EP0977590A1 - Anticorps monoclonaux humains - Google Patents

Anticorps monoclonaux humains

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Publication number
EP0977590A1
EP0977590A1 EP97913749A EP97913749A EP0977590A1 EP 0977590 A1 EP0977590 A1 EP 0977590A1 EP 97913749 A EP97913749 A EP 97913749A EP 97913749 A EP97913749 A EP 97913749A EP 0977590 A1 EP0977590 A1 EP 0977590A1
Authority
EP
European Patent Office
Prior art keywords
antibody
ser
rsv
human
sequence
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.)
Withdrawn
Application number
EP97913749A
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German (de)
English (en)
Other versions
EP0977590A4 (fr
Inventor
Keith Charles Deen
Susan Beth Dillon
Terence Graham Porter
Raymond Whitney Sweet
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication of EP0977590A1 publication Critical patent/EP0977590A1/fr
Publication of EP0977590A4 publication Critical patent/EP0977590A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • 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
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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'

Definitions

  • This invention relates to novel human monoclonal antibodies (mAbs) and to the genes encoding same. More specifically, this invention relates to human monoclonal antibodies specifically reactive with an epitope of the fusion (F) protein of Respiratory Syncytial Virus (RSV). Such antibodies are useful for the therapeutic and/or prophylactic treatment of RSV infection in human patients, particularly infants and young children.
  • mAbs novel human monoclonal antibodies
  • F fusion protein of Respiratory Syncytial Virus
  • Respiratory syncytial virus is the major cause of lower respiratory disease in children, giving rise to predictable annual epidemics of bronchiolitis and pneumonia in children worldwide.
  • the virus is highly contagious, and infections can occur at any age. Immunity to RSV appears to be short-lived, thus reinfections are frequent. Zero to 2 year old infants are the most susceptible and represent the primary affected population. In this group, 1 out of 5 will develop lower respiratory (below larynx) disease upon infection and this ratio stays the same upon reinfection.
  • hospitalization is required in 1-3% of cases of RSV infection and is usually of long duration (up to 3 weeks).
  • monoclonal antibodies have been employed.
  • the advantages of such an approach include: a higher concentration of specific antibody can be achieved thereby reducing the amount of globulin required to be given; the reliance on direct blood products can be eliminated; the levels of antibody in the preparation can be more uniformly controlled and the routes of administration can be extended.
  • passive immunotherapy employing monoclonal antibodies from a heterologous species (e.g., murine) has been suggested (See: PCT Application
  • PCT/US94/08699, Publication No. WO 95/04081) one alternative to reduce the risk of an undesirable immune response on the part of the patient directed against the foreign antibody is to employ "humanized" antibodies. These antibodies are substantially of human origin, with only the Complementarity Determining Regions (CDRs) being of non-human origin. Particularly useful examples of this approach are disclosed in PCT Application PCT/GB91/01554, Publication No. WO 92/04381 and PCT Application PCT/GB93/00725, Publication No. WO93/20210.
  • RSV belonging to the family paramyoxoviridae, is a negative-strand unsegmented RNA virus with properties similar to those of the paramyxoviruses. It has, however been placed in a separate genus Pneumovirus, based on morphologic differences and lack of hemagglutinin and neuraminidase activities. RSV is pleomorphic and ranges in size from 150-300 nm in diameter.
  • the virus matures by budding from the outer membrane of a cell and virions appear as membrane-bound particles with short, closely spaced projections or "spikes".
  • the RNA genome encodes 10 unique viral polypeptides ranging in size from 9.5 kDa to 160 kDa (Huang, Y. T. and G. W.Wertz, J. Virol. 43: 150-157 (1982)). Seven proteins (F, G, N, P, L, M, M2) are present in RSV virions and at least three proteins (F, G, and SH) are expressed on the surface of infected cells.
  • the F protein has been conclusively identified as the protein responsible for cell fusion since specific antibodies to this protein inhibit syncytia formation in vitro and cells infected with vaccinia virus expressing recombinant F protein form syncytia in the absence of other RSV virus proteins. In contrast, antibodies to the G protein do not block syncytia formation but prevent attachment of the virus to cells.
  • RSV can be divided into two antigenically distinct subgroups, (A & B)
  • RSV is distributed worldwide.
  • Other respiratory viruses cause epidemics at irregular intervals or exhibit a mixed endemic/epidemic pattern, but RSV is the only respiratory viral pathogen that produces a sizable epidemic every year in large urban centers.
  • RSV epidemics In the temperate areas of the world, RSV epidemics have occurred primarily in the late fall, winter or spring but never during the summer.
  • the occurrence and spread of infection within a community is characteristic and easily diagnosed, leading to sharp rises in cases of bronchiolitis and pediatric pneumonia and the number of hospital admissions of young children with acute lower respiratory tract disease.
  • Other respiratory viral agents that occur in outbreaks are rarely present at the same time as RSV .
  • bronchiolitis and pneumonia is most common in patients under six months of age. Infants who have apparently recovered completely from this illness may display symptomatic respiratory abnormalities for years (recurrent wheezing, decreased pulmonary function, recurrent cough, asthma, and bronchitis).
  • the mechanisms by which the immune system protects against RSV infection and reinfection are not well understood. It is clear, however, that immunity is only partially protective since reinfection is common at all ages, and sometimes occurs in infants only weeks after recovery from a primary infection.
  • Both serum and secretory antibodies (IgA) have been detected in response to RSV infection in adults as well as in very young infants.
  • the titers of serum antibodies to the viral F or G glycoprotein, as well as of neutralizing antibodies found in infants (1-8 months of age) are 15-25% of those found in older subjects. These reduced titers may contribute to the increased incidence of serious infection in younger children.
  • mice can be cured by adoptive transfer of primed T cells (Cannon, M. J. et al., Immunology 62: 133-138 (1987)).
  • This invention relates to the use of human mABs specific for the F protein of RSV virus to passively treat or prevent infection.
  • passive antibody therapy in humans is well documented and is being used to treat other infectious diseases such as hepatitis and cytomegalovirus.
  • Clinical trials are also on-going to evaluate the efficacy of humanized antibodies for treatment of RSV infection in young children.
  • Studies in animals have clearly demonstrated that polyclonal and monoclonal antibody against both F and G glycoprotein can confer passive protection in RSV virus infection when given prophylactically or therapeutically (Prince, et al., supra). In these studies, passive transfer of neutralizing F or G mAbs to mice, cotton rats or monkeys, significantly reduce or completely prevent replication of the RSV virus in the lungs.
  • the induction of neutralizing antibodies to RSV virus appears to be limited to the F and G surface glycoproteins.
  • the F protein is the major target for cross-reactive neutralizing antibodies associated with protection against different strains of RSV virus.
  • experimental vaccination of mice or cotton rats with F protein also results in cross protection.
  • the antigenic relatedness of the F protein across strains and subgroups of the virus is reflected in its high degree of homology at the amino acid level.
  • antigenic dimorphism was linked primarily to the G glycoprotein.
  • the F protein has a predicted molecular weight of 68-70 kDa; a signal peptide at its N-terminus; a membrane anchor domain at its C terminus; and is cleaved proteolytically in the infected cell prior to virion assembly to yield disulfide linked ⁇ 2 and F1 .
  • Five neutralizing epi topes have been identified within the F protein sequence and map to residues 205-225; 259-278; 289-299: 483-488 and 417-438.
  • Studies to determine the frequency of sequence diversion in the F protein showed that the majority of the neutralizing epitopes were conserved in all of the 23 strains of RSV virus isolated in Australia, Europe, and regions of the U.S. over a period of thirty years.
  • This invention relates to fully human monoclonal antibodies and functional fragments thereof specifically reactive with an F protein epitope of RSV and capable of neutralizing RSV infection.
  • the present invention provides modifications to neutralizing Fab fragments or F(ab') 2 fragments specific for the F protein of RSV produced by random combinatorial cloning of human antibody sequences and isolated from a filamentous phage Fab display library.
  • a reshaped human antibody containing human heavy and light chain constant regions from a first human donor and heavy and light chain variable regions or the CDRs thereof derived from human neutralizing monoclonal antibodies for the F protein of RSV derived from a second human donor.
