法
WO2009126934A2 (fr) 2008-04-11 2009-10-15 Seattle Genetics, Inc. Détection et traitement du cancer du pancréas, des ovaires et d'autres cancers
GB0807413D0 (en) 2008-04-23 2008-05-28 Ucb Pharma Sa Biological products
CN104530233B (zh) 2008-04-24 2018-01-30 株式会社遗传科技 细胞外基质蛋白的氨基酸序列rgd的特异性人源化抗体及其应用
EP2282769A4 (fr) 2008-04-29 2012-04-25 Abbott Lab Immunoglobulines à double domaine variable et utilisations
CA2723197C (fr) 2008-05-02 2017-09-19 Seattle Genetics, Inc. Procede et compositions pour preparer des anticorps et des derives d'anticorps avec une fucosylation centrale reduite
EP2500361B1 (fr) 2008-05-09 2016-03-30 AbbVie Deutschland GmbH & Co KG Anticorps contre le récepteur pour produits terminaux de glycation avancée (RAGE) et utilisations de ceux-ci
EP2304439A4 (fr) 2008-05-29 2012-07-04 Nuclea Biotechnologies Llc Anticorps anti-phospho-akt
JP5723769B2 (ja) 2008-06-03 2015-05-27 アッヴィ・インコーポレイテッド 二重可変ドメイン免疫グロブリン及びその使用
EP2297209A4 (fr) 2008-06-03 2012-08-01 Abbott Lab Immunoglobulines à deux domaines variables et leurs utilisations
US20110081347A1 (en) 2008-06-04 2011-04-07 Macrogenics, Inc. Antibodies with Altered Binding to FcRn and Methods of Using Same
SG192496A1 (en) 2008-07-08 2013-08-30 Abbott Lab Prostaglandin e2 binding proteins and uses thereof
EP2321422A4 (fr) 2008-07-08 2013-06-19 Abbvie Inc Immunoglobulines à double domaine variable de prostaglandine e2 et leurs utilisations
US8058406B2 (en) 2008-07-09 2011-11-15 Biogen Idec Ma Inc. Composition comprising antibodies to LINGO or fragments thereof
WO2010048615A2 (fr) 2008-10-24 2010-04-29 The Government Of The United States Of America As Represented By The Secretary, Department Of Health & Human Services, Center For Disease Control And Prevention Espèce de virus ebola humain et compositions et procédés associés
BRPI0921845A2 (pt) 2008-11-12 2019-09-17 Medimmune Llc formulação aquosa estéril estável, forma de dosagem unitária farmacêutica, seringa pré-carregada, e, métodos para tratar uma doença ou distúrbio, para tratar ou prevenir rejeição, para esgotar células t que expressam icos em um paciente humano, e para interromper arquitetura central germinal em um órgão linfóide secundário de um primata
BRPI0918122A8 (pt) 2008-12-19 2017-01-24 Macrogenics Inc molécula de diabody, diabody, e mólecula dart
CN102341411A (zh) 2008-12-31 2012-02-01 比奥根艾迪克Ma公司 抗-淋巴细胞毒素抗体
GB0900425D0 (en) 2009-01-12 2009-02-11 Ucb Pharma Sa Biological products
EP2387584A1 (fr) 2009-01-14 2011-11-23 IQ Therapeutics BV Anticorps combinés destinés au traitement et à la prévention d'une maladie provoquée par bacillus anthracis, ainsi que par les bactéries connexes et par leurs toxines
KR101245929B1 (ko) 2009-01-20 2013-03-22 호메이욘 에이치. 자데흐 항체 매개된 골질 재생
AU2010207552A1 (en) 2009-01-21 2011-09-01 Oxford Biotherapeutics Ltd. PTA089 protein
WO2010087927A2 (fr) 2009-02-02 2010-08-05 Medimmune, Llc Anticorps contre le virus respiratoire syncytial et procédés permettant de produire des vaccins contre le virus respirateur syncytial
WO2010093993A2 (fr) 2009-02-12 2010-08-19 Human Genome Sciences, Inc. Utilisation d'antagonistes de la protéine stimulant les lymphocytes b afin de favoriser la tolérance aux greffes
UY32451A (es) 2009-02-17 2010-09-30 Ucb Pharma Sa Moléculas de anticuerpo con especifidad por el ox40 humano
RU2015132478A (ru) 2009-03-05 2015-12-10 Эббви Инк. Связывающие il-17 белки
EP2405920A1 (fr) 2009-03-06 2012-01-18 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Nouvelle thérapie contre l'anxiété
EP2406285B1 (fr) 2009-03-10 2016-03-09 Gene Techno Science Co., Ltd. Génération, expression et caractérisation de l'anticorps monoclonal humanisé k33n
GB0904214D0 (en) 2009-03-11 2009-04-22 Ucb Pharma Sa Biological products
EP2241323A1 (fr) 2009-04-14 2010-10-20 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Tenascine-W et cancers du cerveau
NZ595792A (en) 2009-04-20 2014-01-31 Oxford Biotherapeutics Ltd Antibodies specific to cadherin-17
WO2010122148A1 (fr) * 2009-04-24 2010-10-28 Boehringer Ingelheim International Gmbh Domaine d'anticorps amélioré et fragments d'anticorps et anticorps associés
MX2012000121A (es) 2009-06-22 2012-03-07 Medimmune Llc Regiones fc modificadas para la conjugacion especifica del sitio.
