WO2005003174A1 - Element de liaison a une proteine a d'adhesine de surface pneumocoque - Google Patents

Element de liaison a une proteine a d'adhesine de surface pneumocoque Download PDF

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Publication number
WO2005003174A1
WO2005003174A1 PCT/DK2004/000492 DK2004000492W WO2005003174A1 WO 2005003174 A1 WO2005003174 A1 WO 2005003174A1 DK 2004000492 W DK2004000492 W DK 2004000492W WO 2005003174 A1 WO2005003174 A1 WO 2005003174A1
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Prior art keywords
binding
binding member
seq
antibody
psaa
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PCT/DK2004/000492
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English (en)
Inventor
Anders Per SØRENSEN
Thomas Lars Benfield
Jens Dilling Lundgren
Thomas D. Kempe
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Genesto A/S
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Priority to JP2006517971A priority Critical patent/JP2007527703A/ja
Priority to US10/563,976 priority patent/US20070003561A1/en
Priority to EP04738989A priority patent/EP1646654A1/fr
Publication of WO2005003174A1 publication Critical patent/WO2005003174A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a binding member comprising at least one binding domain capable of specifically binding Streptococcus pneumoniae surface adhesin A (PsaA) protein, in particular to a binding member having at least two binding domains, to the use of said binding members in diagnostic methods as well as for treatment.
  • PsaA Streptococcus pneumoniae surface adhesin A
  • Streptococcus pneumoniae is one of the leading causes of life-threatening bacterial infection. In developing countries it has been estimated that several million children under 5 years of age will die of S. pneumoniae each year (3). In the industrialized world, the incidence of S. pneumoniae pneumonia is 5-10 per 100.000 persons and the case-fatality rate is 5-7%. S. pneumoniae meningitis occurs in 1-2 per 100.000 persons with a case-fatality of 30-40% (14). S. pneumoniae is one of the most frequent causes of bacteremia. S. pneumoniae is the most frequent organism isolated from children with otitis media. App. 75% of all children less than 6 years old will suffer from otitis media.
  • S. pneumoniae is a gram-positive bacteria that grows in pairs or short chains.
  • the surface is composed of three layers: capsule, cell wall and plasma membrane.
  • the capsule is the thickest layer and completely conceals the inner structures of growing S. pneumoniae.
  • Different serotypes contain ribitol, arabitinol or phosphoryl- choline as part of their capsule, resulting in chemical structures that are serotype specific.
  • the cell wall consists of peptidoglycan but also teichoic acid and lipotei- choic acid.
  • the plasma membrane is a double phospholipid membrane that encompasses the cell and anchors various molecules to its surface (2).
  • S. pneumoniae At present 90 different types of S. pneumoniae are recognized based on the diversity of the S. pneumoniae capsule (26).
  • the capsule is pivotal in the pathogenesis of S. pneumoniae infections.
  • Antibodies raised against one capsular type offers protection from infection with this type but not against infection with other capsular types.
  • the current 23-valent polysaccharide vaccine offers protection from more than 60-85% of the most frequent serotypes.
  • the cell wall contains two polysaccarides, C-polysaccharide (C-PS) (teichoic acid and peptidoglycan) and F-antigen (lipoteichoic acid, Forssman antigen) (26).
  • C-PS C-polysaccharide
  • F-antigen Lipoteichoic acid, Forssman antigen
  • Other bacteria than S. pneumoniae contain C-PS, e.g. alpha-streptococci (12).
  • F-antigen cross-reacts with streptococcal group C polysaccharide (29).
  • Several antibodies to capsular polysaccharides cross-react with C-PS presumably because these are covalently linked.
  • the protective role of anti-C-PS antibodies is controversial since some studies find them protective in mice (6) and others not (20;28).
  • C-PS capsular concealment of C-PS.
  • Antibodies to C-PS may protect hosts infected with acapsular strains or bind to decaying S. pneumoniae that shed their capsule. Immunization of mice with the F-antigen does not protect against S. pneumoniae infection (4).
  • IgA to PsaA is detectable in saliva from children less than two years (193 of 261) and adults (17 of 17) (34).
  • Anti-PsaA IgG was detectable by EIA in most children less than two years (872 of 1108) and most adults (262/325) (35).
  • Seroconversion was correlated to carrier status, i.e. children who had had with S. pneumoniae cultured from nasopharyngeal or middle ear specimens were more likely to be anti- PsaA IgG positive. Summary
  • the present invention relates to a binding member comprising at least one binding domain capable of specifically binding Streptococcus pneumoniae surface adhesin
  • a (PsaA) protein wherein the binding member is suitable for use in a pharmaceutical composition for preventing and treating diseases and disorders related to Streptococcus, in particular Streptococcus pneumoniae.
  • the invention in another aspect relates to an isolated binding member comprising at least a first binding domain and a second binding domain, said first binding domain being capable of specifically binding Streptococcus pneumoniae surface adhesin A (PsaA) protein.
  • PsaA Streptococcus pneumoniae surface adhesin A
  • the invention further relates to a kit comprising at least one binding member as defined above, wherein said binding member is labelled, for use in a diagnostic method.
  • the invention relates to the use of a binding member as defined above for the production of a pharmaceutical composition for the treatment or prophylaxis of disorders or diseases associated with Streptococcus pneumoniae, such as pneumonia, meningitis and/or sepsis.
  • the invention relates to a method for treating or preventing an individual suffering from disorders or diseases associated with Streptococcus pneumoniae, such as pneumonia, meningitis and/or sepsis by administering an effective amount of a binding member as defined above.
  • Affinity the strength of binding between receptors and their ligands, for example between an antibody and its antigen.
  • Avidity The functional combining strength of an antibody with its antigen which is related to both the affinity of the reaction between the epitopes and paratopes, and the valencies of the antibody and antigen
  • Amino Acid Residue An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • Antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules of the compositions of this invention, i.e., molecules that contain an antibody combining site or paratope.
  • Exemplary antibody molecules are intact immuno- globulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab') 2 and Fv.
  • a schematic drawing of Fab is shown in Figure 1.
  • antibody as used herein is also intended to include human, single chain and humanized antibodies, as well as binding fragments of such antibodies or modified ver- sions of such antibodies, such as multispecific, bispecific and chimeric molecules having at least one antigen binding determinant derived from an antibody molecule.
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; lgA-1 and lgA-2.
  • the heavy chains constant domains that correspond to the different classes. of immunoglobulins are called alpha ( ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
  • An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen.
  • the term immunore- act in its various forms means specific binding between an antigenic determinant- containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.
  • an antibody combining site is known as an antigen binding site.
  • Binding domain An antigen binding site which specifically binds an antigen.
  • a binding member may be multispecific and contain two or more binding domains which specifically bind two immunologically distinct antigens.
  • Complementary Bases Nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
  • Complementarity determining region or CDR Regions in the V-domains of an antibody that together form the antibody recognizing and binding domain.
  • Complementary Nucleotide Sequence A sequence of nucleotides in a single- stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize to it with consequent hydrogen bonding.
  • a nucleotide sequence is conserved with respect to a preselected (ref- erence) sequence if it non-randomly hybridizes to an exact complement of the preselected sequence.
  • Constant Region or constant domain or C-domain Constant regions are those structural portions of an antibody molecule comprising amino acid residue se- quences within a given isotype which may contain conservative substitutions therein.