  • the present invention provides a pharmaceutical composition which contains one (or more) altered antibodies and a pharmaceutically acceptable carrier.
  • the present invention provides a method for passive immunotherapy of RSV disease in a human by administering to said human an effective amount of the pharmaceutical composition of the invention for the prophylatic or therapeutic treatment of RSV infection.
  • the present invention provides methods for, and components useful in, the recombinant production of human and altered antibodies (e.g., engineered antibodies, CDRs, Fab or F(ab) 2 fragments, or analogs thereof) which are derived from human neutralizing monoclonal antibodies (mAbs) for F protein of RSV.
  • These components include isolated nucleic acid sequences encoding same, recombinant plasmids containing the nucleic acid sequences under the control of selected regulatory sequences which are capable of directing the expression thereof in host cells (preferably mammalian) transfected with the recombinant plasmids.
  • the production method involves culturing a transfected host cell line of the present invention under conditions such that the human or altered antibody is expressed in said cells and isolating the expressed product therefrom.
  • a method to diagnose the presence of RSV in a human which comprises contacting a sample of biological fluid with the human antibodies and altered antibodies of the instant invention and assaying for the occurrence of binding between said human antibody (or altered antibody) and RSV.
  • a pharmaceutical composition comprising at least one dose of an immunotherapeutically effective amount of the monoclonal antibody of this invention in combination with at least one additional monoclonal antibody.
  • the additional monoclonal antibody is an anti-RSV antibody distinguished from the subject antibody of by virtue of being reactive with a different epitope of the RSV F protein antigen.
  • Figure 1 illustrates the cloning strategy used for the construction of the Hu 19A monoclonal antibody.
  • the heavy chain V region was cloned into the PCD derivative vector as a Xhol - Bsp ⁇ 20l fragment.
  • the entire light chain V and C regions were cloned into the PCN derivative vector as a Sacl - Xbal fragment. Details are described in the hereinbelow.
  • Figure 2 provides a comparison of the heavy chain amino acid sequences of various monoclonal antibodies of this invention. The amino acid sequences of the heavy chains for the A, B, C and D constructs are shown (SEQ ID NOS: 5, 6, 7 and 8, respectively).
  • Sequence A has an amino acid insertion between positions 4 and 5 due to the cloning strategy utilized by Barbas et al. ( Proc. Natl Acad. Sci. (USA) 89, 10164-10168 ( 1992), PCT publication WO94/06448).
  • Bold residues correspond to the leader region, and to CDRs 1-3.
  • the underlined sequence in CDR2 identifies the N-linked glycosylation site in versions A and B that was mutated in version C. Residues P14 and G15, marked with an "*" were listed as L and A, respectively in the published sequence (Barbas et al., supra).
  • Figure 3 provides a comparison of the light chain amino acid sequences of various monoclonal antibodies of this invention.
  • the amino acid sequences of the light chains for the A, B, C and D constructs are shown (SEQ ID NOS: 10, 1 1 , 12 and 13). Numbering of the residues in the VK region is based on the germline (GL) gene Dpk9 (SEQ ID NO: 9), beginning at the mature processed amino terminus and ending at CDR3; but for reference to framework 4, the actual numbering is also shown for Hul9ALc.
  • the "-" indicates identity to the preceding sequence.
  • the G at position 97 in framework 4 of Hul9A, marked with an "*" was listed as E in the published sequence (see text).
  • Sequence A has a two amino acid deletion at residues 1 and 2 due to the cloning strategy. Bold residues correspond to the leader region, and to CDRs 1-3. The K constant region is shown for constructs A and B in comparison to the germline gene. The L mutation near the C-terminus was corrected in version C (See; Figure 3, SEQ ID NO: 13).
  • Figure 4 illustrates the DNA sequences of plasmids for the expression of the Hul9 mAB heavy and light chains.
  • Figure 4A is the DNA sequence of Hul9AHcpcd (SEQ ID NO: 14). The start of translation, leader peptide, amino- terminal processing site (SEQ ID NO: 15), carboxy terminus of the 19A heavy chain (SEQ ID NO: 16) and Eco RI restriction endonuclease cleavage site are shown.
  • Figure 4B is the DNA sequence of Hul9ALcpcn (SEQ ID NO: 17), and shows the corresponding features for the light chain and the Xba I restriction site following the end of the coding region for the light chain (SEQ ID NO'S: 18, 19).
  • Figure 4C is the DNA sequence of the coding region of the heavy chain of plasmid Hul9BHcpcd (SEQ ID NO'S 20,21).
  • Figure 4D is the DNA sequence of the coding region for the light chain of plasmid Hul9BLcpcn (SEQ ID NO:22,23 & 24).
  • Figure 4E is the DNA sequence of the coding region of the heavy chain of the plasmid Hul9CHcpcd (SEQ ID NO'S 25,26).
  • Figure 4F is the DNA sequence of the coding sequence of the heavy chain of plasmid Hul9DHcpcd (SEQ ID NO:'S 27,28).
  • Figure 4G is the DNA sequence of the coding region of the light chain of plasmid Hul9CLcpcn (SEQ ID NO'S: 29, 30).
  • Figures 4C-G bolded residues indicate differences from the full vector sequences for Hul9AHcpcd and Hul9ALc shown in Figures 4 A and 4B, respectively.
  • Figure 5 illustrates a Coomassie stained SDS-PAGE gel of Hul9B and
  • Figure 6 illustrates the separation of Hul9 Glycovarients by anion exchange chromatography.
  • Figure 7 illustrates SDS-PAGE analysis of Hul9B Fab glycovarients.
  • This invention provides useful human monoclonal antibodies (and fragments thereof) reactive with the F protein of RSV, isolated nucleic acids encoding same and various means for their recombinant production as well as therapeutic, prophylactic and diagnostic uses of such antibodies and fragments thereof.
  • altered antibody refers to a protein encoded by an altered immunoglobulin coding region, which may be obtained by expression in a selected host cell.
  • altered antibodies are engineered antibodies (e.g., chimeric, humanized, or reshaped or immunologically edited human antibodies) or fragments thereof lacking all or part of an immunoglobulin constant region, e.g., Fv, Fab, or F(ab') 2 and the like.
  • altered immunoglobulin coding region refers to a nucleic acid sequence encoding an altered antibody of the invention or a fragment thereof.
  • Reshaped human antibody refers to an altered antibody in which minimally at least one CDR from a first human monoclonal donor antibody is substituted for a CDR in a second human acceptor antibody. Preferrably all six CDRs are replaced. More preferrably an entire antigen combining region (e.g., Fv, Fab or F(ab')2 ) from a first human donor monoclonal antibody is substituted for the corresponding region in a second human acceptor monoclonal antibody. Most preferrably the Fab region from a first human donor is operatively linked to the appropriate constant regions of a second human acceptor antibody to form a full length monoclonal antibody.
  • an entire antigen combining region e.g., Fv, Fab or F(ab')2
  • Hul9A, Hul9B, Hul9C and Hul9D are defined as reshaped human antibodies comprising a light chain amino acid sequence selected from Sequences 19A, 19B, 19C and 19D of Figure 3 and a heavy chain amino acid sequence selected from Sequences 19A, 19B, 19C and 19D of Figure 2, or functional partial sequences thereof.
  • First immunoglobulin partner refers to a nucleic acid sequence encoding a human framework or human immunoglobulin variable region in which the native (or naturally-occurring) CDR-encoding regions are replaced by the CDR-encoding regions of a donor human antibody.
  • the human variable region can be an immunoglobulin heavy chain, a light chain (or both chains), an analog or functional fragments thereof.
  • Such CDR regions, located within the variable region of antibodies (immunoglobulins) can be determined by known methods in the art. For example Kabat et al. (Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)) disclose rules for locating CDRs.
  • “Second fusion partner” refers to another nucleotide sequence encoding a protein or peptide to which the first immunoglobulin partner is fused in frame or by means of an optional conventional linker sequence (i.e., operatively linked).
  • the fusion partner is an immunoglobulin gene and when so, it is referred to as a "second immunoglobulin partner".
  • the second immunoglobulin partner may include a nucleic acid sequence encoding the entire constant region for the same (i.e., homologous - the first and second altered antibodies are derived from the same source) or an additional (i.e., heterologous) antibody of interest.
  • the second immunoglobulin partner may be an immunoglobulin heavy chain or light chain (or both chains as part of a single polypeptide).
  • the second immunoglobulin partner is not limited to a particular immunoglobulin class or isotype.
  • the second immunoglobulin partner may comprise part of an immunoglobulin constant region, such as found in a Fab, or F(ab) (i.e., a discrete part of an appropriate human constant region or framework region).