EP2464664B1 (fr) 2009-08-13 2015-09-23 Crucell Holland B.V. Anticorps contre le virus respiratoire syncytial (vrs) humain et procédés d'utilisation
EP2292266A1 (fr) 2009-08-27 2011-03-09 Novartis Forschungsstiftung, Zweigniederlassung Traitement du cancer en modulant la copine III
CN102741288B (zh) 2009-08-29 2015-08-19 Abbvie公司 治疗用dll4结合蛋白
SG178602A1 (en) 2009-09-01 2012-04-27 Abbott Lab Dual variable domain immunoglobulins and uses thereof
US20120283415A1 (en) 2009-09-10 2012-11-08 Ucb Pharma S.A. Multivalent Antibodies
WO2011036118A1 (fr) 2009-09-22 2011-03-31 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Traitement du cancer par modulation de mex-3
GB201005063D0 (en) 2010-03-25 2010-05-12 Ucb Pharma Sa Biological products
UY32914A (es) 2009-10-02 2011-04-29 Sanofi Aventis Anticuerpos que se usan específicamente al receptor epha2
EP2470569A1 (fr) 2009-10-13 2012-07-04 Oxford Biotherapeutics Ltd. Anticorps anti-epha10
WO2011045352A2 (fr) 2009-10-15 2011-04-21 Novartis Forschungsstiftung Tyrosine kinase de la rate et cancers du cerveau
WO2011047262A2 (fr) 2009-10-15 2011-04-21 Abbott Laboratories Immunoglobulines à deux domaines variables et utilisations afférentes
US9234037B2 (en) 2009-10-27 2016-01-12 Ucb Biopharma Sprl Method to generate antibodies to ion channels
GB0922434D0 (en) 2009-12-22 2010-02-03 Ucb Pharma Sa antibodies and fragments thereof
GB0922435D0 (en) 2009-12-22 2010-02-03 Ucb Pharma Sa Method
CA2778673A1 (fr) 2009-10-27 2011-05-05 Karen Margrete Miller Modification fonctionnelle des anticorps anti-nav 1.7
UY32979A (es) 2009-10-28 2011-02-28 Abbott Lab Inmunoglobulinas con dominio variable dual y usos de las mismas
US20120213801A1 (en) 2009-10-30 2012-08-23 Ekaterina Gresko Phosphorylated Twist1 and cancer
WO2011053707A1 (fr) 2009-10-31 2011-05-05 Abbott Laboratories Anticorps pour récepteur pour produits terminaux de glycation avancée (rage) et utilisations de ceux-ci
EP2496605A1 (fr) 2009-11-02 2012-09-12 Oxford Biotherapeutics Ltd. Ror1 comme cible thérapeutique et diagnostique
EP2499491B1 (fr) 2009-11-11 2015-04-01 Gentian AS Dosage immunologique pour le dosage d'analytes apparentés d'origine différente
GB0920127D0 (en) 2009-11-17 2009-12-30 Ucb Pharma Sa Antibodies
GB0920324D0 (en) 2009-11-19 2010-01-06 Ucb Pharma Sa Antibodies
ES2562832T3 (es) 2009-12-08 2016-03-08 Abbvie Deutschland Gmbh & Co Kg Anticuerpos monoclonales contra la proteína RGM para su uso en el tratamiento de la degeneración de la capa de fibra nerviosa de la retina
GB201000467D0 (en) 2010-01-12 2010-02-24 Ucb Pharma Sa Antibodies
JP5964249B2 (ja) 2010-03-02 2016-08-03 アッヴィ・インコーポレイテッド 治療用ｄｌｌ４結合タンパク質
EP2542578A1 (fr) 2010-03-05 2013-01-09 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Smoc1, ténascine-c et cancers du cerveau
GB201005064D0 (en) 2010-03-25 2010-05-12 Ucb Pharma Sa Biological products
TR201903279T4 (tr) 2010-03-25 2019-03-21 Ucb Biopharma Sprl Disülfür stabilize edilmiş DVD-IG molekülleri.
JP2013523182A (ja) 2010-04-15 2013-06-17 アボット・ラボラトリーズ アミロイドベータ結合タンパク質
WO2011131611A1 (fr) 2010-04-19 2011-10-27 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Modulation de xrn1
EP2380909A1 (fr) 2010-04-26 2011-10-26 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Protéine PTK-7 impliquée dans le cancer du sein
WO2011137395A1 (fr) 2010-04-30 2011-11-03 Rother Russell P Anticorps anti-c5a et méthodes pour utiliser les anticorps
AR081246A1 (es) 2010-05-14 2012-07-18 Abbott Lab Proteinas de union a il-1
EP2569335B1 (fr) 2010-05-14 2018-08-22 Orega Biotech Méthodes de traitement et/ou de prévention de troubles de prolifération cellulaire à l'aide d'antagonistes de il-17
EP2580239A1 (fr) 2010-06-10 2013-04-17 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Traitement du cancer par modulation de la kinase 3 stérile de mammifère de type 20
WO2012006500A2 (fr) 2010-07-08 2012-01-12 Abbott Laboratories Anticorps monoclonaux contre la protéine de capside du virus de l'hépatite c
NZ703035A (en) 2010-07-09 2016-06-24 Crucell Holland Bv Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
UY33492A (es) 2010-07-09 2012-01-31 Abbott Lab Inmunoglobulinas con dominio variable dual y usos de las mismas
JP2013537415A (ja) 2010-08-03 2013-10-03 アッヴィ・インコーポレイテッド 二重可変ドメイン免疫グロブリンおよびその使用
EP2603524A1 (fr) 2010-08-14 2013-06-19 AbbVie Inc. Protéines de liaison bêta-amyloïdes
ES2665954T3 (es) 2010-08-19 2018-04-30 Zoetis Belgium S.A. Anticuerpos anti-NGF y su uso
GB201014033D0 (en) 2010-08-20 2010-10-06 Ucb Pharma Sa Biological products
CA2809433A1 (fr) 2010-08-26 2012-03-01 Abbvie Inc. Immunoglobulines a deux domaines variables et leurs utilisations
US20130171159A1 (en) 2010-09-10 2013-07-04 Brian Arthur Hemmings Phosphorylated twist1 and metastasis
WO2012065937A1 (fr) 2010-11-15 2012-05-24 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research Agents antifongiques
CN110251668A (zh) 2010-11-30 2019-09-20 霍夫曼-拉罗奇有限公司 低亲和力血脑屏障受体抗体及其用途
BR112013015944A2 (pt) 2010-12-21 2018-06-19 Abbvie Inc imunoglobulinas de domínio duplo variável il-1 alpha e beta biespecífico e seus usos.
WO2012088094A2 (fr) 2010-12-21 2012-06-28 Abbott Laboratories Protéines liant l'il-1
CN103429261A (zh) 2010-12-22 2013-12-04 塞法隆澳大利亚股份有限公司 半寿期改进的修饰抗体
GB201100282D0 (en) 2011-01-07 2011-02-23 Ucb Pharma Sa Biological methods
US10208349B2 (en) 2011-01-07 2019-02-19 Ucb Biopharma Sprl Lipocalin 2 as a biomarker for IL-17 inhibitor therapy efficacy
CA2823812C (fr) 2011-01-14 2017-02-14 Ucb Pharma S.A. Molecules d'anticorps se liant a il-17a et il-17f
DK2668210T3 (da) 2011-01-26 2020-08-24 Celldex Therapeutics Inc Anti-kit antistoffer og anvendelser deraf
EP2699264B1 (fr) 2011-04-20 2018-03-14 Medlmmune, LLC Anticorps et autres molécules qui se lient à b7-h1 et à pd-1
SG195073A1 (en) 2011-05-21 2013-12-30 Macrogenics Inc Deimmunized serum-binding domains and their use for extending serum half-life
EP2717911A1 (fr) 2011-06-06 2014-04-16 Novartis Forschungsstiftung, Zweigniederlassung Protéine tyrosine phosphatase de type non-récepteur 11 (ptpn11), et cancer du sein triple négatif
US9561274B2 (en) 2011-06-07 2017-02-07 University Of Hawaii Treatment and prevention of cancer with HMGB1 antagonists
WO2012170740A2 (fr) 2011-06-07 2012-12-13 University Of Hawaii Biomarqueur d'exposition à l'amiante et mésothéliome
WO2012170807A2 (fr) 2011-06-10 2012-12-13 Medimmune, Llc Molécules de liaison anti-psl de pseudomonas et leurs utilisations
JP6247209B2 (ja) 2011-06-28 2017-12-13 オックスフォード ビオトヘラペウトイクス エルティーディー． Ａｄｐリボシルシクラーゼ２に対する抗体
JP6113721B2 (ja) 2011-06-28 2017-04-12 オックスフォード ビオトヘラペウトイクス エルティーディー． 治療標的及び診断標的
KR20140061403A (ko) 2011-07-13 2014-05-21 애브비 인코포레이티드 항―ｉｌ―１３ 항체를 이용하여 천식을 치료하기 위한 방법 및 조성물
GB201112056D0 (en) 2011-07-14 2011-08-31 Univ Leuven Kath Antibodies
JP6120848B2 (ja) 2011-08-15 2017-04-26 メディミューン，エルエルシー 抗ｂ７−ｈ４抗体およびその使用
WO2013034579A1 (fr) 2011-09-05 2013-03-14 Rheinische Friedrich-Wilhelms-Universität Bonn Groupe de gènes biosynthétiques pour la production d'analogues de peptides/de protéines
US9447192B2 (en) 2011-09-09 2016-09-20 Medimmune Limited Anti-Siglec-15 antibodies and uses thereof
MX2014004977A (es) 2011-10-24 2014-09-11 Abbvie Inc Inmunoaglutinantes dirigidos contra esclerostina.