  • Exemplary heavy chain immunoglobulin constant regions are those portions of an immunoglobulin molecule known in the art as CH1 , CH2, CH3, CH4 and CH5.
  • An exemplary light chain immunoglobulin constant region is that portion of an immunoglobulin molecule known in the art as C L .
  • Diabodies This term refers to a small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Kd Dissociation constant
  • Downstream Further along a DNA sequence in the direction of sequence transcription or read out, that is travelling in a 3'- to 5'-direction along the non-coding strand of the DNA or 5'- to 3'-direction along the RNA transcript.
  • Duplex DNA A double-stranded nucleic acid molecule comprising two strands of substantially complementary polynucleotides held together by one or more hydrogen bonds between each of the complementary bases present in a base pair of the duplex. Because the nucleotides that form a base pair can be either a ribonucleotide base or a deoxyribonucleotide base, the phrase "duplex DNA” refers to either a DNA-DNA duplex comprising two DNA strands (ds DNA), or an RNA-DNA duplex comprising one DNA and one RNA strand.
  • Fusion Polypeptide A polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous polypeptide.
  • the two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.
  • Gene A nucleic acid whose nucleotide sequence codes for an RNA or polypeptide.
  • a gene can be either RNA or DNA.
  • Human antibody framework A molecule having an antigen binding site and essentially all remaining immunoglobulin-derived parts of the molecule derived from a hu- man immunoglobulin.
  • Immunoglobulin The serum antibodies, including IgG, IgM, IgA, IgE and IgD.
  • Immunologically distinct refers to the ability to distinguish between two polypeptides on the ability of an antibody to specifically bind one of the polypeptides and not specifically bind the other polypeptide.
  • the subject is an organism possessing leukocytes capable of responding to antigenic stimulation and growth factor stimula- tion.
  • the subject is a mammal, including humans and non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice.
  • the subject is a human.
  • Infectious disease a disorder caused by one or more species of Streptococcus, in particular Streptococcus pneumoniae.
  • Monoclonal Antibody in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a differ- ent antigen, e.g., a bispecific monoclonal antibody.
  • Multimeric A polypeptide molecule comprising more than one polypeptide.
  • a mul- timer may be dimeric and contain two polypeptides and a multimer may be trimeric and contain three polypeptides.
  • Multimers may be homomeric and contain two or more identical polypeptides or a multimer may be heteromeric and contain two or more nonidentical polypeptides.
  • Nucleic Acid A polymer of nucleotides, either single or double stranded.
  • Nucleotide A monomeric unit of DNA or RNA consisting of a sugar moiety (pen- tose), a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide.
  • a sequence of operatively linked nucleotides is typically referred to herein as a
  • base sequence or “nucleotide sequence”, and their grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5'-terminus to 3'-terminus.
  • Nucleotide Analog A purine or pyrimidine nucleotide that differs structurally from A, T, G, C, or U, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
  • Pneumococcus is used synonymously with Streptococcus pneumoniae.
  • Polyclonal antibody is a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen.
  • Polynucleotide A polymer of single or double stranded nucleotides. As used herein
  • polynucleotide and its grammatical equivalents will include the full range of nucleic acids.
  • a polynucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art.
  • the polynucleotides of the present inven- tion include primers, probes, RNA/DNA segments, oligonucleotides or "oligos" (relatively short polynucleotides), genes, vectors, plasmids, and the like.
  • Polypeptide refers to a molecule comprising amino acid residues which do not contain linkages other than amide linkages between adjacent amino acid residues.
  • Recombinant DNA (rDNA) molecule A DNA molecule produced by operatively linking two DNA segments.
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.
  • rDNA's not having a common biological origin, i.e., evolutionarily different, are said to be "heterologous”.
  • Specificity refers to the number of potential antigen binding sites which immunoreact with (specifically bind to) a given antigen in a polypeptide.
  • the polypeptide may be a single polypeptide or may be two or more polypeptides joined by disulfide bonding.
  • a polypeptide may be monospecific and contain one or more antigen binding sites which specifically bind an antigen or a polypeptide may be bispecific and contain two or more antigen binding sites which specifically bind two immunologically distinct antigens.
  • a polypeptide may contain a plurality of antigen binding sites which specifically bind the same or different antigens.
  • Serotype Identification of bacteria within species of Streptococcus, that consist of many strains differing from one another in a variety of characteristics. Commonly used characteristics defining serotypes are particular antigenic molecules.
  • Single Chain Antibody or scFv refers to a single polypeptide comprising one or more antigen binding sites.
  • H and L chains of an Fv fragment are encoded by separate genes, they may be linked either directly or via a peptide, for example a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain anti- body, sAb; Bird et al. 1988 Science 242:423-426; and Huston et al. 1988 PNAS
  • antibody single chain antibodies are also encompassed within the term "antibody”, and may be utilized as binding determinants in the design and engineering of a multispecific binding molecule.
  • Upstream In the direction opposite to the direction of DNA transcription, and therefore going from 5' to 3' on the non-coding strand, or 3' to 5' on the mRNA.
  • Valency refers to the number of potential antigen binding sites, i.e. binding domains, in a polypeptide.
  • a polypeptide may be monovalent and con- tain one antigen binding site or a polypeptide may be bivalent and contain two antigen binding sites. Additionally, a polypeptide may be tetravalent and contain four antigen binding sites. Each antigen binding site specifically binds one antigen. When a polypeptide comprises more than one antigen binding site, each antigen binding site may specifically bind the same or different antigens. Thus, a polypeptide may contain a plurality of antigen binding sites and therefore be multivalent and a polypeptide may specifically bind the same or different antigens.
  • V-domain Variable domain are those structural portions of an antibody molecule comprising amino acid residue sequences forming the antigen binding sites.
  • An ex- emplary light chain immunoglobulin variable region is that portion of an immunoglobulin molecule known in the art as V L .
  • Vector A rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as "expression vectors".
  • Particularly important vectors allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.
  • the present invention relates to binding members, in particular antibodies or fragments thereof capable of specifically recognising and binding to a Streptococcus pneumoniae protein, more specifically to Pneumococcus surface adhesin A protein, PsaA.
  • the binding members according to the invention are particularly useful in the treatment of diseases caused by Streptococcus pneumoniae, as well as for being employed in diagnostic methods and kits for detecting the bacteria.
  • the Penumococcus surface adhesin A protein is preferably a polypeptide having the amino acid sequence shown Figure 15 (SEQ ID NO: 50) or SEQ ID NO: 56..
  • the binding member according to the invention should preferably be immunologically active, for example as an antibody, such as being capable of binding to an antigen and presenting the antigen to immunoactive cells, thereby facilitating phagocytosis of said antigen.
  • the binding member is an antibody, such as any suitable antibody known in the art, in partucular antibodies as defined herein, such as antibodies or immunologically active fragments of antibodies, or single chain antibodies.
  • Antibody molecules are typically Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains.
  • the C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains.
  • the N-terminal regions, also known as V-domains are variable in sequence and are responsible for the antibody specificity.
  • the antibody specifically recognizes and binds to an antigen mainly through six short complementarity-determining regions located in their V-domains (see Fig. 1).
  • the antibodies according to the invention are especially useful, since they have a strong affinity towards the PsaA.
  • the binding members according to the invention have a binding domain having a dissociation constant K d for PsaA which is less than 1 x 10 "6 M. More preferably the dissociation constant K d for PsaA which is less than 1 x 10 "7 M, more pref- erably less than 1 x 10 "8 M, more preferably less than 5 x 10 "8 M, more preferably less than 1 x 10 "9 M, more preferably less than 5 x 10 "9 M more preferably less than 1 x 10 "10 M.