  • a second fusion partner may also comprise a sequence encoding an integral membrane protein exposed on the outer surface of a host cell, e.g., as part of a phage display library, or a sequence encoding a protein for analytical or diagnostic detection, e.g., horseradish peroxidase, ⁇ -galactosidase, etc.
  • Fv, Fc, Fd, Fab, or F(ab') 2 are used with their standard meanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).
  • an “engineered antibody” describes a type of altered antibody, i.e., a full-length synthetic antibody (e.g., a chimeric, humanized, reshaped or immunologically edited human antibody as opposed to an antibody fragment) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope.
  • a full-length synthetic antibody e.g., a chimeric, humanized, reshaped or immunologically edited human antibody as opposed to an antibody fragment
  • a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope.
  • such molecules may include antibodies characterized by a humanized heavy chain associated with an unmodified light chain (or chimeric light chain), or vice versa.
  • Engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain framework regions in order to retain donor antibody binding specificity. These antibodies can comprise replacement of one or more CDRs (preferably all) from the acceptor antibody with CDRs from a donor antibody described herein.
  • a “chimeric antibody” refers to a type of engineered antibody which contains naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody from a heterologous species.
  • a "humanized antibody” refers to a type of engineered antibody having its
  • CDRs derived from a non-human donor immunoglobulin the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
  • framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al., Proc. Natl Acad. Sci. USA, 86: 10029-10032 (1989), Hodgson et a , Biotechnology, 9:421 (1991)).
  • an "immunologically edited antibody” refers to a type of engineered antibody in which changes are made in donor and/or acceptor sequences to edit regions in respect of cloning artifacts, germ line enhancements, etc. aimed at reducing the likelihood of an immunological response to the antibody on the part of a patient being treated with the edited antibody.
  • donor antibody refers to an antibody (monoclonal, or recombinant) which contributes the nucleic acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
  • One donor antibody suitable for use in this invention is a Fab fragment of a human neutralizing monoclonal antibody designated as Fab Hul9.
  • Fab Hul9 is defined as a having the variable light chain DNA and amino acid sequences Hu 19A as shown in Figures 2, 3, 4A and 4B.
  • acceptor antibody refers to an antibody (monoclonal, or recombinant) from a source genetically unrelated to the donor antibody, which contributes all (or any portion, but preferably all) of the nucleic acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
  • a human antibody is the acceptor antibody.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et aL, Sequences of Proteins of Immunological Interest, 4th Ed., U.S.
  • CDRs refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate). CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
  • CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
  • Fab Hul9 may be characterized by a certain level of antigen affinity
  • a CDR encoded by a nucleic acid sequence of Fab Hul9 in an appropriate structural environment may have a lower, or higher affinity. It is expected that CDRs of Fab Hu 19 in such environments will nevertheless recognize the same epitope(s) as does the intact Fab Hul9.
  • a "functional fragment” is a partial heavy or light chain variable sequence (e.g., minor deletions at the amino or carboxy terminus of the immunoglobulin variable region) which retains the same antigen binding specificity and/or neutralizing ability as the antibody from which the fragment was derived.
  • an “analog” is an amino acid sequence modified by at least one amino acid, wherein said modification can be chemical or a substitution or a rearrangement of a few amino acids (i.e., no more than 10), which modification permits the amino acid sequence to retain the biological characteristics, e.g., antigen specificity and high affinity, of the unmodified sequence.
  • (silent) mutations can be constructed, via substitutions, when certain endonuclease restriction sites are created within or surrounding CDR-encoding regions. Analogs may also arise as allelic variations.
  • An "allelic variation or modification” is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention. Such variations or modifications may be due to degeneracy in the genetic code or may be deliberately engineered to provide desired characteristics. These variations or modifications may or may not result in alterations in any encoded amino acid sequence.
  • effector agents refers to non-protein carrier molecules to which the altered antibodies, and/or natural or synthetic light or heavy chains of the donor antibody or other fragments of the donor antibody may be associated by conventional means.
  • non-protein carriers can include conventional carriers used in the diagnostic field, e.g., polystyrene or other plastic beads, polysaccharides, e.g., as used in the BIAcore (Pharmacia) system, or other non-protein substances useful in the medical field and safe for administration to humans and animals.
  • Other effector agents may include a macrocycle, for chelating a heavy metal atom, or radioisotopes. Such effector agents may also be useful to increase the half-life of the altered antibodies, e.g., polyethylene glycol.
  • Combinatorial cloning is disclosed generally in PCT Publication No. WO90/14430. Simply stated, the goal of combinatorial cloning is to transfer to a population of bacterial cells the immunological genetic capacity of a human cell, tissue or organ. It is preferred to employ cells, tissues or organs which are immunocompetent. Particularly useful sources include, without limitation, spleen, thymus, lymph nodes, bone marrow, tonsil and perpherial blood lymphocytes. The cells may be optionally RSV stimulated in vitro, or selected from donors which are known to have produced an immune response or donors who are HIV+ but asymptomatic. The genetic information isolated from the donor cells can be in the form of
  • DNA or RNA is conveniently amplified by Polymerase Chain Reaction (PCR) or similar techniques.
  • PCR Polymerase Chain Reaction
  • the genetic information is preferably converted into cDNA by reverse transcription prior to amplification.
  • the amplification can be generalized or more specifically tailored. For example, by a careful selection of PCR primer sequences, selective amplification of immunoglobulin genes or subsets within that class of genes can be achieved.
  • the light and heavy chain genes are associated in random combinations to form a random combinatorial library.
  • Various recombinant DNA vector systems have been described to facilitate combinatorial cloning (see: PCT Publication No. WO90/14430 supra, Scott and Smith, Science 249:386-406 (1990) or U. S. Patent 5,223.409). Having generated the combinatorial library, the products can, after expression, be conveniently screened by biopanning with RSV F protein or, if necessary, by epitope blocked biopanning as described in more detail below.
  • Fab fragments of mAbs for combinatorial cloning and screening and then to convert the Fabs to full length mAbs after selection of the desired candidate molecules.
  • single chain antibodies can also be used for cloning and screening.
  • the present invention contemplates the use of Fab fragments or F(ab') 2 fragments to derive full-length mAbs directed against the F protein of RSV.
  • these fragments may be independently useful as protective and therapeutic agents in vivo against RSV-mediated conditions or in vitro as part of an RSV diagnostic, they are employed herein as a component of a reshaped human antibody.
  • a Fab fragment contains the entire light chain and amino terminal portion of the heavy chain; and an F(ab') 2 fragment is the fragment formed by two Fab fragments bound by additional disulfide bonds.
  • RSV binding monoclonal antibodies provide sources of Fab fragments and F(ab') 2 fragments and can be obtained via combinatorial phage library (see, e.g., Winter et aL, Ann. Rev. Immunol., 12:433- 455 (1994) or Barbas et al. ( Proc. Natl Acad. Sci. (USA) 89, 10164-10168 (1992)) which are both hereby incorporated by reference in their entirety).
  • Fab Hul9 or other antibodies described herein may contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR sequences, functional fragments, and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obtaining various altered antibodies which are characterized by the antigen binding specificity of the donor antibody.
  • the present invention thus provides variable light chain and variable heavy chain sequences from the RSV human Fab Hul9A-D and sequences derived therefrom.
  • nucleic acid sequences of this invention, or fragments thereof, encoding the variable light chain and heavy chain peptide sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incorporation of the resulting modified or fusion nucleic acid sequence into a plasmid for expression.
  • silent substitutions in the nucleotide sequence of the framework and CDR- encoding regions can be used to create restriction enzyme sites which would facilitate insertion of mutagenized CDR (and/or framework) regions.