ES2697674T3 (es) 2011-11-01 2019-01-25 Bionomics Inc Procedimientos para bloquear el crecimiento de células madre cancerosas
WO2013067057A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps anti-gpr49
CN104053671A (zh) 2011-11-01 2014-09-17 生态学有限公司 治疗癌症的抗体和方法
EP2773667A1 (fr) 2011-11-01 2014-09-10 Bionomics, Inc. Anticorps anti-gpr49
WO2013068431A1 (fr) 2011-11-08 2013-05-16 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research Traitement inédit contre les maladies neurodégénératives
WO2013068432A1 (fr) 2011-11-08 2013-05-16 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research Diagnostic précoce de maladies neurodégénératives
US9803004B2 (en) 2011-11-11 2017-10-31 Ucb Biopharma Sprl Albumin binding antibodies and binding fragments thereof
JP6342812B2 (ja) 2011-12-14 2018-06-13 アッヴィ・ドイチュラント・ゲー・エム・ベー・ハー・ウント・コー・カー・ゲー 鉄関連障害を診断および治療するための組成物および方法
CA2855570A1 (fr) 2011-12-14 2013-06-20 AbbVie Deutschland GmbH & Co. KG Composition et methode pour le diagnostic et le traitement de troubles lies au fer
ES2721882T3 (es) 2011-12-23 2019-08-06 Pfizer Regiones constantes de anticuerpo modificadas por ingeniería genética para conjugación específica de sitio y procedimientos y usos de las mismas
US9120870B2 (en) 2011-12-30 2015-09-01 Abbvie Inc. Dual specific binding proteins directed against IL-13 and IL-17
US20140363448A1 (en) 2012-01-02 2014-12-11 Novartis Ag Cdcp1 and breast cancer
GB201201332D0 (en) 2012-01-26 2012-03-14 Imp Innovations Ltd Method
CN107880124B (zh) 2012-01-27 2021-08-13 艾伯维德国有限责任两合公司 用于诊断和治疗与神经突变性相关的疾病的组合物和方法
EP2812702B1 (fr) 2012-02-10 2019-04-17 Seattle Genetics, Inc. Diagnostic et prise en charge des cancers exprimant le CD30
PL2814842T3 (pl) 2012-02-15 2018-12-31 Novo Nordisk A/S Przeciwciała wiążące białko 1 rozpoznające peptydoglikan
US9550830B2 (en) 2012-02-15 2017-01-24 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
EP3196214B1 (fr) 2012-02-15 2019-07-31 Novo Nordisk A/S Anticorps liant et bloquant un récepteur de déclenchement exprimé sur des cellules-1 myéloïdes (trem-1)
GB201203071D0 (en) 2012-02-22 2012-04-04 Ucb Pharma Sa Biological products
GB201203051D0 (en) 2012-02-22 2012-04-04 Ucb Pharma Sa Biological products
WO2013142808A1 (fr) 2012-03-23 2013-09-26 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Isolats de phlebovirus pathogènes, ainsi que des compositions et des méthodes d'utilisation
WO2013144240A1 (fr) 2012-03-29 2013-10-03 Friedrich Miescher Institute For Biomedical Research Inhibition de l'interleukine 8 et/ou de ses récepteurs cxcrl dans le traitement du cancer du sein surexprimant her2/her3
WO2013151649A1 (fr) 2012-04-04 2013-10-10 Sialix Inc Composés d'interaction avec des glycanes
CA2873623C (fr) 2012-05-14 2021-11-09 Biogen Idec Ma Inc. Antagonistes de lingo-2 pour le traitement d'affections impliquant des neurones moteurs
BR112014028306A2 (pt) 2012-05-15 2018-04-17 Morphotek, Inc. métodos para tratamento de câncer gástrico.
WO2013184871A1 (fr) 2012-06-06 2013-12-12 Zoetis Llc Anticorps anti-ngf caninisés et procédés associés
US10048253B2 (en) 2012-06-28 2018-08-14 Ucb Biopharma Sprl Method for identifying compounds of therapeutic interest
EP2866831A1 (fr) 2012-06-29 2015-05-06 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Traitement de maladies par modulation d'un isoforme spécifique de mkl-1
US20150184154A1 (en) 2012-07-05 2015-07-02 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Resear New treatment for neurodegenerative diseases
WO2014006115A1 (fr) 2012-07-06 2014-01-09 Novartis Ag Combinaison d'un inhibiteur de phosphoinositide 3-kinase et d'un inhibiteur de l'interaction il-8/cxcr
US9670276B2 (en) 2012-07-12 2017-06-06 Abbvie Inc. IL-1 binding proteins
GB201213652D0 (en) 2012-08-01 2012-09-12 Oxford Biotherapeutics Ltd Therapeutic and diagnostic target
WO2014055442A2 (fr) 2012-10-01 2014-04-10 The Trustees Of The University Of Pennsylvania Compositions et procédés de ciblage de cellules stromales pour le traitement du cancer
CA2887528C (fr) 2012-10-12 2023-08-29 The Brigham And Women's Hospital, Inc. Renforcement de la reponse immunitaire
BR112015009961B1 (pt) 2012-11-01 2020-10-20 Abbvie Inc. proteína de ligação capaz de se ligar a dll4 e vegf, bem como composição que a compreende como composição que a compreende
JP6359031B2 (ja) 2012-12-21 2018-07-18 メディミューン，エルエルシー 抗ｈ７ｃｒ抗体
GB201223276D0 (en) 2012-12-21 2013-02-06 Ucb Pharma Sa Antibodies and methods of producing same
US9550986B2 (en) 2012-12-21 2017-01-24 Abbvie Inc. High-throughput antibody humanization
AU2013202668B2 (en) 2012-12-24 2014-12-18 Adelaide Research & Innovation Pty Ltd Inhibition of cancer growth and metastasis
GB201302447D0 (en) 2013-02-12 2013-03-27 Oxford Biotherapeutics Ltd Therapeutic and diagnostic target
JP6505076B2 (ja) 2013-03-14 2019-04-24 アボット・ラボラトリーズＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Ｈｃｖ抗原−抗体組み合わせアッセイおよびこれに使用するための方法および組成物
MX2015012825A (es) 2013-03-14 2016-06-10 Abbott Lab Anticuerpos monoclonales del dominio de union de lipido del núcleo del virus de la hepatitis c vhc.