  • the binding member is preferably an isolated binding member as defined above, and more preferably an isolated, pure binding member.
  • the high binding strength is caused by incorporating into the binding domain an amino acid sequence having one or more of the following motifs of the sequences shown below.
  • binding domain preferably comprises a CDR1 region comprising a sequence selected from
  • SEQ ID NO 4 CDR2 OF AMINO ACID SEQUENCE IN FlG. 16A
  • SEQ ID NO 12 CDR2 OF AMINO ACID SEQUENCE IN FlG. 16B
  • SEQ ID NO 20 CDR2 OF AMINO ACID SEQUENCE IN FIG. 17A
  • binding domain preferably comprises a CDR3 region comprising a sequence selected from
  • variable part of the binding domain comprises a sequence selected from
  • SEQ ID NO 32 AMINO ACID SEQUENCE IN FlG. 17B
  • SEQ ID NO 40 AMINO ACID SEQUENCE IN FlG. 18A
  • SEQ ID NO 48 AMINO ACID SEQUENCE IN FlG. 18B or a homologue thereof, wherein a homologue is as defined elsewhere herein.
  • identity shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequen- ce identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madi- son, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
  • sequence analysis software e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madi- son, Wis. 53705
  • a homologue of one or more of the sequences specified herein may vary in one or more amino acids as compared to the sequences defined, but is capable of per- forming the same function, i.e. a homologue may be envisaged as a functional equivalent of a predetermined sequence.
  • a homologue of any of the predetermined sequences herein may be defined as:
  • homologues comprising an amino acid sequence capable of recognising an antigen also being recognised by the predetermined amino acid sequence, and/or
  • homologues comprising an amino acid sequence capable of binding selec- tively to an antigen, wherein said antigen is also bound selectively by a predetermined sequence, and/or
  • homologues having a substantially similar or higher binding affinity to PsaA as a binding domain comprising a predetermined sequence, such as SEQ ID NO 8.
  • homologues comprises one or more conservative amino acid substitutions including one or more conservative amino acid substitutions within the same group of predetermined amino acids, or a plurality of conservative amino acid sub- stitutions, wherein each conservative substitution is generated by substitution within a different group of predetermined amino acids.
  • Homologues may thus comprise conservative substitutions independently of one another, wherein at least one glycine (Gly) of said homologue is substituted with an amino acid selected from the group of amino acids consisting of Ala, Val, Leu, and lie, and independently thereof, homologues, wherein at least one of said alanines (Ala) of said homologue thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Val, Leu, and lie, and independently thereof, homologues, wherein at least one valine (Val) of said homologue thereof is substi- tuted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Leu, and lie, and independently thereof, homologues thereof, wherein at least one of said leucines (Leu) of said homologue thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and lie, and independently thereof, homologues thereof, wherein at least one isoleucine (lie) of said homologue
  • Conservative substitutions may be introduced in any position of a preferred prede- termined sequence. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.
  • a non-conservative substitution leading to the formation of a functionally equivalent homologueof the sequences herein would for example i) differ substantially in polar- ity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, lie, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gin or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ sub- stantialiy in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric
  • Substitution of amino acids may in one embodiment be made based upon their hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.
  • Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the binding domain comprises a homologue having an amino acid sequence at least 60 % homologous to a sequence selected from SEQ ID NO 2.
  • SEQ ID NO 4 amino acid sequence at least 60 % homologous to a sequence selected from SEQ ID NO 2.
  • SEQ ID NO 4 amino acid sequence at least 60 % homologous to a sequence selected from SEQ ID NO 2.
  • SEQ ID NO 6 amino acid sequence at least 60 % homologous to a sequence selected from SEQ ID NO 8.
  • the homology is at least 65 %, such as at least 70 % homologous, such as at least 75 % homologous, such as at least 80 % homologous, such as at least 85 % homologous, such as at least 90 % homologous, such as at least 95 % homologous, such as at least 98 % homologous to a sequence selected from SEQ ID NO 2.
  • SEQ ID NO 4 SEQ ID NO 6 and SEQ ID NO 8.
  • the percentages mentioned above relates to the identity of the sequence of a homologue as compared to a sequence selected from SEQ ID NO 2.
  • SEQ ID NO 4 SEQ ID NO 6 and SEQ ID NO 8.
  • the antibodies according to the present invention preferably recognize and bind to an epitope localised in the N-terminal part of PsaA corresponding to N-terminal amino acid residues 1-150 of SEQ ID NO 50 or SEQ ID NO: 56.
  • the epitope is localised in the N-terminal part of PsaA corresponding to N-terminal amino acid residues 1-100 of SEQ ID NO 50 or SEQ ID NO: 56, more preferably in the N- terminal part of PsaA corresponding to N-terminal amino acid residues 1-65 of SEQ ID NO 50 or SEQ ID NO: 56.
  • the antibody recognizes and binds to a fragment selected from the group of fragments having a sequence corresponding to SEQ ID NO 51 , SEQ ID NO 52 or SEQ ID NO 53.
  • the antibody according to the invention recognizes and binds to an epitope wherein said epitope is also recognized by an antibody having a variable part comprising a sequence selected from the group of
  • SEQ ID NO 8 AMINO ACID SEQUENCE IN FlG. 16A
  • SEQ ID NO 16 AMINO ACID SEQUENCE IN FlG. 16B
  • SEQ ID NO 24 AMINO ACID SEQUENCE IN FlG. 17A
  • SEQ ID NO 32 AMINO ACID SEQUENCE IN FlG. 17B
  • SEQ ID NO 40 AMINO ACID SEQUENCE IN FlG. 18A
  • SEQ ID NO 48 AMINO ACID SEQUENCE IN FlG. 18B
  • the binding member according to this invention is capable of binding PsaA from two or more different Pneumococcus serotypes, such as from three or more different Pneumococcus serotypes, such as from four or more different Pneumococcus serotypes, such as from five or more different Pneumococcus serotypes. Most preferably the binding member according to the invention is capable of recognising and binding Pneumococcus from essentially all serotypes. Monoclonal/polyclonal antibodies
  • the binding member is an antibody, wherein the antibody may be a polyclonal or a monoclonal antibody derived from a mammal or mixtures of monoclonal antibodies. In a preferred embodiment the binding member is a monoclonal antibody or a fragment thereof.
  • the antibody may be any kind of antibody, however it is preferably a IgG antibody. More preferably the antibody is a lgG1 antibody or a fragment thereof.
  • Monoclonal antibodies are antibodies, wherein every antibody molecule is similar and thus recognises the same epitope. Monoclonal antibodies are in general produced by a hybridoma cell line. Methods of making monoclonal antibodies and antibody-synthesizing hybridoma cells are well known to those skilled in the art. Antibody-producing hybridomas may for example be prepared by fusion of an anti- body-producing B lymphocyte with an immortalized cell line.
  • a monoclonal antibody can be produced by the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as described above for preparation of a polyclonal antibody. The immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response.
  • an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response.
  • the mammal is a rodent such as a rabbit, rat or mouse.
  • the mammal is then maintained on a booster schedule for a time period sufficient for the mammal to generate high affinity antibody molecules as described.
  • a suspension of antibody-producing cells is removed from each immunized mammal secreting the desired antibody.