  • These CDR- encoding regions may be used in the construction of reshaped human antibodies of this invention.
  • variable heavy and light chain amino acid sequences may be constructed which encode the variable heavy and light chain amino acid sequences, and CDR sequences of the invention as well as functional fragments and analogs thereof which share the antigen specificity of the donor antibody.
  • the isolated nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs can be used to produce altered antibodies, e.g., chimeric or humanized antibodies, or other engineered antibodies of this invention when operatively combined with a second immunoglobulin partner.
  • nucleic acid sequences encoding portions of the altered antibody and antibodies described herein
  • other such nucleic acid sequences are encompassed by the present invention, such as those complementary to the native CDR-encoding sequences or complementary to the modified human framework regions surrounding the CDR-encoding regions.
  • sequences include all nucleic acid sequences which by virtue of the redundancy of the genetic code are capable of encoding the same amino acid sequence as given in Figures 2 and 3.
  • Other useful DNA sequences encompassed by this invention include those sequences which hybridize under stringent hybridization conditions (See: T.
  • Altered immunoglobulin coding regions encode altered antibodies which include engineered antibodies such as chimeric antibodies, humanized, reshaped and immunologically edited human antibodies.
  • a desired altered immunoglobulin coding region contains CDR-encoding regions in the form of Fab regions that encode peptides having the antigen specificity of an RSV antibody, preferably a high affinity antibody such as provided by the present invention, inserted into an acceptor immunoglobulin partner.
  • the acceptor when the acceptor is an immunoglobulin partner, as defined above, it includes a sequence encoding a second antibody region of interest, for example an Fc region.
  • Immunoglobulin partners may also include sequences encoding another immunoglobulin to which the light or heavy chain constant region is fused in frame or by means of a linker sequence. Engineered antibodies directed against functional fragments or analogs of RSV may be designed to elicit enhanced binding with the same antibody.
  • the immunoglobulin partner may also be associated with effector agents as defined above, including non-protein carrier molecules, to which the immunoglobulin partner may be operatively linked by conventional means.
  • Fusion or linkage between the immunoglobulin partners, e.g., antibody sequences, and the effector agent may be by any suitable means, e.g., by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional cross- linkers, e.g., carbodiimide, glutaraldehyde, and the like.
  • suitable means e.g., by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional cross- linkers, e.g., carbodiimide, glutaraldehyde, and the like.
  • linker sequences which simply provide for a desired amount of space between the second immunoglobulin partner and the effector agent may also be constructed into the altered immunoglobulin coding region.
  • the design of such linkers is well known to those of skill in the art.
  • signal sequences for the molecules of the invention may be modified to enhance expression.
  • the reshaped human antibody having the signal sequence and CDRs derived from the Fab Hul9 heavy chain sequence may have the original signal peptide replaced with another signal sequence such as the Campath leader sequence (Page, M. J. et al., BioTechnology 9:64-68(1991)).
  • An exemplary altered antibody a reshaped human antibody, contains a variable heavy and the entire light chain peptide or protein sequence having the antigen specificity of Fab Hul9, fused to the constant heavy regions CH_ I -CH_3 derived from a second human antibody.
  • the engineered antibody of the invention may have attached to it an additional agent.
  • the procedure of recombinant DNA technology may be used to produce an engineered antibody of the invention in which the Fc fragment or CH2 CH3 domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule (i.e., a polypeptide effector or reporter molecule).
  • Another desirable protein of this invention may comprise a complete antibody molecule, having full length heavy and light chains, or any discrete fragment thereof, such as the Fab or F(ab'), fragments, a heavy chain dimer, or any minimal recombinant fragments thereof such as an F v or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor Fab Hul9.
  • Such protein may be used in the form of an altered antibody, or may be used in its unfused form.
  • an engineered antibody results.
  • Engineered antibodies can comprise immunoglobulin (Ig) constant regions and variable framework regions from one source, e.g., the acceptor antibody, and one or more (preferably all) CDRs from the donor antibody, e.g., the anti-RSV antibody described herein.
  • alterations, e.g., deletions, substitutions, or additions, of the acceptor mAb light and/or heavy variable domain framework region at the nucleic acid or amino acid levels, or the donor CDR regions may be made in order to retain donor antibody antigen binding specificity or to reduce potential immunogenicity.
  • a preferred mutation is the alteration of the consensus N-linked glycosylation site in CDR2 of the Hul9A and Hul9B heavy chain, as exemplified in the heavy chains of Hul9C and Hul9D (Fig. 2) (SEQ ID NO'S 7 and 8).
  • Such engineered antibodies are designed to employ one (or both) of the variable heavy and/or light chains of the RSV mAb (optionally modified as described) or one or more of the below-identified heavy or light chain CDRs.
  • the engineered antibodies of the invention are neutralizing, i.e., they desirably inhibit virus growth in vitro and in vivo in animal models of RSV infection.
  • Such engineered antibodies may include a reshaped human antibody containing the human heavy and light chain constant regions fused to the RSV antibody functional fragments.
  • a suitable human (or other animal) acceptor antibody may be one selected from a conventional database, e.g., the KABAT® database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • heterologous framework and constant regions are selected from human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA and IgE.
  • the Fc domains are not limited to native sequences, but include mutant variants known in the art that alter function. For example, mutations have been described in the Fc domains of certain IgG antibodies that reduce Fc-mediated complement and Fc receptor binding (see, e.g., A. R. Duncan et al., Nature 332:563- 564 (1988); A. R. Duncan and G. Winter, Nature 332:738-740 (1988); M.-L. Alegre et aL, J. Immunol.
  • the acceptor antibody need not comprise only human immunoglobulin protein sequences.
  • a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding a non-immunoglobulin amino acid sequence such as a polypeptide effector or reporter molecule.
  • the altered antibody thus preferably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use, e.g., treatment of RSV mediated diseases in man, or for diagnostic uses.
  • variable domain amino acids may be further modified by changes in variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody (i.e., an analog). It is anticipated that heavy and light chain amino acids may be substituted by other amino acids either in the variable domain frameworks or CDRs or both. Particularly preferred is the immunological editing of such reconstructed sequences as illustrated in the examples herein.
  • variable or constant region may be altered to enhance or decrease selective properties of the molecules of the instant invention, as described above.
  • dimerization binding to Fc receptors, or the ability to bind and activate complement (see, e.g., Angal et aL, Mol. Immunol, 30: 105-108 (1993), Xu et al., J. Biol. Chem, 269:3469-3474 (1994), Winter et al., EP 307,434-B).
  • Such antibodies are useful in the prevention and treatment of RSV mediated disorders, as discussed below.
  • the resulting reshaped human antibodies of this invention can be expressed in recombinant host cells, e.g., COS, CHO or myeloma cells.
  • a conventional expression vector or recombinant plasmid is produced by placing these coding sequences for the altered antibody in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell.
  • Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences, which can be derived from other known antibodies.
  • a second expression vector can be produced having a DNA sequence which encodes a complementary antibody light or heavy chain.
  • this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed.
  • the heavy and light chain coding sequences for the altered antibody may reside on a single vector.
  • a selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains.
  • the transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention.
  • the production of the antibody which includes the association of both the recombinant heavy chain and light chain is measured in the culture by an appropriate assay, such as ELISA or RIA.
  • Similar conventional techniques may be employed to construct other altered antibodies and molecules of this invention.
  • Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art.
  • the conventional pUC series of cloning vectors may be used.
  • One vector used is pUC19, which is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).