JP2016512241A (ja) 2013-03-14 2016-04-25 アボット・ラボラトリーズＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ 改良された抗体検出のためのｈｃｖｎｓ３組換え抗原およびこの突然変異体
BR112015023520A2 (pt) 2013-03-15 2017-10-24 Abbvie Inc purificação do conjugado de anticorpo-fármaco (adc)
WO2014144280A2 (fr) 2013-03-15 2014-09-18 Abbvie Inc. Protéines de liaison spécifiques à domaines variables doubles dirigées contre il -1β et/ou il -17
US9469686B2 (en) 2013-03-15 2016-10-18 Abbott Laboratories Anti-GP73 monoclonal antibodies and methods of obtaining the same
TWI689313B (zh) 2013-03-15 2020-04-01 德商艾伯維德國有限及兩合公司 抗-ｅｇｆｒ抗體藥物結合物調配物
AU2014262843B2 (en) 2013-05-06 2017-06-22 Scholar Rock, Inc. Compositions and methods for growth factor modulation
KR20160129698A (ko) 2013-05-24 2016-11-09 메디뮨 엘엘씨 항-b7-h5 항체 및 이의 용도
SG10201708143QA (en) 2013-06-06 2017-11-29 Pierre Fabre Médicament Anti-c10orf54 antibodies and uses thereof
CA2914566A1 (fr) 2013-06-07 2014-12-11 Duke University Inhibiteurs du facteur h du complement
WO2015019286A1 (fr) 2013-08-07 2015-02-12 Friedrich Miescher Institute For Biomedical Research Nouvelle méthode de criblage pour le traitement de l'ataxie de friedreich
US9475874B2 (en) 2013-08-26 2016-10-25 MabVax Therapeutics, Inc. Nucleic acids encoding human antibodies to sialyl-lewisa
AU2014342528A1 (en) 2013-10-28 2016-04-28 Dots Technology Corp. Allergen detection
US10684289B2 (en) 2013-12-09 2020-06-16 Adimab, Llc Polyclonal mixtures of antibodies, and methods of making and using them
US9914769B2 (en) 2014-07-15 2018-03-13 Kymab Limited Precision medicine for cholesterol treatment
US9067998B1 (en) 2014-07-15 2015-06-30 Kymab Limited Targeting PD-1 variants for treatment of cancer
US8992927B1 (en) 2014-07-15 2015-03-31 Kymab Limited Targeting human NAV1.7 variants for treatment of pain
US8986694B1 (en) 2014-07-15 2015-03-24 Kymab Limited Targeting human nav1.7 variants for treatment of pain
US9045545B1 (en) 2014-07-15 2015-06-02 Kymab Limited Precision medicine by targeting PD-L1 variants for treatment of cancer
GB201403775D0 (en) 2014-03-04 2014-04-16 Kymab Ltd Antibodies, uses & methods
US9738702B2 (en) 2014-03-14 2017-08-22 Janssen Biotech, Inc. Antibodies with improved half-life in ferrets
US9546214B2 (en) 2014-04-04 2017-01-17 Bionomics, Inc. Humanized antibodies that bind LGR5
EP3131929B1 (fr) * 2014-04-16 2022-06-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps pour la prévention ou le traitement d'épisodes de saignement
WO2015175874A2 (fr) 2014-05-16 2015-11-19 Medimmune, Llc Molécules présentant une altération de liaison de récepteur fc néonatal ayant des propriétés thérapeutiques et diagnostiques améliorées
KR102433464B1 (ko) 2014-05-28 2022-08-17 아게누스 인코포레이티드 항-gitr 항체 및 이의 사용 방법
GB201409558D0 (en) 2014-05-29 2014-07-16 Ucb Biopharma Sprl Method
KR102226248B1 (ko) 2014-06-04 2021-03-12 바이오엔테크 리서치 앤드 디벨롭먼트 인코포레이티드 강글리오사이드 ｇｄ２에 대한 사람 단클론 항체
US20170137824A1 (en) 2014-06-13 2017-05-18 Indranil BANERJEE New treatment against influenza virus
EP3157535A1 (fr) 2014-06-23 2017-04-26 Friedrich Miescher Institute for Biomedical Research Méthodes de déclenchement de la formation de novo de l'hétérochromatine et/ou d'inhibition épigénétique avec des petits arn
GB201411320D0 (en) 2014-06-25 2014-08-06 Ucb Biopharma Sprl Antibody construct
WO2016001830A1 (fr) 2014-07-01 2016-01-07 Friedrich Miescher Institute For Biomedical Research Combinaison d'un inhibiteur de braf v600e et d'un inhibiteur de mertk pour le traitement du mélanome
US9139648B1 (en) 2014-07-15 2015-09-22 Kymab Limited Precision medicine by targeting human NAV1.9 variants for treatment of pain
GB201412658D0 (en) 2014-07-16 2014-08-27 Ucb Biopharma Sprl Molecules
GB201412659D0 (en) 2014-07-16 2014-08-27 Ucb Biopharma Sprl Molecules
RS65136B1 (sr) 2014-07-17 2024-02-29 Novo Nordisk As Mutageza usmerena na lokaciju trem-1 antitela za smanjenje viskoziteta
EP3197557A1 (fr) 2014-09-24 2017-08-02 Friedrich Miescher Institute for Biomedical Research Lats et cancer du sein
US9982061B2 (en) 2014-10-01 2018-05-29 Medimmune Limited Antibodies to ticagrelor and methods of use
DK3218005T5 (da) 2014-11-12 2024-10-14 Seagen Inc Glycan-interagerende forbindelser og anvendelsesfremgangsmåder
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
US10766959B2 (en) 2014-12-11 2020-09-08 Pierre Fabre Medicament Anti-C10ORF54 antibodies and uses thereof
WO2016094881A2 (fr) 2014-12-11 2016-06-16 Abbvie Inc. Protéines de liaison à lrp-8
ES2862701T3 (es) 2014-12-22 2021-10-07 Univ Rockefeller Anticuerpos agonistas anti-MERTK y usos de los mismos
CA2973266A1 (fr) 2015-01-08 2016-07-14 Biogen Ma Inc. Antagonistes de lingo-1 et leurs utilisations pour le traitement de troubles de demyelinisation
BR112017015880A2 (pt) 2015-03-03 2018-07-31 Kymab Ltd anticorpos, usos e métodos
ES2904573T3 (es) 2015-03-27 2022-04-05 Univ Southern California Terapia con células T dirigida a LHR para el tratamiento de tumores sólidos
GB201506869D0 (en) 2015-04-22 2015-06-03 Ucb Biopharma Sprl Method