  • the animal e.g., mouse
  • antibody-producing lymphocytes are obtained from one or more of the lymph nodes, spleens and peripheral blood.
  • Spleen cells are preferred, and can be mechanically separated into individual cells in a physiological medium using methods well known to one of skill in the art.
  • the antibody-producing cells are immortalized by fusion to cells of a mouse myeloma line.
  • Mouse lymphocytes give a high percentage of stable fusions with mouse homologous myelomas, however rat, rabbit and frog somatic cells can also be used.
  • Spleen cells of the desired antibody-producing animals are immortalized by fusing with myeloma cells, generally in the presence of a fusing agent such as polyethyl- ene glycol.
  • a fusing agent such as polyethyl- ene glycol.
  • Any of a number of myeloma cell lines suitable as a fusion partner are used with to standard techniques, for example, the P3-NS1/1-Ag4-1 , P3-x63- Ag8.653 or Sp2/O-Ag14 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, Md.
  • Monoclonal antibodies can also be generated by other methods well known to those skilled in the art of recombinant DNA technology.
  • An alternative method referred to as the "combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies.
  • Polyclonal antibodies is a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen.
  • polyclonal antibodies are purified from serum of a mammal, which previously has been immunized with the antigen.
  • Polyclonal antibodies may for example be prepared by any of the methods described in Antibodies: A Laboratory Manual, By Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, 1988.
  • Polyclonal antibodies may be derived from any suitable mammalian species, for example from mice, rats, rabbits, donkeys, goats, and sheep.
  • the monospecific binding member may be monovalent, i.e. having only one binding domain.
  • the immunoglobulin constant domain amino acid residue sequences comprise the structural portions of an antibody molecule known in the art as CH1 , CH2, CH3 and CH4. Preferred are those binding members which are known in the art as _C .
  • Preferrred C L polypeptides are selected from the group consisting Of Ckappa. and C
  • the constant domain can be either a heavy or light chain constant domain (C H or C L , respectively)
  • a variety of monovalent binding member compositions are contemplated by the present invention.
  • light chain constant domains are capable of disulfide bridging to either another light chain constant domain, or to a heavy chain constant domain.
  • a heavy chain constant domain can form two independent disulfide bridges, allowing for the possibility of bridging to both another heavy chain and to a light chain, or to form polymers of heavy chains.
  • the invention contemplates a composition comprising a monovalent polypeptide wherein the constant chain domain C has a cysteine resi- due capable of forming at least one disulfide bridge, and where the composition comprises at least two monovalent polypeptides covalently linked by said disulfide bridge.
  • the constant chain domain C can be either CL or CH.
  • C is C
  • the CL polypeptide is preferably selected from the group consisting of
  • the invention contemplates a binding member composition comprising a monovalent polypeptide as above except where C is CL having a cys- teine residue capable of forming a disulfide bridge, such that the composition contains two monovalent polypeptides covalently linked by said disulfide bridge.
  • the binding member is a multivalent binding member having at least two binding domains.
  • the binding domains may have specificity for the same ligand or for different ligands. Multispecificity, including bispecificity
  • the instant invention relates to multispecific binding members, which have affinity for and are capable of binding at least two different entities.
  • Multispecific binding members can include bispecific binding members.
  • the multispecific molecule is a bispecific antibody (BsAb), which carries at least two different binding domains, at least one of which is of antibody origin.
  • BsAb bispecific antibody
  • the V H is linked to a (the corresponding) VL .
  • the multispecific molecules described above can be made by a number of methods. For example, all specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the multi- specific molecule is a mAb X mAb, mAb X Fab, Fab X F(ab') 2 or ligand X Fab fusion protein.
  • the invention offers several advantages as compared to monospecific/monovalent binding members.
  • the at least one other binding domain may be used for spe- cifically binding a mammalian cell, such as a human cell. It is preferred that the at least other binding domain is capable of binding an immunoactive cell, such as a leucocyte, a macrophage, a lymphocyte, a basophilic cell, and/or an eosinophilic cell, in order to increase the effect of the binding member in a therapeutic method. This may be accomplished by establishing that the at least one other binding do- main is capable of specifically binding a mammalian protein, such as a human protein, such as a protein selected from any of the cluster differentiation proteins (CD), in particular CD64 and/or CD89.
  • CD cluster differentiation proteins
  • Humanised antibodies are in general chimeric antibodies comprising regions derived from a human antibody and regions derived from a non-human antibody, such as a rodent antibody.
  • Humanisation also called Reshaping or CDR-grafting
  • mAbs monoclonal antibodies
  • xenogeneic sources commonly rodent
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • One method for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody, preferably a human antibody.
  • Methods for carrying out such chimerisation procedures are for example described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023 (Genen- tech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University) and EP-A-0 194 276 (Celltech Limited).
  • a more complex form of humanisa- tion of an antibody involves the re-design of the variable region domain so that the amino acids constituting the non-human antibody binding site are integrated into the framework of a human antibody variable region (Jones et al., 1986).
  • the humanized antibody of the present invention may be made by any method capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized , antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly incorporated by reference.
  • the human CDRs may be replaced with non-human CDRs using oligonucleotide site-directed mutagenesis as described in the examples below.
  • humanized antibody of the present invention may be made as described in the brief explanation below.
  • the humanized antibodies of the present invention may be produced by the following process:
  • the host cell may be cotransfected with the two vectors of the invention, the first vector containing an operon encoding a light chain derived polypeptide and the second vector containing an operon encoding a heavy chain derived polypeptide.
  • the two vectors contain different selectable markers, but otherwise, apart from the antibody heavy and light chain coding sequences, are preferably identical, to ensure, as far as possible, equal expression of the heavy and light chain polypeptides.
  • a single vector may be used, the vector including the sequences encoding both the light and the heavy chain polypeptides.
  • the coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both.
  • the host cell used to express the altered antibody of the invention may be either a bacterial cell such as Escherichia coli, or a eukaryotic cell.
  • a mammalian cell of a well defined type for this purpose such as a myeloma cell or a Chinese hamster ovary cell may be used.
  • transfection methods required to produce the host cell of the invention and culture methods required to produce the antibody of the invention from such host cells are all conventional techniques.
  • the humanized antibodies of the invention may be purified according to standard procedures as described below.
  • Human mAb antibodies directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al.
  • transgenic mice are available from Abgenix, Inc., Fremont, Calif., and
  • the antibodies are produced by the method described in Example 1.
  • the binding member is a fragment of an antibody, preferably an antigen binding fragment or a variable region.
  • anti- body fragments useful with the present invention include Fab, Fab', F(ab') 2 and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc 1 ).
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • the antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site.
  • Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the VH of an Fv (Cumber et al., 1992), or at the C-terminus of the VL of an scFv (Pack and Pluck- thun, 1992), or through the hinge cysteine residues of Fab's (Carter et al., 1992).
  • Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor.
  • Some preferred fragments are defined as follows: (1) Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule.
  • a Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • Fab' is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. Fab' fragments differ from Fab fragments by the addition of a few resi- dues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • (Fab') 2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction.
  • F(ab') 2 is a di- mer of two Fab' fragments held together by two disulfide bonds.
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the antibody is a single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable poly- peptide linker as a genetically fused single chain molecule.
  • SCA single chain antibody
  • Such single chain antibodies are also refered to as "single-chain Fv” or “sFv” antibody fragments.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • the antibody fragments according to the invention may be produced in any suitable manner known to the person skilled in the art.
  • microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli (Better et al., 1988, Skerra and Pluckthun, 1988, Carter et al., 1992), yeast (Horwitz et al., 1988), and the filamentous fungus Trichoderma reesei (Nyyssonen et al., 1993) has been described.
  • the recombinant protein yields in these alternative systems can be relatively high (1-2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation, see Carter et al., 1992), or at a lower level, e.g.
  • the fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a VH and a VL can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv.
  • the invention relates to an isolated nucleic acid molecule encoding at least a part of the binding member as defined above.
  • the nucleic acid molecule encodes a light chain and another nucleic acid encodes a heavy chain.
  • the two nucleic acid molecule may be separate or they may be fused into one nucleic acid molecule, optionally spaced apart by a linker sequence.
  • the nucleic acid molecule may encode the whole binding member, however dependant on the design of the binding member this may also be relevant for some larger binding members.
  • the nucleic acid molecule preferably is a DNA sequence, more preferably a DNA sequence comprising in its up- stream end regulatory elements promoting the expression of the binding member once the nucleic acid molecule is arranged in a host cell.
  • the invention relates to a polynucleotide selected from the group consisting of i) a polynucleotide comprising a sequence selected from the nucleotide sequence of Figure 16a, Figure 16b, Figure 17a, Figure 17b, Figure 18a, and Figure 18b, ii) a polynucleotide encoding a binding member comprising one or more of the amino acid sequence selected from the group of Figure 16a, Figure 16b, Figure 17a, Figure 17b, Figure 18a, and Figure 18b, iii) a polynucleotide encoding a fragment of a polypeptide encoded by poly- nucleotides i), wherein said fragment a) is capable of recognising an antigen also being recognised by the binding member of ii), and/or b) is capable of binding selectively to an antigen, wherein said antigen is also bound selectively by by the binding member of ii),, and/or c) has a substantially
  • the invention further relates to a vector comprising the nucleic acid molecule as defined above, either one vector per nucleic acid, or two or more nucleic acids in the same vector.
  • the vector preferably comprises a nucleotide sequence which regu- lates the expression of the antibody encoded by the nucleic acid molecule.
  • the invention relates to a host cell comprising the nucleic acid molecule as defined above.
  • the invention relates to a cell line engineered to express the binding member as defined above, this cell line for example being a hybridoma of a murine lymphocyte and an immortalised cell line.
  • the cell line may be any suitable cell line, however the cell line P3 is preferred. In another embodiment a CHO cell line is preferred.
  • the binding members according to the invention are preferably purified.
  • the method of purification used is dependent upon several factors including the purity required, the source of the antibody, the intended use for the antibody, the species in which the antibody was produced, the class of the antibody and, when the antibody is a monoclonal antibody, the subclass of the antibody.
  • Any suitable conventional methods of purifying polypeptides comprising antibodies include precipitation and column chromatography and are well known to one of skill in the purification arts, including cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography, gel electrophoresis and the like may be used.
  • the eluant from the first anti-immunoglobulin antibody is then specifically bound to a second anti- immunoglobulin antibody.
  • the non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted.
  • the antibody is sequentially purified by a first and second anti- immunoglobulin antibody selected from the group consisting of antibodies which specifically bind heavy and light chain constant regions.
  • affinity chromatography in which the antibody to be purified is bound by protein A, protein G or by an anti-immunoglobulin antibody.
  • Another method of affinity chromatography which is well known to those of skill in the art, is the specific binding of the antibody to its respective antigen.
  • a bispecific antibody comprising two or more variable regions is purified by sequential purification by specifically binding the antibody to a first antigen in a first purification step and to a second antigen in a second purification step.
  • the method of purifying an antibody with an anti-immunoglobulin antibody can be either a single purification procedure or a sequential purification procedure. Methods of single and sequential purification are well known to those in the purification arts.
  • a single-step purification procedure the antibody is specifically bound by a single anti-immunoglobulin antibody. Non-specifically bound molecules are removed in a wash step and the specifically bound molecules are specifically eluted.
  • a sequential purification procedure the antibody is specifically bound to a first anti- immunoglobulin antibody, non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted.
  • the antibody is sequentially purified by a first and second anti- immunoglobulin antibody selected from the group consisting of antibodies which specifically bind heavy and light chain constant regions.
  • the antibody is sequentially purified by a first and second anti-immunoglobulin antibody selected from the group consisting of antibodies which specifically bind the heavy chain constant region of IgG and light chain constant regions of kappa and lambda.
  • the anti-immunoglobulin antibody is selected from the group consisting of antibodies which specifically bind the light chain constant regions of kappa and lambda.
  • the present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of Streptococcus, in particular Streptococcus pneumoniae, in a biological sample where it is desirable to detect the presence, and preferably the amount, of bacteria in a sample according to the diagnostic methods described herein.
  • a diagnostic system preferably in kit form, for assaying for the presence of Streptococcus, in particular Streptococcus pneumoniae, in a biological sample where it is desirable to detect the presence, and preferably the amount, of bacteria in a sample according to the diagnostic methods described herein.
  • the diagnostic system includes, in an amount sufficient to perform at least one assay, a binding member composition according to the present invention, preferably as a separately packaged reagent, and more preferably also instruction for use.
  • the biological sample can be a tissue, tissue extract, fluid sample or body fluid sample, such as blood, plasma or serum.
  • Packaged refers to the use of a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a binding member of the present invention.
  • a package can be a glass vial used to contain milligram quantities of a contemplated labelled binding member preparation, or it can be a microtiter plate well to which microgram quantities of a contemplated binding member has been operatively affixed, i.e., linked so as to be capable of binding a ligand.
  • Instructions for use typically include a tangible expression describing the reagent concentration or, at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
  • a diagnostic system of the present invention preferably also includes a label or indi- eating means capable of signaling the formation of a binding reaction complex containing a binding member complexed with the preselected ligand.
  • the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like.
  • HRP horseradish peroxidase
  • glucose oxidase or the like.
  • additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
  • additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
  • An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).
  • labels i.e., labeling of, polypeptides and proteins or phage
  • proteins can be labelled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
  • the diagnostic systems can also include a specific binding agent, preferably as a separate package.
  • a "specific binding agent” is a molecular entity capable of selectively binding a binding member species of the present invention or a complex containing such a species, but is not itself a binding member of the present invention.
  • Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like.
  • the specific binding agent binds the binding member species when that species is present as part of a complex.
  • the diagnostic kits of the present invention can be used in an "ELISA” format to detect the quantity of a preselected ligand in a fluid sample.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample and is readily applicable to the present methods.
  • a binding member of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the sub- ject diagnostic systems.
  • a reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption meth- ods are described herein.
  • Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from
  • binding member species labelled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a sepa- rate package of a system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • the present invention also contemplates various assay methods for determining the presence, and preferably amount, of a Streptococcus, in particular Streptococcus pneumoniae, typically present in a biological sample.
  • the present invention relates to a method of detecting or diagnosing a disease or disorder associated with Pneumococcus in an individual comprising
  • the bound binding members may be detected either directly or indirectly, to the ⁇ amount of the Streptococcus in the sample.
  • binding reagent of this invention can be used to form an binding reaction product whose amount relates to the amount of the ligand in a sample.
  • exemplary assay methods are described herein, the invention is not so limited.