  • any vector which is capable of replicating readily has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning.
  • the selection of the cloning vector is not a limiting factor in this invention.
  • the vectors employed for expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vectors.
  • Preferred vectors include for example plasmids pCD or pCN.
  • the vectors also contain selected regulatory sequences (such as CMV promoters) which direct the replication and expression of heterologous DNA sequences in selected host cells.
  • These vectors contain the above described DNA sequences which code for the engineered antibody or altered immunoglobulin coding region.
  • the vectors may incorporate the selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation.
  • the expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR).
  • DHFR mammalian dihydrofolate reductase gene
  • Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro).
  • BGH bovine growth hormone
  • betaglopro betaglobin promoter sequence
  • replicons e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • selection genes e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
  • the present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or altered immunoglobulin molecules thereof.
  • Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. coli are used for replication of the cloning vectors and other steps in the construction of altered antibodies of this invention.
  • Suitable host cells or cell lines for the expression of the engineered antibody or altered antibody of the invention are preferably mammalian cells such as CHO, COS, a fibroblast cell (e.g., 3T3), and myeloid cells, and more preferably a CHO or a myeloid cell.
  • Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns.
  • other eukaryotic cell lines may be employed.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., cited above.
  • Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs of the present invention (see, e.g., Pl ⁇ ckthun, A., Immunol. Rev., 130: 151-188 (1992)).
  • the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form does not pose as great a concern as Fabs are not normally glycosylated and can be engineered for exported expression thereby reducing the high concentration that facilitates misfolding. Nevertheless, any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability.
  • bacterial cell If the molecule expressed by the bacterial cell was produced and exported in a properly folded form, that bacterial cell would be a desirable host.
  • E. coli used for expression are well-known as host cells in the field of biotechnology.
  • Various strains of 5. subtilis, Streptomyces, other bacilli and the like may also be employed in this method.
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.
  • the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the altered antibody of the invention from such host cell are all conventional techniques.
  • the altered antibodies of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention.
  • Yet another method of expression of resphaped antibodies may utilize expression in a transgenic animal, such as described in U. S. Patent No. 4,873,316.
  • This relates to an expression system using the animal's casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.
  • the engineered antibody is then examined for in vitro activity by use of an appropriate assay.
  • Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the altered antibody to RSV.
  • other in vitro assays and in vivo animal models may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the altered antibody in the body despite the usual clearance mechanisms.
  • This invention also relates to a method of treating humans experiencing RSV-related symptoms which comprises administering an effective dose of antibodies including one or more of the altered antibodies described herein or fragments thereof.
  • the therapeutic response induced by the use of the molecules of this invention is produced by the binding to RSV and thus subsequently blocking RSV propagation.
  • the molecules of the present invention when in preparations and formulations appropriate for therapeutic use, are highly desirable for those persons experiencing RSV infection. For example, longer treatments may be desirable when treating seasonal episodes or the like.
  • the dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient.
  • the altered antibodies, antibodies and fragments thereof of this invention may also be used alone or in conjunction with other antibodies, particularly human or humanized mAbs reactive with other epitopes on the F protein or other RSV target antigens as prophylatic agents.
  • the mode of administration of the therapeutic and prophylatic agents of the invention may be any suitable route which delivers the agent to the host.
  • the altered antibodies, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously, or intranasally.
  • Therapeutic and prophylactic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the altered antibody of the invention as an active ingredient in a pharmaceutically acceptable carrier.
  • compositions for parenteral administration will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved in an pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • an aqueous carrier e.g. 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration).
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • the concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 80 mg or more preferably, about 5 mg to about 75 mg of an engineered antibody of the invention.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 75 and preferably 5 to about 50 mg/ml of an engineered antibody of the invention.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania.
  • the therapeutic and prophylactic agents of the invention when in a pharmaceutical preparation, be present in unit dose forms.
  • the appropriate therapeutically effective dose can be determined readily by those of skill in the art.
  • one dose of approximately 0.1 mg to approximately 20 mg per 70 kg body weight of a protein or an antibody of this invention should be administered parenterally, preferably i.v. or i.m. (intramuscularly).
  • Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.
  • the altered antibodies and engineered antibodies of this invention may also be used in diagnostic regimens, such as for the determination of RSV mediated disorders or tracking progress of treatment of such disorders.
  • these altered antibodies may be conventionally labeled for use in ELISAs and other conventional assay formats for the measurement of RSV levels in serum, plasma or other appropriate tissue, or the release by human cells in culture.
  • the nature of the assay in which the altered antibodies are used are conventional and do not limit this disclosure.
  • the antibodies, altered antibodies or fragments thereof described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.
  • the heavy chain variable region and the light chain variable and constant regions from the Fab clone 19 plasmid (C. Barbas III et aL, Proc. Natl Acad. Sci. (USA) 89, 10164-10168 (1992) and PCT application Publication No. WO 94/06448, Application No. PCT/US93/08786 Cell Line Clone 19 referenced therein as ATCC Accession No. 69072) herein designated Hul9 Fab, were cloned into derivatives of plasmid PCDN (Nambi, A. etal., Molecular and Cellular Biochemistry 131:75-86 (1994), in which the expression of the antibody chain is driven by the CMV promoter.
  • Plasmid PCD-HC68B is used for cloning and expressing full length heavy chains and plasmid PCN-HuLC, for cloning and expressing full length light chains (Figure 1 shows the strategy for cloning of version A of the Hul9 mAb).
  • the variable region was extracted from the Hul9 Fab plasmid (C. Barbas III et al., Proc. Natl Acad. Sci. (USA) 89, 10164-10168 (1992)) as an Xho ⁇ -Bspl20l fragment and introduced into the same sites in PCD-HC68B.
  • the Xho ⁇ site was introduced at the amino terminus by the PCR primer and, when cloned into PCD- HC68B at the same site is preceded in frame by the Campath leader sequence (Page, J.M.
  • EVQLLEE (Fig. 2 SEQ ID NO 5, AMINO ACIDS 20 - 26), where the residues LE are encoded by the nucleotide sequence for the Xhol cloning site.
  • the complete nucleotide sequence for the plasmid Hul9AHcpcd is shown in Fig. 4A (SEQ ID NO 14).
  • sequence analysis revealed base differences from the published sequence (C. Barbas III et al., Proc. Natl Acad. Sci. (USA) 89, 10164-10168 (1992), PCT publication WO94/06448) within the heavy chain region from the Hul9 Fab plasmid.
  • variable and constant regions of the Hul9 Fab plasmid were cloned as a Sacl/Xbal fragment into the same sites in the pCN-
  • Both restriction sites correspond to restriction sites introduced by the primers used in the PCR amplification.
  • the Sacl site is introduced at the amino terminus by the PCR primer and, when cloned into pCN-HuLC at the same site, is preceded in frame by the Campath leader sequence (Page, J.M. et al., Biotechnology 9:64-68 (1991). The first 2 amino acids of the mature light chain are therefore deleted.
  • Hul9ALcpcn the first 2 amino acids immediately following the leader are EL (Fig. 3, part A), where the residues EL are encoded by the nucleotide sequence for the Sacl cloning site .
  • the PCR primer used at the carboxy terminus of the constant region introduces a nucleotide substitution which changes the amino acid at position 202 of the mature light chain, from a serine to a leucine (Fig 3, part B).