GB201506870D0 (en) 2015-04-22 2015-06-03 Ucb Biopharma Sprl Method
EP3288542B1 (fr) 2015-04-29 2021-12-29 University Of South Australia Compositions et méthodes pour l'administration d'anticorps
EP3291836A4 (fr) 2015-05-06 2018-11-14 Janssen Biotech, Inc. Agents de liaison bispécifique à l'antigène membranaire spécifique de la prostate (psma) et utilisations de ceux-ci
AU2016256911B2 (en) 2015-05-07 2022-03-31 Agenus Inc. Anti-OX40 antibodies and methods of use thereof
MA47395A (fr) 2015-05-27 2019-12-11 Ucb Biopharma Sprl Méthode pour le traitement d'une maladie neurologique
MA53355A (fr) 2015-05-29 2022-03-16 Agenus Inc Anticorps anti-ctla-4 et leurs procédés d'utilisation
DK3303395T3 (da) 2015-05-29 2020-01-27 Abbvie Inc Anti-cd40-antistoffer og anvendelser deraf
AU2016270474A1 (en) 2015-06-04 2018-01-04 University Of Southern California Lym-1 and Lym-2 targeted CAR cell immunotherapy
TW201710286A (zh) 2015-06-15 2017-03-16 艾伯維有限公司 抗ｖｅｇｆ、ｐｄｇｆ及／或其受體之結合蛋白
GB201510758D0 (en) 2015-06-18 2015-08-05 Ucb Biopharma Sprl Novel TNFa structure for use in therapy
GB201601073D0 (en) 2016-01-20 2016-03-02 Ucb Biopharma Sprl Antibodies
GB201601077D0 (en) 2016-01-20 2016-03-02 Ucb Biopharma Sprl Antibody molecule
GB201601075D0 (en) 2016-01-20 2016-03-02 Ucb Biopharma Sprl Antibodies molecules
PE20181322A1 (es) 2015-09-01 2018-08-14 Agenus Inc Anticuerpo anti-pd1 y sus metodos de uso
KR20180053744A (ko) 2015-09-23 2018-05-23 사이토이뮨 테라퓨틱스 엘엘씨 면역 요법을 위한 flt3 유도된 car 세포
KR20180067676A (ko) 2015-10-27 2018-06-20 유씨비 바이오파마 에스피알엘 항-il-17a/f 항체를 사용한 치료 방법
US20180348224A1 (en) 2015-10-28 2018-12-06 Friedrich Miescher Institute For Biomedical Resear Ch Tenascin-w and biliary tract cancers
IL302822A (en) 2015-11-12 2023-07-01 Seagen Inc Compounds interacting with glycans and methods of use
US11253590B2 (en) 2015-12-02 2022-02-22 Stsciences, Inc. Antibodies specific to glycosylated BTLA (B- and T- lymphocyte attenuator)
WO2017096051A1 (fr) 2015-12-02 2017-06-08 Stcube & Co., Inc. Anticorps et molécules se liant de manière immunospécifique à btn1a1 et leurs utilisations thérapeutiques
GB201521391D0 (en) 2015-12-03 2016-01-20 Ucb Biopharma Sprl Antibodies
GB201521382D0 (en) 2015-12-03 2016-01-20 Ucb Biopharma Sprl Antibodies
GB201521389D0 (en) 2015-12-03 2016-01-20 Ucb Biopharma Sprl Method
GB201521383D0 (en) 2015-12-03 2016-01-20 Ucb Biopharma Sprl And Ucb Celltech Method
GB201521393D0 (en) 2015-12-03 2016-01-20 Ucb Biopharma Sprl Antibodies
EP3386542B1 (fr) 2015-12-10 2020-11-18 Katholieke Universiteit Leuven Anticorps anti-adamts13 et leur utilisation pour le traitement ou la prévention de troubles hémorragiques provoqués par un dispositif d'assistance ventriculaire
GB201602413D0 (en) 2016-02-10 2016-03-23 Nascient Ltd Method
KR20240017421A (ko) 2016-03-10 2024-02-07 비엘라 바이오, 인크. Ilt7 결합 분자 및 이의 사용 방법
CN108697799A (zh) 2016-03-22 2018-10-23 生态学有限公司 抗lgr5单克隆抗体的施用
SG11201809437TA (en) * 2016-05-03 2018-11-29 Sqz Biotechnologies Co Intracellular delivery of biomolecules to induce tolerance
CN116333130A (zh) 2016-05-24 2023-06-27 英斯梅德股份有限公司 抗体及其制备方法
NZ749355A (en) 2016-05-27 2023-04-28 Agenus Inc Anti-tim-3 antibodies and methods of use thereof
EP3481864A1 (fr) 2016-07-08 2019-05-15 Staten Biotechnology B.V. Anticorps anti-apoc3 et leurs méthodes d'utilisation
EP3487521A4 (fr) 2016-07-21 2020-07-01 Emory University Anticorps contre le virus ebola et agents de liaison dérivés de ceux-ci
GB201616596D0 (en) 2016-09-29 2016-11-16 Nascient Limited Epitope and antibodies
BR112019006710A2 (pt) 2016-10-03 2019-06-25 Abbott Lab métodos aprimorados para avaliação do estado da uch-l1 em amostras de pacientes
CA3037380A1 (fr) 2016-10-11 2018-04-19 Agenus Inc. Anticorps anti-lag-3 et leurs procedes d'utilisation
CN110300600A (zh) 2016-11-02 2019-10-01 伊缪诺金公司 利用抗体-药物缀合物和parp抑制剂的组合治疗
EP3534947A1 (fr) 2016-11-03 2019-09-11 Kymab Limited Anticorps, combinaisons comprenant des anticorps, biomarqueurs, utilisations et procédés
AU2017353939A1 (en) 2016-11-07 2019-06-06 Neuracle Science Co., Ltd. Anti-family with sequence similarity 19, member A5 antibodies and method of use thereof
EP3541847A4 (fr) 2016-11-17 2020-07-08 Seattle Genetics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
SI3551660T1 (sl) 2016-12-07 2024-02-29 Agenus Inc. Protitelesa proti antictla-4 in načini njihove uporabe
BR112019011582A2 (pt) 2016-12-07 2019-10-22 Agenus Inc. anticorpos e métodos de utilização dos mesmos
GB201621635D0 (en) 2016-12-19 2017-02-01 Ucb Biopharma Sprl Crystal structure
KR20240044544A (ko) 2017-03-03 2024-04-04 씨젠 인크. 글리칸-상호작용 화합물 및 사용 방법
JP7346300B2 (ja) 2017-03-23 2023-09-19 アボット・ラボラトリーズ 早期バイオマーカーであるユビキチンカルボキシ末端ヒドロラーゼｌ１を使用する、ヒト対象における外傷性脳損傷の程度の診断及び決定の一助となるための方法
KR102629972B1 (ko) 2017-04-13 2024-01-29 아게누스 인코포레이티드 항-cd137 항체 및 이의 사용 방법
AU2018250688B2 (en) 2017-04-15 2024-07-04 Abbott Laboratories Methods for aiding in the hyperacute diagnosis and determination of traumatic brain injury in a human subject using early biomarkers