  • the detecting step can be directed, as is well known in the immunologieal arts, to either the complex or the binding reagent (the receptor component of the complex).
  • a secondary binding reagent such as an antibody specific for the receptor may be utilized.
  • the complex may be detectable by virtue of having used a labelled receptor molecule, thereby making the complex labelled. Detection in this case comprises detecting the label present in the complex.
  • a further diagnostic method may utilize the multivalency of a binding member composition of one embodiment of this invention to cross-link ligand, thereby forming an aggregation of multiple ligands and polypeptides, producing a precipitable aggregate.
  • This embodiment is comparable to the well-known methods of immune precipitation.
  • This embodiment comprises the steps of admixing a sample with a bind- ing member composition of this invention to form a binding admixture under binding conditions, followed by a separation step to isolate the formed binding complexes. Typically, isolation is accomplished by centrifugation or filtration to remove the aggregate from the admixture. The presence of binding complexes indicates the presence of the preselected ligand to be detected.
  • compositions of the present invention contemplates pharmaceutical compositions useful for practising the therapeutic methods described herein.
  • Pharmaceutical compositions of the present invention contain a physiologically tolerable carrier together with at least one species of binding member as described herein, dissolved or dispersed therein as an active ingredient.
  • the pharmaceutical composition is not immunogenic when administered to a human individual for therapeutic purposes, unless that purpose is to induce an immune response.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • compositions that contains active ingredients dissolved or dispersed therein are well understood in the art.
  • compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the preparation can also be emulsified.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, which enhance the effectiveness of the active ingredient.
  • the pharmaceutical composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phospho- ric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclu- sion of water.
  • additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.
  • the invention also relates to a method for preparing a medicament or pharmaceuti- cal composition comprising an antibody of the invention, the medicament being used for immunotherapy of a disease or disorder associated with Streptococcus, in particular Streptococcus pneumoniae, such as pneumonia, meningitis and sepsis, comprising admixing at least one binding member as defined above with a physiologically acceptable carrier.
  • the pharmaceutical composition may also be a kit-in-part further including an antibiotic agent, such as antibiotics selected from ⁇ -lactams, cephalosporins, penicilins and aminoglycosides, and/or include an immunostimulating agent, such as cytokines, interferons, growth factors, for example GCSF or GM-CSF.
  • an antibiotic agent such as antibiotics selected from ⁇ -lactams, cephalosporins, penicilins and aminoglycosides
  • an immunostimulating agent such as cytokines, interferons, growth factors, for example GCSF or GM-CSF.
  • the kit-in-part may be used for simultaneous, sequential or separate administration.
  • the pharmaceutical composition may include the binding member according to the invention in combination with the Streptococcus protein PsaA, in particular as a vaccine. It has been found that by combining the binding member ac- cording to the invention with the protein PsaA, the immunising properties of the combination product is better than for the protein PsaA alone. This may be due to the fact that the protein PsaA is presented to the immune system by the binding member.
  • the binding members according to the present invention are particular useful in therapeutic methods due to their high affinity and specificity. Accordingly, the binding members can be used immunotherapeutically towards a disease or disorder associated with Streptococcus, in particular Streptococcus pneumoniae, such as pneumonia, meningitis and sepsis.
  • Streptococcus pneumoniae such as pneumonia, meningitis and sepsis.
  • binding members denotes both prophylactic as well as therapeutic administration.
  • the binding members can be administered to high- risk patients in order to lessen the likelihood and/or severity of disease, administered to patients already evidencing active infection, or administered to patients at risk of infection.
  • the dosage ranges for the administration of the binding members of the invention are those large enough to produce the desired effect in which the symptoms of the disease are ameliorated or the likelihood of infection decreased.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the indi- vidual physician in the event of any complication.
  • a therapeutically effective amount of an binding member of this invention is typically an amount of antibody .such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 micro- gram ( ⁇ g) per miUiliter (ml) to about 100 ⁇ g/ml, preferably from about 1 ⁇ g/ml to about 5 ⁇ g/ml, and usually about 5 ⁇ g/ml.
  • the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.
  • the binding members of the invention can be administered parenterally by injection or by gradual infusion over time.
  • the infection may be systemic and therefore most often treated by intravenous administration of pharmaceutical compositions, other tissues and delivery means are contemplated where there is a likelihood that targeting a tissue will result in a lessening of the disease.
  • antibodies of the invention can be administered parenterally, such as intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic means.
  • compositions containing a binding member of this invention are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a pharmaceutical composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calcu- lated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • mice having the ability of making fully human antibodies were used.
  • the mice were HuMAb-Mouse, obtained from Medarex, Inc.
  • His6-psaA protein (wherein the PsaA protein has the sequence depicted in Figure 15) was used as antigen for the immunization experiments. The concentrations of these proteins were calculated by spectrophotometer, and their purity was ascertained by SDS-PAGE after silver staining. Proteins were prepared for immunization using complete Freund's, incomplete Freund's, and RIBI as an adjuvant, as appropriate. Groups of 12 to 18 male HuMAb mice were used for all immunization ex- periments. Each mouse was immunized by intraperitoneal (i.p.) and subcutaneous injection of 20-50 ⁇ g of His ⁇ -psaA (PsaA amino acid sequence is shown in Figure 15) at three-week intervals.
  • mice received 5x10e8 heat-inactivated Streptococcus pneumoniae R6 cells i.p. Sera were collected from mice by retro-orbital bleeding. Titers to rpsaA were determined by ELISA. Mice were boosted intrave- nously prior to sacrifice.
  • Hydridoma supernatants are tested in 3 dilutions against S.pneumococus R6 in a sandwich enzyme linked immunosorbent assay with reagent excess.
  • H202 H2S04 Coating Buffer Na2C03 0.05 M, pH 9.6, expire two weeks after production, storage at 4°C.
  • Dilution and wash buffer PBS with 0.1 % Tween20, expire one week after production, storage at 4°C Secondary Antibody: Dilute 1 :1500 in dilution buffer, freshly made every day.
  • Stop reagent 1.2 M H2S04, expire 1 year after production, storage at RT.
  • Example 2 The monoclonal antibodies obtained in Example 1 were tested in vitro against Pneumococcus of various strains identifying antibodies against whole bacteria (ELISA) see method 1 , and using Western blot, see method 2, below.
  • ELISA identifying antibodies against whole bacteria
  • Transfer Buffer 500 ml of Transfer Buffer was prepared by adding 25 ml 20X NuPAGE® Transfer Buffer and 100 ml Ethanol 96% to 375 ml Milli-Q grade H 2 0. Blotting pads were soaked in 350 ml of Transfer Buffer. PVDF membrane was soaked in 96% Ethanol for 30 sec. and wash in transfer buffer for 2 min, and the filter paper was briefly soaked in Transfer Buffer.
  • a piece of pre-soaked filter paper was placed on top of the gel (adhered to the bottom plate) and any trapped air bubbles were removed.
  • the plate was turned over so the gel and filter paper were facing downwards over a gloved hand or clean flat surface.
  • the pre-soaked transfer membrane was placed on the gel and trapped air bubbles removed.
  • the blot module was filled with Transfer Buffer until the gel/membrane assembly was covered.
  • the Outer Buffer Chamber was filled with 650 ml Milli-Q grade H 2 0, the lid placed on the unit and the electrical leads connected to the power supply.