  • the Xba ⁇ restriction site introduced by the same PCR primer, lies outside the coding region and has no effect on the final amino acid sequence of the mature light chain.
  • the complete nucleotide sequence of the plasmid Hul9Apcn is shown in Fig. 4B.
  • the heavy chain there was a sequence discrepancy for the light chain between the published sequence (C. Barbas III et al., Proc. Natl Acad. Sci.
  • Glycine but not glutamic acid, is encoded at this position in a human J germline J mini-gene and glutamic acid was not observed among a large collection of human antibody sequences (Kabat et al., "Sequences of Proteins of Immunological Interest", fifth edition, NIH Publication No. 91-3242, 1991). Also as for the heavy chain, the glycine encoding sequence was observed for 3 separate clonings from the original Fab 19 vector (Barbas et al., Proc. Natl Acad. Sci. (USA) 89, 10164-10168 (1992), PCT publication WO94/06448). These results demonstrate that the originally published sequence for Fab 19 light chain is in error. The Hul9AHcpcd and Hul9ALcpcn set of vectors were used to produce antibody Hu 19A in COS cells and in CHO cells.
  • variable region of the Fab 19 heavy chain In cloning the variable region of the Fab 19 heavy chain, non-consensus amino acid changes relative to the predicted germline sequence were introduced at the amino terminus by the PCR primer (C. Barbas III et al., Proc. Natl Acad. Sci. (USA) 89, 10164-10168 (1992)). To determine the likely amino terminus of the heavy chain, the peptide sequence of the variable region of the Fab 19 heavy chain was aligned with all known human germline heavy chain sequences. Based on the results of this alignment, the germline amino terminus is predicted to be either QVQLVE or EVQLVE rather than the sequence EVQLLEE present in version A.
  • the original Fab clone 19 heavy chain peptide was aligned with human heavy chain sequences previously cloned at SmithKline Beecham.
  • a clone designated 97B27 which was obtained via PCR amplification from the beginning of its leader sequence, had the acceptable N-terminus of QVQLVE and was used to replace this region in the Fab 19 heavy chain.
  • the Fab 19 heavy chain in the Hul9 Fab plasmid was PCR amplified using a constant region primer which spanned the naturally occurring BspI201 site at the beginning of CHI, and a variable region primer which created a Pvull site (corresponding to the site naturally occurring in clone 97B27) at amino acids 3 and 4 of the mature protein.
  • This primer also introduced changes in the coding sequence at the amino terminus of the Fab 19 heavy chain, coding for the amino acid sequence of QLVE for amino acids 3-6 instead of QLLEE, as in the version A construct.
  • Hul9BHcpcd contained the leader and first 3 amino acids of the variable region of clone 97B27 and coded for the consensus sequence QVQLVE at its amino terminus (Fig. 2).
  • the nucleotide sequence of Hul9BHcpcd is shown in Fig. 4C (SEQ ID NO: 20) for the region encoding the heavy chain. Sequences differing from Hu 19 AHcpcd are bolded.
  • variable region of the Fab clone 19 light chain changes were introduced at the amino terminus for cloning purposes, by the PCR primer, such that the first 4 amino acids of the Fab 19 light chain are EIEL.
  • the peptide sequence of the variable region of the Fab 19 light chain was aligned with all known human germline kappa chain sequences. Based on the results of this alignment, the germline amino terminus is predicted to be DIQM.
  • Fab 19 light chain was aligned with human kappa chain sequences previously cloned at SB.
  • a Clone designated AG1-37 which is the kappa chain obtained from cell line AG1-37 obtained by PCR amplification from the middle of its leader sequence, had the desired N-terminus and was used to introduce the corrections into the Fab 19 light chain.
  • the N-terminal portion of the leader sequence was provided by the expression vector and was the consensus sequence for this family of leader regions.
  • the light chain coding region was excised from the Hul9 Fab vector (Fig. 1) as a HinfiJXba I fragment. Hinfl recognizes a site which spans amino acids 18 an 19 of the mature protein and is also present in clone AG1-37.
  • the HinfllXbal fragment of the Fabl9 light chain was ligated to the Hinfl site in clone AG1-37.
  • the final construct consisted of the leader and first 18 amino acids of the AG1-37 variable region linked to the variable and constant regions of the Fab 19 light chain, beginning at amino acid 19 of the V-region.
  • the resulting clone designated Hul9BLcpcn, is altered only in the region encoding the first four amino acids of the variable region, coding for the consensus sequence DIQM (SEQ ID NO: 11, AMINO ACIDS 21 - 24) instead of EIEL present in version A (Fig. 3A).
  • Fig. 4D The nucleotide sequence for plasmid Hul9BLcpcn is shown in Fig. 4D (SEQ ID NO: 22) for the region encoding the light chain. Sequences differing from Hul9ALcpcn are bolded.
  • Hul9BHcpcd and Hul9BLcpcn were used to produce antibody Hul9B in COS cells and in CHO cells.
  • N-linked glycosylation site is encoded within the CDR2 loop of the heavy chain. This glycosylation adds the potential for heterogeneity in the mAb produced in eucaryotic cells and for interference in binding antigen.
  • mutations were introduced separately at two different residues via PCR overlap technology. For the first mutation the serine at position 61 of the mature Hul9B heavy chain was substituted with alanine, to create Hul9C heavy chain. For the second substitution, the asparagine at position 59 was changed to glutamine, to create Hul9D heavy chain.
  • the mutations were introduced via the PCR overlap technique using one set of primers encoding the mutation and a second set of primers annealing to sequences within the CMV promoter and the CH2 constant region in plasmid Hul9Bpcd, as the outside 5' and 3' primers, respectfully.
  • the final PCR product was digested with restriction enzymes, EcoRl and BspllO ⁇ , and cloned into the Hul9BHcpcd vector at the same sites to create Hul9CHcpcd (Ser to Ala mutation) and Hul9DLcpcd (Asp to Gin mutation ) (Fig. 2).
  • the final constructs were sequenced to verify that the mutations were present.
  • Plasmid Lcvector4 a pucl8 derivative containing a normal human kappa constant region with a Xbal site just distal to the coding region, was cut with Bbsl and Xbal and a 321 bp fragment containing the nucleotide sequence coding for the entire kappa constant region beginning at amino acid 6 was isolated. This fragment contains the naturally occurring Sacl site near the end of the carboxy terminus and codes for a serine at position 202. Plasmid Hul9BLcpcn was also cut with EcoRl and Xba 1 and a 4947 bp fragment, containing the remainder of the vector sequence from plasmid Hul9BLcpcn, was isolated.
  • Hul9CLcpcn The three fragments were ligated together to create Hul9CLcpcn.
  • the amino acid sequence of the Hul9C light chain is shown in Figs. 3 A and 3B (S ⁇ Q ID NO'S 1 1 and 12) and the nucleotide sequence of the light chain region is shown in Fig 4G (S ⁇ Q ID NO: 29). Differences from Hul9ALcpcn are bolded.
  • the vector Hul9CLcpcn was used with Hul9CHcpcd or Hul9DHcpcd to produce antibody Hul9C and Hul9D, respectively, in COS cells and in CHO cells.
  • Hul9A heavy and light chain Hul9B heavy and light chain
  • Hul9C heavy and light chain Hul9D heavy with Hul9C light chain
  • Hul9D heavy with Hul9C light chain were expressed in COS cells essentially as described in Current Protocols in Molecular Biology (edited by F. M. Ausubel et al. 1988, John Wiley & Sons, vol. 1, section 9.1).
  • the culture growth medium was replaced with a serum-free medium which was changed on day 3.
  • the serum-free medium was a proprietary formulation but satisfactory results are obtained using DMEM supplemented with ITSTM Premix (insulin, transferrin, selenium mixture - Collaborative Research, Bedford, MA) and 1 mg/ml BSA.
  • the mAb was prepared from the day 3 + day 5 conditioned medium by standard protein A affinity chromatography methods (e.g., as described in Protocols in Molecular Biology) using, for example, Prosep A affinity resin (Bioprocessing Ltd., UK). To produce larger quantities of the Hul9 mAb (100-200 mgs), the vectors were introduced into a proprietary CHO cell system. However, similar results will be obtained using dhfr CHO cells as previously described (P.
  • the antibody was purified from conditioned medium by standard procedures using protein A affinity chromatography (Protein A sepharose, Pharmacia) followed by size exclusion chromatography (Superdex 200, Pharmacia). The concentration and the antigen binding activity of the eluted antibody are measured by ELISA. The antibody containing fractions are pooled and further purified by size exclusion chromatography. As expected for any such antibody, by SDS-PAGE, the predominant protein product migrated at approximately 150 kDa under non-reducing conditions and as 2 bands of 50 and 25 kDa under reducing conditions. For antibody produced in CHO cells, the purity was > 90%, as judged by SDS-PAGE, and the concentration was accurately determined by amino acid analysis.
  • Example F Preparation of Fab from Hul9B mAb: Samples with and without glycosylation in heavy chain CDR2
  • mAb was purified using an essentially similar purification procedure that is detailed here for mAb 19B.