MA50958A (fr) 2017-04-21 2020-10-14 Staten Biotechnology B V Anticorps anti-apoc3 et leurs méthodes d'utilisation
AU2018256845B2 (en) 2017-04-28 2024-03-14 Abbott Laboratories Methods for aiding in the hyperacute diagnosis and determination of traumatic brain injury using early biomarkers on at least two samples from the same human subject
US11021537B2 (en) 2017-05-01 2021-06-01 Agenus Inc. Anti-TIGIT antibodies and methods of use thereof
US10865238B1 (en) 2017-05-05 2020-12-15 Duke University Complement factor H antibodies
JOP20190256A1 (ar) 2017-05-12 2019-10-28 Icahn School Med Mount Sinai فيروسات داء نيوكاسل واستخداماتها
WO2018218169A1 (fr) 2017-05-25 2018-11-29 Abbott Laboratories Procédés d'aide à la détermination de la réalisation ou non d'une imagerie sur un sujet humain ayant subi ou susceptible d'avoir subi une lésion à la tête à l'aide de biomarqueurs précoces
BR112019025387A2 (pt) 2017-05-30 2020-07-07 Abbott Laboratories métodos para auxiliar no diagnóstico e avaliação de uma lesão traumática cerebral branda em um indivíduo humano com o uso de troponina cardíaca i e biomarcadores precoces
EP3630834A1 (fr) 2017-05-31 2020-04-08 STCube & Co., Inc. Méthodes de traitement du cancer à l'aide d'anticorps et de molécules se liant de manière immunospécifique à btn1a1
KR20200015602A (ko) 2017-05-31 2020-02-12 주식회사 에스티큐브앤컴퍼니 Btn1a1에 면역특이적으로 결합하는 항체 및 분자 및 이의 치료적 용도
CN110997724A (zh) 2017-06-06 2020-04-10 斯特库伯株式会社 使用结合btn1a1或btn1a1-配体的抗体和分子治疗癌症的方法
GB201709379D0 (en) 2017-06-13 2017-07-26 Univ Leuven Kath Humanised ADAMTS13 binding antibodies
WO2019010131A1 (fr) 2017-07-03 2019-01-10 Abbott Laboratories Procédés améliorés de mesure de niveaux d'hydrolase à terminaison carboxy d'ubiquitine l1 dans le sang
CN111278858B (zh) 2017-07-11 2024-07-23 指南针制药有限责任公司 结合人cd137的激动剂抗体及其用途
EP3684811A2 (fr) 2017-08-17 2020-07-29 Massachusetts Institute of Technology Agents de liaison à spécificité multiple de chimiokines cxc et leurs utilisations
BR112020003533A2 (pt) 2017-08-25 2020-11-17 Five Prime Therapeutics, Inc. anticorpos b7-h4 e métodos de uso dos mesmos
US11707522B2 (en) 2017-10-13 2023-07-25 Boehringer Ingelheim International Gmbh Human antibodies to Tn antigen
US11718679B2 (en) 2017-10-31 2023-08-08 Compass Therapeutics Llc CD137 antibodies and PD-1 antagonists and uses thereof
CN111315772A (zh) 2017-10-31 2020-06-19 斯塔顿生物技术有限公司 抗apoc3抗体及其使用方法
WO2019089594A1 (fr) 2017-10-31 2019-05-09 Immunogen, Inc. Polythérapie avec des conjugués anticorps-médicament et de la cytarabine
CN111587123A (zh) 2017-11-09 2020-08-25 品通治疗有限公司 用于生成和使用人源化构象特异性磷酸化的τ抗体的方法和组合物
US11851497B2 (en) 2017-11-20 2023-12-26 Compass Therapeutics Llc CD137 antibodies and tumor antigen-targeting antibodies and uses thereof
JP7165193B2 (ja) 2017-11-27 2022-11-02 パーデュー、ファーマ、リミテッド、パートナーシップ ヒト組織因子を標的とするヒト化抗体
BR112019028254A2 (pt) 2017-12-09 2020-07-14 Abbott Laboratories métodos para ajudar no diagnóstico e avaliação de um paciente que sofreu uma lesão ortopédica e que sofreu ou pode ter sofrido uma lesão na cabeça, tal como uma lesão cerebral traumática (lct) leve, usando a proteína ácida fibrilar glial (gfap) e/ou a hidrolase carbóxi-terminal da ubiquitina l1 (uch-l1)
CA3067055A1 (fr) 2017-12-09 2019-06-13 Abbott Laboratories Procedes d'aide au diagnostic et a l'evaluation d'un lesion cerebrale traumatique chez un sujet humain au moyen d'une combinaison de gfap et d'uch-l1
GB201802486D0 (en) 2018-02-15 2018-04-04 Ucb Biopharma Sprl Methods
AU2019228600A1 (en) 2018-03-02 2020-09-24 Five Prime Therapeutics, Inc. B7-H4 antibodies and methods of use thereof
BR112020017701A2 (pt) 2018-03-12 2020-12-29 Zoetis Services Llc Anticorpos anti-ngf e métodos dos mesmos
IL301295A (en) 2018-03-14 2023-05-01 Surface Oncology Inc Antibodies that bind CD39 and uses thereof
CN112512571A (zh) 2018-03-22 2021-03-16 表面肿瘤学公司 抗il-27抗体及其用途
AU2019248547A1 (en) 2018-04-02 2020-09-10 Bristol-Myers Squibb Company Anti-TREM-1 antibodies and uses thereof
WO2019200357A1 (fr) 2018-04-12 2019-10-17 Surface Oncology, Inc. Biomarqueur pour agents thérapeutiques ciblant cd47 et leurs utilisations
EP3784274A1 (fr) 2018-04-27 2021-03-03 Fondazione Ebri Rita Levi-Montalcini Anticorps dirigé contre un peptide neurotoxique dérivé de tau et ses utilisations
JP7530638B2 (ja) 2018-05-10 2024-08-08 ニューラクル サイエンス カンパニー リミテッド 配列類似性１９、メンバーａ５抗体を有する抗ファミリー及びその使用方法
WO2019222130A1 (fr) 2018-05-15 2019-11-21 Immunogen, Inc. Traitement combiné avec des conjugués anticorps-médicament et des inhibiteurs flt3
WO2019226617A1 (fr) 2018-05-21 2019-11-28 Compass Therapeutics Llc Compositions et procédés pour améliorer la destruction de cellules cibles par des lymphocytes nk
WO2019226658A1 (fr) 2018-05-21 2019-11-28 Compass Therapeutics Llc Compositions multispécifiques de liaison à l'antigène et procédés d'utilisation
MX2020013808A (es) 2018-06-18 2021-05-27 UCB Biopharma SRL Antagonista de gremlina-1 para la prevencion y el tratamiento del cancer.