  • PVDF membranes with electrophoretically transferred proteins, bacterial lysate(s) or lipopolysaccharide Materials:
  • Part A 1 ml Blocker/Diluent (Part B)
  • Dried PVDF membranes are re-wetted in Ethanol 96% and rinsed two times for 5 min each in 20 ml of Milli-Q grade H20, proceed to step 4.
  • Method to immobilize Protein G via amines on a CM5 chip (Amine coupling method) a. Normalize the chip at least twice with appropriate buffer b. A 0.5 ⁇ g/mL dilution of Protein-G is made in 10mM sodium acetate buffer of pH 2.9 c. Activate the CM5 chip for 7 minutes, by flowing freshly mixed EDC & NHS at a flow rate of 5 ⁇ L/min, according to the method mentioned in Biacore Handbook. d. Inject Protein-G sample for 22 minutes over this activated surface. e. Deactivate by flowing 1 M ethonolamine-HCI for 10 minutes. f. This method couples about 10000 RUs of Protein-G on the activated surface. g. For the blank surface, the same activation and deactivation procedure is followed without the injection of Protein-G.
  • CM5 chip Method to immobilize PsaA via amines on a CM5 chip (Amine coupling method) a.
  • a CM5 chip was normalized as above b.
  • PsaA dilutions are made in concentrations ranging from 50 to 150 ⁇ g/mL, in sodium 10mM acetate buffer of pH 4.0 c.
  • the chip is activated for 7 minutes by flowing freshly mixed EDC and NHS.
  • d. Inject PsaA, made in acetate buffer, by manual injection until the amount captured on the chip reaches the desired level (in our case, 350 and 800 RUs)
  • Deactivate the chip by injecting 1 M ethanolamine-HCI for 10 minutes
  • the blank surface is generated in the same method, but without the step of injecting the protein.
  • Chip used Coupling Buffer: 10mM Acetate, pH 4.0
  • Fc1 & 3 Blank
  • Fc2 PsaA: 353RUs
  • Fc4 PsaA: 824RUs
  • Antibody Cone 5, 4, 3, 2 & 1ug/mL (66.7, 53.36, 40.02, 26.68 &13.34nM)
  • Running buffer HBS-EP Flow rate: 30uL/min
  • Regeneration Buffer 100mM HCI+150mMNaCI Flow rate: 10OuL/min Regeneration Time: 1 min Results:
  • Antibody Cone 4, 3, 2, 1 & 0.5ug/mL (53.36, 40.02, 26.68, 13.34 & 6.67nM)
  • Running buffer HBS-EP Flow rate: 25uL/min Association Time: 5 min. Dissociation Time: 45min
  • Regeneration Buffer 100mM HCI+150mM NaCl
  • Antibody Cone 3, 2, 1 , 0.75 & 0.50ug/mL (40.02, 26.68, 13.34, 10.0, 6.67nM)
  • Running buffer HBS-EP Flow rate: 30ulJmin Association Time: 8 min.
  • Dissociation Time: 30-40min Regeneration Buffer 100mM HCI+150mMNaCI Flow rate: 100uL/min Regeneration Time: 45sec-1 min
  • Dav-1 Pn.-strain is seeded on a 4 x 5% bloodplate, and Incubated overnight at 35°C.
  • the Pneumococcus strain is suspended in filtered broth to 10 8 CFU/ml (cf. MU/F074-01), and diluted to 1x 10 6 CFU/ml (50 ⁇ l 10 8 CFU/ml i 4.95 ml PBS) and further diluted with antibody (see scheme).
  • the bacteria/antibody mixture is shaken and incubated for 10 min. at 35° and then the mice are inoculated with 0.5 ml i.p.
  • Serum and peritoneal fluid was frozen at -20°C for antibody concentration meas- urements
  • mice were sedated with CO 2 . A cut was made in the axilla, and blood collected in tube glass 0. 100 ⁇ l was transferred to glass 1 and mixed thoroughly, whereafter 100 ⁇ l was spread on a bloodplate with a glass rod. Then CFU determination was conducted. The rest of the blood was centrifuged at 2000 x G for 7 min. and serum transferred to another tube, stored at -20°C.
  • mice were sacrificed, 2 ml sterile saline was injected into the abdomen, and 10 see later the fluid was withdrawn with a sterile Pasteur pipette and transferred to an Eppendorf tube. CFU determination was conducted. The rest of the peritoneal fluid was stored at -20°C.
  • F(ab') 2 fragments of each of the HuMAbs, anti-CD64 (88.53), and anti-PsaA 5-9A7 were generated by pepsin digestion and purified to homogeneity by Superdex 200 gel filtration chromatography. Size exclusion HPLC was performed and profiles are depicted for each of the F(ab') 2 in Figure 2.
  • F(ab') 2 fragments were >95% pure.
  • a Fab' fragment of the 88.53 was generated by mild reduction of the inter-heavy chain disulfide bonds of the F(ab') 2 fragment with mercaptoethanolamine (MEA). The exact reducing conditions were determined prior to conjugation in small-scale experiments. Size exclusion HPLC was performed and the profile is depicted for the Fab' in Figure 3. By this type of analysis the 88.53 Fab' was >90% pure.
  • the Fab' fragment of the 88.53 was separated from free MEA by G-25 column chromatography.
  • the Fab' fragment was incubated with dinitrothiobenzoate (DTNB) to generate a Fab-TNB conjugate.
  • DTNB dinitrothiobenzoate
  • a Fab' fragment of the 5-9A7 was generated by mild reduction of the inter-heavy chain disulfide bonds of the F(ab') 2 fragment with mercaptoethanolamine (MEA). The exact reducing conditions were determined prior to conjugation in small-scale experiments. Size exclusion HPLC was performed and the profile is depicted for the Fab'in Figure 4. By this type of analysis the 5-9A7 Fab' was >90% pure.
  • the Fab' fragment of the 5-9A7 was separated from free MEA by G-25 column chromatography and mixed with 88.53 Fab-TNB at a 1 :1 molar ratio overnight at room temperature.
  • the HPLC profile depicted in Figure 5 represents a profile of the conjugation mixture after 18 hours of incubation and before purification. This profile shows a mixture of bispecific antibody as well as unconjugated Fab' molecules.
  • the bispecifc antibody was purified from contaminating Fab' molecules by Superdex
  • the Fab' fragment of the 88.53 was separated from free MEA by G-25 column chromatography.
  • the Fab' fragment was incubated with dinitrothiobenzoate (DTNB) 16a and 16b to generate a Fab-TNB conjugate.
  • DTNB dinitrothiobenzoate
  • a Fab' fragment of the 14A8 was generated by mild reduction of the inter-heavy chain disulfide bonds of the F(ab') 2 fragment with mercaptoethanolamine (MEA). The exact reducing conditions were determined prior to conjugation in small-scale experiments. Size exclusion HPLC was performed and the profile is depicted for the
  • the bispecific antibody was purified from contaminating Fab' molecules by Superdex 200 size exclusion chromatography and the purified molecule was analyzed by HPLC. As shown in Figure 11 the 88.53 x 14A8 bispecific antibody was purified to near homogeneity.
  • ELISA plates were coated recombinant PsaA, 50 ⁇ l/well, 5 ⁇ g/ml and incubated overnight at 4°C. 2. The plates were blocked with 5% BSA in PBS. 3. Titrations of the bispecific antibody were added to the plate. Controls included the anti-CD64 x anti-CD89 bispecific (control bispecific) and the F(ab') 2 fragments of the anti-CD64 Ab, 88.53 or of the anti-PsaA Ab, 5-9A7. 4. The plates were then incubated with a supernatant containing a fusion pro- tein consisting of soluble CD64 linked to the Fc portion of human IgM. 5. The plates were finally incubated with an alkaline phosphatase labelled goat anti-human IgM antibody. Positive wells were detected with the alkaline phosphatase substrate.