  • Conditioned media (2L) from a 6 day culture was harvested, sterile filtered and applied to a 2.5 X 5.1cm Protein A (Pharmacia, fast flow) equilibrated in 20mM sodium phosphate, 150mM sodium chloride, pH 7
  • mAb 19B resolved as 2 major bands at 52 kDa and 28kDa corresponding to the heavy and light chains of IgG respectively, with an additional band at 59 kDa representing about 7% of the total protein (Fig. 5).
  • LC/mass spectrometry analysis of the two heavy chains following excision from an SDS- PAGE and proteolytic digestion confirmed that the 59 kDa species represented an additional glycoform of mAb 19B that contained carbohydrate at the predicted VJJ glycosylation site.
  • reduced SDS-PAGE analysis of mAb 19C (Fig. 5), in which this VJJ glycosylation site is removed, showed that this mAb contains only the lower molecular weight (52 kDa) heavy chain species, as expected.
  • the Hul9B construct contains an additional consensus sequence for N-linked glycosylation in the variable region of the heavy chain, -Asn 59 -Tyr-Ser-, in addition to the normal glycosylation site in the C H 2 domain of the heavy chain, -Asn 299 -Ser- Thr-.
  • Analysis of both heavy chain bands by liquid chromatography, electrospray mass spectrometry (LC-ELMS) following reduction, alkylation, and tryptic digestion revealed that the 59 kDa band contains a variant that is glycosylated at Asn 59 in addition to being glycosylated at Asn 299 .
  • the carbohydrate at Asn 59 is predominantly biantennary, core fucosylated carbohydrates having two sialic acid residues. This is a common carbohydrate structure found in CHO-expressed glycoproteins (such as sCR-1 and sCD4), but it differs from the carbohydrate found at the Asn299 site which lacks sialic acid altogether.
  • glycosylated and unglycosylated Fab from cleaved mAb 19B was dialyzed against 20 mM sodium acetate, pH 4.5 and applied (4mg) to a 0.5 X 5cm Mono S column (Pharmacia) at 300cm/h equilibrated with 20mM sodium acetate buffer, pH 4.5. The column was then washed with equilibration buffer and eluted isocratically with the equilibration buffer containing 100 mM NaCl. Glycosylated Fab eluted after 5 column volumes whereas the unglycosylated FAb was retained longer, eluting after 6 column volumes.
  • Binding of the various antibody constructs to recombinant F protein was measured in a standard solid phase ELISA.
  • Antigen diluted in PBS pH 7.0 was adsorbed onto polystyrene round-bottom microplates (Dynatech, Immunolon II) for 18 hours. Wells were then aspirated and blocked with 0.5% boiled casein (BC) in PBS containing 1% Tween 20 (PBS/0.05% BC) for 2 hours.
  • Antibodies (50 ⁇ l/well) were diluted to varying concentrations in PBS/0.5% BC containing 0.025% Tween 20 and incubated in antigen coated wells for one hour.
  • the antigen binding epitope of Fab 19 and mAb construct 19B were examined in a competition ELISA.
  • the test antibody construct was mixed with increasing concentrations of RSMU19 or B4 and added to F protein-coated wells.
  • the epitope regions recognized by mAbs RSMU19 and B4 have been previously described in Arbiza et al., J. Gen'l Virol. 73:2225-34 (1992).
  • the concentration of Fab 19 or mAb 19B used in competition studies was determined previously to give 90% maximal binding to F antigen. Binding of Fab 19 or mAb 19B in the presence of other mAbs was detected using HRP-labelled goat anti-human IgG. The reaction was developed as stated above.
  • Fab 19 and amAb constructs 19A or 19B demonstrated equivalent binding to rF protein based on molar concentrations. Binding of Fabl9 or mAb 19B to rF (recombinant F) protein was inhibited by mAb B4 but not by RSMU19 indicating that the epitope region recognized by these constructs is localized to region aa 255- 275 of the F protein (Table 1). Table 1: Viral F Protein Epitope Recognized by mAb 19B
  • the mAb 19B also showed specific binding to RSV infected cells indicating recognition of the F protein as displayed in its native form.
  • VERO cells infected with approximately 50 TCID50 RS Long virus were fixed in 90% methanol when CPE reached > 90% and were used as antigen in the ELISA format described above. Binding of biotinylated mAb 19B was detected with HRP-labelled -Streptavidin. In this assay, the EC50 for mAb 19B was 34 +/- ng/ml.
  • Fab fragments to inhibit virus-induced cell fusion was determined using a modification of the in vitro microneutralization assay described by Beeler et al (J. of Virology 63: 2941-2950 (1989)).
  • 50 ul of RS Long strain virus (approximately 100 TCID5Q/well; American Type Culture Collection ATCC VR-26) were mixed with 0.1 ml VERO cells (5 x 10 /well; ATCC CCL-81) in Minimum Essential Media (MEM) containing 2% FCS, for 4 hours at 37°C, 5% CO2- Serial two-fold dilution (in duplicate) of test samples (50 ul) were then added to wells containing virus-infected cells.
  • MEM Minimum Essential Media
  • Control cultures contained cells incubated with virus only (positive virus control) or cells incubated with media alone. Cultures were incubated at 37°C in 5% CO2 for 6 days at which time cytopathic effects (CPE) in virus control wells were > 90%. Neutralization assays were performed as described above except that serial dilutions of test samples were mixed with 100 TCID50 of RS virus (50 ul each) for 2 hours at 37°C in 5% CO2 before the addition of VERO cells (5 x 10 3 ).
  • Fusion-inhibition or neutralization titers were defined as the reciprocal dilution of test sample, or concentration of antibody, which caused a 50% reduction in ELISA signal (ED50) as compared to virus controls. Based on the curve generated in the ELISA by the standard virus titration, a 50% reduction in O.D.450 in wells corresponded to > 90% reduction in virus titer. To determine the ED50, mean absorbance for replicate cultures (per dilution of test sample) was plotted against dilution of sample. Calculation of the 50% point, defined as (mean absorbance virus-infected cells + mean absorbance uninfected cells)/2, was based on regression analysis of the dose titration.
  • SB 209763 is a humanized derivative of RSMU19 as described in P. R. Tempest et al., Biotechnology 9, 266-271 (1991). To determine the effects of coadministration of mAbl9B and SB 209763 on in vitro fusion-inhibition, the antibodies were titrated alone and in combination. Antibody interactions were analyzed using MacSynergy TM II software.
  • the antiviral titers of the mAb constructs were approximately 5 to 10-fold lower than the titers obtained with the corresponding Fab constructs - Fab 19, Fab 19 A or B (Table 2).
  • Fab 19 is the original Fab protein produced directly in E. coli from the clone 19 plasmid,whereas Fabl9A and Fabl9B were derived by papain cleavage from the coresponding full length mAbs. Removal of the N-linked glycoslation site encoded within the CDR2 loop of the heavy chain by cloning had no effect on the overall antiviral activity of the mAb (Table 2; construct C compared to A and B).
  • mice (5/group) were inoculated intraperitoneally with doses ranging from 0.06 mg/kg to 5 mg/kg of mAb 19B either 24 hours prior (prophylaxis) or 4 days after (therapy) intranasal infection with 10 ⁇ PFU of the A2 strain of human RSV. Mice were sacrificed 5 days after infection. Sera was obtained to determine antibody levels and lungs were homogenized to determine virus titers. Virus was undetectable in the lungs of mice treated prophylactically with > 1.25 ug mAb 19B, and corresponding serum concentrations of > 5 ug/ml (Table 5). Higher doses of mAb 19B were required for complete viral clearance when mAb was administered therapeutically (5 mg/kg). Table 5: mAb 19B Prophylaxis and Therapy in Balb/c Mice
  • Hul9 antibodies have potent antiviral activity in vitro against a broad range of native RSV isolates of both type A and B, and show prophylactic and therapeutic efficacy in vivo in animal models.
  • the Hul9 antibodies most preferably Hul9C or Hul9D, are candidates for therapeutic, prophylactic, and diagnostic application in man.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 610-270-5968 (B) TELEFAX: 610-270-5090
  • Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
  • Tyr Tyr Cys Ala Thr Ala Pro lie Ala Pro Pro Tyr Phe Asp His Trp 115 120 125 Gly Gin Gly Thr Leu Val Thr Val Ser Ser
  • CTAGTTGCCA GCCATCTGTT GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG 2520
  • CTTTAGAGAC AGTGTTCTCT GCACAGATAA GGACAAACAT TATTCAGAGG GAGTACCCAG 2880 AGCTGAGACT CCTAAGCCAG TGAGTGGCAC AGCATTCTAG GGAGAAATAT GCTTGTCATC 2940
  • TCGCGCCAAA CTTGACGGCA ATCCTAGCGT GAAGGCTGGT AGGATTTTAT CCCCGCTGCC 3180 ATCATGGTTC GACCATTGAA CTGCATCGTC GCCGTGTCCC AAAATATGGG GATTGGCAAG 3240
  • CTCAAAGAAC CACCACGAGG AGCTCATTTT CTTGCCAAAA GTTTGGATGA TGCCTTAAGA 3480 CTTATTGAAC AACCGGAATT GGCAAGTAAA GTAGACATGG TTTGGATAGT CGGAGGCAGT 3540
  • MOLECULE TYPE protein
  • GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGC TTTGCATACT TCTGCCTGCT 300 GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACAT TCCACAGAAT TAATTCCCGG 360
  • TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT 3420 AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT 3480
  • CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG
  • GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC 5280 GACCGAGTTG CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT 5340
  • CGCCAGGCTC CAGGGAAGGG GCTGGAGTGG GTCTCATCCA TTACTGGAGG TAGCAACTTC 240
  • CGCCAGGCTC CAGGGAAGGG GCTGGAGTGG GTCTCATCCA TTACTGGAGG TAGCAACTTC 240
  • CGCCAGGCTC CAGGGAAGGG GCTGGAGTGG GTCTCATCCA TTACTGGAGG TAGCAACTTC 240