WO2019244107A1 (fr) 2018-06-21 2019-12-26 Daiichi Sankyo Company, Limited Compositions comprenant des fragments de liaison à l'antigène cd3 et leurs utilisations
WO2020003172A1 (fr) 2018-06-26 2020-01-02 Mor Research Applications Anticorps anti-transthyrétine et leurs utilisations
BR112021000934A2 (pt) 2018-07-20 2021-04-27 Pierre Fabre Medicament receptor para vista
EP3833442A2 (fr) 2018-08-09 2021-06-16 Compass Therapeutics LLC Anticorps qui se lient à cd277 et leurs utilisations
WO2020033925A2 (fr) 2018-08-09 2020-02-13 Compass Therapeutics Llc Anticorps qui se lient à cd277 et leurs utilisations
WO2020033923A1 (fr) 2018-08-09 2020-02-13 Compass Therapeutics Llc Agents de liaison à l'antigène qui se lient à cd277 et leurs utilisations
US20210380675A1 (en) 2018-09-28 2021-12-09 Kyowa Kirin Co., Ltd. Il-36 antibodies and uses thereof
MA53811A (fr) 2018-10-03 2022-03-16 Staten Biotechnology B V Anticorps spécifiques à l'apoc3 humaine et du cynomolgus et procédés pour leur utilisation
GB201817309D0 (en) 2018-10-24 2018-12-05 Ucb Biopharma Sprl Antibodies
GB201817311D0 (en) 2018-10-24 2018-12-05 Ucb Biopharma Sprl Antibodies
BR112021008795A2 (pt) 2018-11-13 2021-08-31 Compass Therapeutics Llc Construtos de ligação multiespecíficos contra moléculas de ponto de verificação e seus usos
US20220289857A1 (en) 2018-12-20 2022-09-15 Kyowa Kirin Co., Ltd. Fn14 antibodies and uses thereof
CN113474371A (zh) 2019-01-16 2021-10-01 指南针制药有限责任公司 与人cd137结合的抗体的制剂及其用途
GB201900732D0 (en) 2019-01-18 2019-03-06 Ucb Biopharma Sprl Antibodies
MX2021010003A (es) 2019-02-26 2021-12-10 Inspirna Inc Anticuerpos anti-mertk de alta afinidad y usos de los mismos.
WO2020185763A1 (fr) 2019-03-11 2020-09-17 Memorial Sloan Kettering Cancer Center Anticorps cd22 et leurs procédés d'utilisation
EP3947442A2 (fr) 2019-03-28 2022-02-09 Danisco US Inc. Anticorps modifiés
EP4013506A1 (fr) 2019-08-12 2022-06-22 Aptevo Research and Development LLC Protéines de liaison à 4-1bb et ox40 et compositions et méthodes associées, anticorps dirigés contre 4-1bb, anticorps dirigés contre ox40
WO2021042019A1 (fr) 2019-08-30 2021-03-04 Agenus Inc. Anticorps anti-cd96 et procédés d'utilisation de ces derniers
KR20220088847A (ko) 2019-09-04 2022-06-28 주식회사 와이바이오로직스 항-vsig4 항체 또는 항원-결합 단편 및 이들의 용도
TW202124444A (zh) 2019-09-16 2021-07-01 美商表面腫瘤學公司 抗ｃｄ３９抗體組合物及方法
JP2022549854A (ja) 2019-09-25 2022-11-29 サーフィス オンコロジー インコーポレイテッド 抗ｉｌ－２７抗体及びその使用
EP4041767A1 (fr) 2019-09-26 2022-08-17 StCube & Co. Anticorps spécifiques dirigés contre ctla-4 glycosylée et leurs procédés d'utilisation
WO2021072277A1 (fr) 2019-10-09 2021-04-15 Stcube & Co. Anticorps spécifiques de lag3 glycosylé et leurs procédés d'utilisation
US11459389B2 (en) 2019-10-24 2022-10-04 Massachusetts Institute Of Technology Monoclonal antibodies that bind human CD161
JP2023502968A (ja) 2019-11-15 2023-01-26 ユニバーシティ オブ テネシー リサーチ ファウンデーション アミロイド沈着物を標的化するための修飾免疫グロブリン
GB201917480D0 (en) 2019-11-29 2020-01-15 Univ Oxford Innovation Ltd Antibodies
GB201919061D0 (en) 2019-12-20 2020-02-05 Ucb Biopharma Sprl Multi-specific antibody
GB201919062D0 (en) 2019-12-20 2020-02-05 Ucb Biopharma Sprl Antibody
GB201919058D0 (en) 2019-12-20 2020-02-05 Ucb Biopharma Sprl Multi-specific antibodies
TW202138388A (zh) 2019-12-30 2021-10-16 美商西根公司 以非海藻糖苷化抗－ｃｄ７０抗體治療癌症之方法
IL294330A (en) 2020-01-06 2022-08-01 Vaccinex Inc Anti-ccr8 antibodies and their uses
CN115397848A (zh) 2020-02-05 2022-11-25 拉利玛生物医药公司 Tat肽结合蛋白及其用途
WO2021160268A1 (fr) 2020-02-13 2021-08-19 UCB Biopharma SRL Anticorps bispécifiques contre cd9
WO2021160267A1 (fr) 2020-02-13 2021-08-19 UCB Biopharma SRL Anticorps bispécifiques dirigés contre cd9 et cd7
EP4103610A1 (fr) 2020-02-13 2022-12-21 UCB Biopharma SRL Anticorps bispécifiques anti-cd44-ctla4
WO2021160265A1 (fr) 2020-02-13 2021-08-19 UCB Biopharma SRL Anticorps bispécifiques contre cd9 et cd137
US20230192900A1 (en) 2020-02-13 2023-06-22 UCB Biopharma SRL Bispecific antibodies binding hvem and cd9
CN115515615A (zh) 2020-02-18 2022-12-23 艾莱克特有限责任公司 Pilra抗体及其使用方法
KR20220151195A (ko) 2020-03-06 2022-11-14 오엔에이 테라퓨틱스 에스.엘. 항-cd36 항체 및 암을 치료하기 위한 이의 용도
KR20220167276A (ko) 2020-03-10 2022-12-20 매사추세츠 인스티튜트 오브 테크놀로지 NPM1c-양성 암의 면역치료를 위한 조성물 및 방법
US20230203191A1 (en) 2020-03-30 2023-06-29 Danisco Us Inc Engineered antibodies
EP4136459A1 (fr) 2020-04-13 2023-02-22 Abbott Laboratories Procédés, complexes et kits pour détecter ou déterminer une quantité d'un anticorps anti-ss-coronavirus dans un échantillon
AR122111A1 (es) 2020-05-17 2022-08-17 Astrazeneca Uk Ltd ANTICUERPOS CONTRA EL SARS-CoV-2 Y MÉTODOS DE SELECCIÓN Y USO DE LOS MISMOS
IL299757A (en) 2020-07-20 2023-03-01 Astrazeneca Uk Ltd SARS-COV-2 proteins, antibodies against SARS-COV-2, and methods of using them
WO2022031804A1 (fr) 2020-08-04 2022-02-10 Abbott Laboratories Méthodes et kits améliorés pour détecter une protéine sars-cov-2 dans un échantillon
KR20230048439A (ko) 2020-08-18 2023-04-11 세파론 엘엘씨 항-par-2 항체 및 이의 사용 방법
JP2023547795A (ja) 2020-10-15 2023-11-14 ユーシービー バイオファルマ エスアールエル Ｃｄ４５を多量体化する結合分子
EP4229081A1 (fr) 2020-10-15 2023-08-23 The United States of America, as represented by The Secretary, Department of Health and Human Services Anticorps spécifique du domaine de liaison du récepteur du sars-cov-2 et procédés thérapeutiques
WO2022087274A1 (fr) 2020-10-21 2022-04-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps qui neutralisent l'activité de l'interféron de type i (ifn)
WO2022119976A1 (fr) 2020-12-01 2022-06-09 Aptevo Research And Development Llc Anticorps hétérodimétriques bispécifiques se liant à cd3 et psma