  • the anti-PsaA x anti-CD64 bispecific showed dose-dependent binding in this assay
  • Blood was taken from CD64 transgenic mice or from non-transgenic littermates, and was incubated with the 88.53 x 5-9A7 bispecific antibody at a concentration of 30 ⁇ g/ml for 30 minutes at room temperature.
  • CD64 Human CD64 is expressed on monocytes and, to a lesser extent, neutrophils of CD64 transgenic mice. As in humans, CD64 is not expressed by lymphocytes of the transgenic mice.
  • the data in Figure 14 show that the bispecific antibody binds to CD64 transgenic monocytes and neutrophils, but not to any cell populations derived from non-transgenic mice.
  • bispecific antibodies anti-CD64 x anti-PsaA and anti-CD64 x anti- CD89, were generated and purified to homogeneity.
  • the 88.53 x 5-9A7 bispecific antibody was shown to bind simultaneously to CD64 and to PsaA.
  • this bispecific antibody binds to CD64 expressed by human CD64 transgenic mice.
  • Antibodies shown in the affinity table in Example 3b were sequenced by conventional methods, and the variable regions of the antibodies are shown in the Figures.
  • McDaniel LS, et al. PspA a surface protein of Streptococcus pneumoniae, is capable of eliciting protection against pneumococci of more than one capsular type. Infect.lmmun.1991 Jan. 59:222-228. 19. Mitchell, T.J., F. Mendez, J.C. Paton, P.W. Andrew, and G.J. Boulnois. 1990. Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae type 1 and 2. Nuclei.Acids.Res. 18:4010

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Abstract

L'invention concerne un élément de liaison comprenant au moins un domaine de liaison permettant la liaison spécifique à une protéine A d'adhésine de surface de pneumonie streptocoque (PsaA), notamment un élément de liaison possédant au moins deux domaines de liaison, ainsi que l'utilisation de ces éléments de liaison dans des procédés de diagnostic, ainsi que pour des traitements. Dans un mode réalisation préféré, l'élément de liaison consiste en un anticorps, notamment un anticorps humain, ou un fragment de celui-ci, et peut également consister en un anticorps bispécifique.
PCT/DK2004/000492 2003-07-08 2004-07-08 Element de liaison a une proteine a d'adhesine de surface pneumocoque WO2005003174A1 (fr)

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US10/563,976 US20070003561A1 (en) 2003-07-08 2004-07-08 Binding member towards pneumococcus surface adhesin a protein (psaa)
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WO2006127020A1 (fr) * 2005-05-19 2006-11-30 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention Epitopes fonctionnels d'antigene psaa de streptococcus pneumoniae et leurs utilisations
WO2009009951A1 (fr) 2007-07-16 2009-01-22 Zhengzhou University 2'-fluoro nucléosides substitués en position 4', leur préparation et leur utilisation
US8859209B2 (en) 2006-01-12 2014-10-14 Carviar Aps Reimmunization and antibody design
US8859233B2 (en) 2006-05-02 2014-10-14 Carviar Aps Method for immunizing an avian species
WO2018222741A1 (fr) * 2017-05-31 2018-12-06 Oklahoma Medical Research Foundation Anticorps bispécifiques pour le traitement de la pneumonie à streptocoques
US10683356B2 (en) 2015-02-03 2020-06-16 Als Therapy Development Institute Methods of treating a CD40L associated disease or disorder by administering anti-CD40L antibodies
WO2020186588A1 (fr) 2019-03-15 2020-09-24 河南真实生物科技有限公司 Forme cristalline a de l'analogue i de nucléoside 2'-fluoro-4'-substitué, son procédé de préparation et son utilisation
US11384152B2 (en) 2017-05-24 2022-07-12 Als Therapy Development Institute Therapeutic anti-CD40 ligand antibodies

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WO2002092017A2 (fr) * 2001-05-16 2002-11-21 Albert Einstein College Of Medicine Of Yeshiva University Anticorps anti-pneumocoques humains provenant d'animaux non humains

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WO2002079254A1 (fr) * 2001-03-29 2002-10-10 Janoff Edward N Anticorps monoclonaux humains contre des polysaccharides capsulaires de streptococcus pneumoniae
WO2002092017A2 (fr) * 2001-05-16 2002-11-21 Albert Einstein College Of Medicine Of Yeshiva University Anticorps anti-pneumocoques humains provenant d'animaux non humains

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J. SAMPSON ET AL.: "Immunologic characterization of a monoclonal antibody to Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) protein.", ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 99, 1999, USA, pages 273, XP008027694 *
N. SRIVASTAVA ET AL.: "Selection of an immunogenic and protective epitope of the PsaA protein of Streptococcus pneumoniae using a phage display library.", HYBRIDOMA, vol. 19, no. 1, February 2000 (2000-02-01), New York, NY, USA, pages 23 - 31, XP001064205 *
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006127020A1 (fr) * 2005-05-19 2006-11-30 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention Epitopes fonctionnels d'antigene psaa de streptococcus pneumoniae et leurs utilisations
US7919104B2 (en) 2005-05-19 2011-04-05 The United States Of America As Represented By The Department Of Health And Human Services, Centers For Disease Control And Prevention Functional epitopes of Streptococcus pneumoniae PsaA antigen and uses thereof
EP2371843A1 (fr) * 2005-05-19 2011-10-05 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Services Épitopes fonctionnels d'antigène psa A de streptococcus pneumoniae et leurs utilisations
US9938337B2 (en) 2006-01-12 2018-04-10 Carviar Aps Reimmunization and antibody design
US8859209B2 (en) 2006-01-12 2014-10-14 Carviar Aps Reimmunization and antibody design
US9914766B2 (en) 2006-05-02 2018-03-13 Carviar Aps Method for immunizing an avian species
US8859233B2 (en) 2006-05-02 2014-10-14 Carviar Aps Method for immunizing an avian species
WO2009009951A1 (fr) 2007-07-16 2009-01-22 Zhengzhou University 2'-fluoro nucléosides substitués en position 4', leur préparation et leur utilisation
US10683356B2 (en) 2015-02-03 2020-06-16 Als Therapy Development Institute Methods of treating a CD40L associated disease or disorder by administering anti-CD40L antibodies
US11014990B2 (en) 2015-02-03 2021-05-25 Als Therapy Development Institute Anti-CD40L antibodies
US11692040B2 (en) 2015-02-03 2023-07-04 Als Therapy Development Institute Anti-CD40L antibodies and methods for treating CD40L-related diseases or disorders
US11384152B2 (en) 2017-05-24 2022-07-12 Als Therapy Development Institute Therapeutic anti-CD40 ligand antibodies
WO2018222741A1 (fr) * 2017-05-31 2018-12-06 Oklahoma Medical Research Foundation Anticorps bispécifiques pour le traitement de la pneumonie à streptocoques
WO2020186588A1 (fr) 2019-03-15 2020-09-24 河南真实生物科技有限公司 Forme cristalline a de l'analogue i de nucléoside 2'-fluoro-4'-substitué, son procédé de préparation et son utilisation

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