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention se rapporte à de nouveaux anticorps monoclonaux humains (mAbs) et aux gènes codant pour lesdits mAbs. Plus particulièrement, l'invention concerne des anticorps monoclonaux humains qui réagissent spécifiquement avec un épitope de la protéine de fusion (F) du virus respiratoire syncytial (VRS). Ces anticorps peuvent être utilisés dans le traitement thérapeutique et/ou prophylactique des patients infectés par le VRS, en particulier des nourrissons et de jeunes enfants.
EP97913749A 1996-11-01 1997-10-23 Anticorps monoclonaux humains Withdrawn EP0977590A4 (fr)

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US3014996P 1996-11-01 1996-11-01
US30149P 1996-11-01
PCT/US1997/019203 WO1998019704A1 (fr) 1996-11-01 1997-10-23 Anticorps monoclonaux humains

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Cited By (1)

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US9963500B2 (en) 2015-10-29 2018-05-08 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus

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CO5280147A1 (es) * 1999-05-18 2003-05-30 Smithkline Beecham Corp Anticuerpo humano monoclonal
EP1278545A1 (fr) * 2000-05-03 2003-01-29 Medimmune, Inc. Polytherapie de maladies respiratoires utilisant des anticorps
CA2466025A1 (fr) * 2001-11-02 2003-08-07 Centocor, Inc. Proteines du vrs, anticorps, compositions, procedes et utilisations
GB0210121D0 (en) 2002-05-02 2002-06-12 Celltech R&D Ltd Biological products
US7070786B2 (en) 2003-06-06 2006-07-04 Centocor, Inc. RSV proteins, antibodies, compositions, methods and uses
SI2285408T1 (sl) 2008-06-05 2019-02-28 Ablynx N.V. Aminokislinska zaporedja usmerjena proti proteinom ovojnicam virusa in polipeptidi, ki zaporedja vsebujejo za zdravljenje virusnih bolezni
JP5575377B2 (ja) 2008-07-18 2014-08-20 シスメックス株式会社 抗ｒｓウイルスモノクローナル抗体を用いたｒｓウイルス検出用キット及びイムノクロマトグラフィー用試験具、並びに新規な抗ｒｓウイルスモノクローナル抗体
ES2643034T3 (es) 2009-06-05 2017-11-21 Ablynx N.V. Construcciones de Nanobody contra el virus respiratorio sincicial humano (VRSH) trivalentes para la prevención y/o el tratamiento de infecciones de las vías respiratorias
WO2011064382A1 (fr) 2009-11-30 2011-06-03 Ablynx N.V. Séquences d'acides aminés améliorées dirigées contre le virus syncytial respiratoire humain (hrsv) et polypeptides comprenant celles-ci pour la prévention et/ou le traitement d'infections du tractus respiratoire

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WO1994006448A1 (fr) * 1992-09-16 1994-03-31 The Scripps Research Institute Anticorps monoclonaux neutralisateurs humains contre le virus respiratoire syncytial

Patent Citations (1)

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WO1994006448A1 (fr) * 1992-09-16 1994-03-31 The Scripps Research Institute Anticorps monoclonaux neutralisateurs humains contre le virus respiratoire syncytial

Non-Patent Citations (2)

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Title
See also references of WO9819704A1 *
WYDE P R ET AL: "Evaluation of the protective efficacy of reshaped human monoclonal antibody RSHZ19 against respiratory syncytial virus in cotton rats." PEDIATRIC RESEARCH, (1995 OCT) 38 (4) 543-50. , XP000972993 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9963500B2 (en) 2015-10-29 2018-05-08 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US10072072B2 (en) 2015-10-29 2018-09-11 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US10323079B2 (en) 2015-10-29 2019-06-18 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US10358480B2 (en) 2015-10-29 2019-07-23 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US11008380B2 (en) 2015-10-29 2021-05-18 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US11566065B2 (en) 2015-10-29 2023-01-31 Merck Sharp & Dohme Llc Antibody neutralizing human respiratory syncytial virus
US11981726B2 (en) 2015-10-29 2024-05-14 Merck Sharp & Dohme Llc Antibody neutralizing human respiratory syncytial virus

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