WO2022119841A1 (fr) 2020-12-01 2022-06-09 Abbott Laboratories Utilisation d'un ou plusieurs biomarqueurs pour déterminer un traumatisme crânien (tbi) chez un sujet soumis à un balayage de tomodensitométrie assistée par ordinateur de la tête à tbi négatif
WO2023102384A1 (fr) 2021-11-30 2023-06-08 Abbott Laboratories Utilisation d'un ou de plusieurs biomarqueurs pour déterminer un traumatisme crânien (tbi) chez un sujet ayant été soumis à un balayage de tomodensitométrie assistée par ordinateur de la tête ne démontrant par de tbi
WO2022147147A1 (fr) 2020-12-30 2022-07-07 Abbott Laboratories Procédés pour déterminer un antigène sras-cov-2 et anticorps anti-sras-cov-2 dans un échantillon
AU2022207708A1 (en) 2021-01-13 2023-07-27 Daiichi Sankyo Company, Limited Antibody-pyrrolobenzodiazepine derivative conjugate
US20240115721A1 (en) 2021-01-13 2024-04-11 Memorial Sloan Kettering Cancer Center Anti-dll3 antibody-drug conjugate
US11639396B2 (en) 2021-02-16 2023-05-02 Janssen Biotech, Inc. Antibody binding to a linker peptide
EP4301786A1 (fr) 2021-03-03 2024-01-10 Pierre Fabre Medicament Anticorps anti-vsig4 ou fragment de liaison à l'antigène et ses utilisations
EP4067381A1 (fr) 2021-04-01 2022-10-05 Julius-Maximilians-Universität Würzburg Nouvelles molécules de liaison tnfr2
AU2022270170A1 (en) 2021-05-07 2023-09-21 Surface Oncology, LLC Anti-il-27 antibodies and uses thereof
WO2022245920A1 (fr) 2021-05-18 2022-11-24 Abbott Laboratories Procédés d'évaluation d'une lésion cérébrale chez un sujet en pédiatrie
EP4348263A1 (fr) 2021-05-28 2024-04-10 Alexion Pharmaceuticals, Inc. Procédés de détection de biomarqueurs cm-tma
CA3218481A1 (fr) 2021-06-14 2022-12-22 argenx BV Anticorps anti-il-9 et leurs procedes d'utilisation
JP2024521476A (ja) 2021-06-14 2024-05-31 アボット・ラボラトリーズ 音響エネルギー、電磁エネルギー、過圧波及び／又は爆風により引き起こされる脳損傷の診断法又は診断の一助となる方法
JP2024527551A (ja) 2021-06-29 2024-07-25 シージェン インコーポレイテッド 非フコシル化抗ｃｄ７０抗体及びｃｄ４７アンタゴニストの組み合わせを用いるがんを処置する方法
WO2023285878A1 (fr) 2021-07-13 2023-01-19 Aviation-Ophthalmology Procédés de détection, de traitement et de prévention de maladies, troubles et états oculaires induits par gpr68
IL309934A (en) 2021-07-30 2024-03-01 Ona Therapeutics S L Anti-CD36 antibodies and their use for cancer treatment
CA3230038A1 (fr) 2021-08-31 2023-03-09 Hongwei Zhang Methodes et systemes de diagnostic de lesion cerebrale
CN118715440A (zh) 2021-08-31 2024-09-27 雅培实验室 诊断脑损伤的方法和系统
CA3232176A1 (fr) 2021-09-30 2023-04-06 Beth MCQUISTON Methodes et systemes de diagnostic de lesion cerebrale
EP4177266A1 (fr) 2021-11-05 2023-05-10 Katholieke Universiteit Leuven Neutralisation d'anticorps humains anti-sars-cov-2
WO2023114978A1 (fr) 2021-12-17 2023-06-22 Abbott Laboratories Systèmes et procédés de détermination d'uch-l1, de gfap et d'autres biomarqueurs dans des échantillons de sang
AU2023216317A1 (en) 2022-02-04 2024-09-05 Abbott Laboratories Lateral flow methods, assays, and devices for detecting the presence or measuring the amount of ubiquitin carboxy-terminal hydrolase l1 and/or glial fibrillary acidic protein in a sample
TW202342510A (zh) 2022-02-18 2023-11-01 英商Ｒｑ生物科技有限公司 抗體
WO2023192436A1 (fr) 2022-03-31 2023-10-05 Alexion Pharmaceuticals, Inc. Dosage monoplex ou multiplex pour marqueurs de complément dans des échantillons biologiques frais
GB202205200D0 (en) 2022-04-08 2022-05-25 Ucb Biopharma Sprl Combination with chemotherapy
GB202205203D0 (en) 2022-04-08 2022-05-25 UCB Biopharma SRL Combination with inhibitor
WO2023209177A1 (fr) 2022-04-29 2023-11-02 Astrazeneca Uk Limited Anticorps contre le sars-cov-2 et leurs procédés d'utilisation
WO2023240124A1 (fr) 2022-06-07 2023-12-14 Regeneron Pharmaceuticals, Inc. Particules virales pseudotypées pour le ciblage de cellules exprimant un tcr
WO2024006876A1 (fr) 2022-06-29 2024-01-04 Abbott Laboratories Systèmes et analyses magnétiques hors laboratoire pour déterminer une gfap dans des échantillons biologiques
WO2024015953A1 (fr) 2022-07-15 2024-01-18 Danisco Us Inc. Procédés de production d'anticorps monoclonaux
WO2024013727A1 (fr) 2022-07-15 2024-01-18 Janssen Biotech, Inc. Matériau et procédés d'appariement amélioré par génie biologique de régions variables de liaison à l'antigène
WO2024050354A1 (fr) 2022-08-31 2024-03-07 Washington University Molécules de liaison à l'antigène d'alphavirus et leurs utilisations
WO2024050524A1 (fr) 2022-09-01 2024-03-07 University Of Georgia Research Foundation, Inc. Compositions et procédés de direction de l'apolipoprotéine l1 en vue d'induire la mort de cellules de mammifère
WO2024054436A1 (fr) 2022-09-06 2024-03-14 Alexion Pharmaceuticals, Inc. Profils de biomarqueurs de diagnostic et de pronostic chez des patients ayant une microangiopathie thrombotique associée à une greffe de cellules souches hématopoïétiques (hsct-tma)
WO2024059708A1 (fr) 2022-09-15 2024-03-21 Abbott Laboratories Biomarqueurs et méthodes de différenciation entre une lésion cérébrale traumatique légère et très légère
WO2024194685A2 (fr) 2023-03-17 2024-09-26 Oxitope Pharma B.V. Anticorps anti-phosphocholine et leurs procédés d'utilisation
WO2024194686A2 (fr) 2023-03-17 2024-09-26 Oxitope Pharma B.V. Anticorps anti-phosphocholine et leurs procédés d'utilisation
WO2024211475A1 (fr) 2023-04-04 2024-10-10 Abbott Laboratories Utilisation de biomarqueurs pour détermination ou suspicion d'une lésion cérébrale acquise subaiguë chez un sujet

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9515982A3 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101563366B (zh) * 2006-10-19 2012-10-03 健泰科生物技术公司 抗notch3激动性抗体及其在制备治疗notch3相关疾病的药物中的用途

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