AU679440B2 - Antibodies for treatment and prevention of respiratory syncytial virus infection - Google Patents

Antibodies for treatment and prevention of respiratory syncytial virus infection Download PDF

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AU679440B2
AU679440B2 AU39000/93A AU3900093A AU679440B2 AU 679440 B2 AU679440 B2 AU 679440B2 AU 39000/93 A AU39000/93 A AU 39000/93A AU 3900093 A AU3900093 A AU 3900093A AU 679440 B2 AU679440 B2 AU 679440B2
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Edward James Stott
Geraldine Taylor
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SCOTGEN Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C07K2317/30—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Description

CORRECED
VERSION 6Mi LT aYO/9 PCf WORLD INTELLECTUAL PROPERTY ORGANIZATION P fr Innntr i nntrin i Rot I pages 108-131; description, and pages 132-139, claims, added INTERNATIONAL APPLICATION PUBLISHbU UNIUbK I Hh PAlN I LUUrV eATION TREATY (PCT) (51) International Patent Classification 5 International Publication Nutniber: WO 93/20210 C12N 15/62, 15/13, C07K 13/00 Al A61K 39/42, C12P 21/08 (43) International Publication Date: 14 October 1993 (14.10.93) (21) International Application Number: PCT/GB93/00725 (74) Agent: VALENTINE, Jill, Barbara; SmithKline Beecham, Corporate Patents, Great Burgh, Yew Tree Bottom (22) International Filing Date: 6 April 1993 (06.04.93) Road, Epsom, Surrey KT18 5XQ (GB).
Priority data: (81) Designated States: AU, CA, JP, KR, NZ, US, European 9207479.8 6 April 1992 (06.04.92) GB patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).
(71) Applicant (for all designated States except US): SCOTGEN LIMITED [GB/GBJ; Queen's House, 2 Holly Road, Published Twickenham, Middlesex TWI 4EG With international search report.
Before the expiration of the time limit for amending the (72) Inventors; and claims and to be republished in the event of the receipt of Inventors/Applicants (for US only) TAYLOR, Geraldine amendments.
[GB/GB]; Robingrove, Wallingford Road, Compton, Berkshire RG16 OPT STOTT, Edward, James [GB/GB]; New Henrietta Farm, Mophetts Lane, Down End, Chievely, Berkshire RG16 8TN (GB).
(54)Title: ANTIBODIES FOR TREATMENT AND PREVENTION OF RESPIRATORY SYNCYTIAL VIRUS INFEC-
TION
(57) Abstract The present invention provides variable light chain and variable heavy chain sequences derived from b"':ine anti.RSV F protein monoclonal antibodies (mAbs), B4 and B13/B 14, and CDR peptides therefrom, which may be employed in the design of fusion proteins (including altered antibodies) which are charaterized by the antigen binding specificity of these mAbs. Also described is a humanized antibody containing bovine variable chain sequences. Methods for producing and using these compositions, including pharmaceutical compositions are disclosed.
(eerdcd to In PCT CauiIw No. 06/1994, S".1ion II) WO 93/20210 PerGB93/007255 ANTIBODIES FOR TREATMENT AND PREVENTION OF RESPIRATORY SYNCYTIAL VIRUS INFECTION Field of the Invention This invention relates generally to the field of monoclonal, and recombinant, humanized antibodies, and specifically, to antibodies directed to epitopes on Respiratory Syncytial Virus.
Background of the Invention Respiratory syncytial virus (RSV) is a pneumovirus of the family Paramyxoviridae and is the major cause of severe lower respiratory tract infections in children and calves during the first year of life [Kim et al., Amer. J.
EpidemiQl., l.:216-225 (1973); Stott et al., J. Hygiene, 5.:257-270 (1980); McIntosh and Chanock, in B. N. Fields et al. (eds), YMilDJg, Raven Press, New York (1990)]. Human and bovine strains of RSV are antigenically distinct, but closely related, and two subgroups (A and B) of both human and bovine strains have been identified [Lerch et al., Jl Vir~Oa £:833-840 (1989); Anderson et al., J. Tnfect. Dis., 1i.: 626-633 (1985)].
The use of anti-RSV antibodies for treatment of RSV in murine and bovine species has been suggested. However, the treatment of non-murine or non-bovine species is potentially limited by the immune response of these species to the foreign murine or bovine antibodies. For example, immune responses in humans against murine antibodies have been shown to both immunoglobulin constant and variable regions.
There remains a need in the art to identify specifically the protective epitopes on RSV proteins AiMd the immune effector mechanisms that protect against infection, and to produce and characterize RSV antigens, epitopes and antibodies thereto for use in safe, effective RSV vaccines.
Summary of the Tnvention SUBSTITUTE SHEET WO 93/20210 PC/GB93/00725 2 The present invention provides a variety of anti-RSV antibodies, functional fragments thereof including CDRs.
These antibodies and fragments are useful in the construction of fusion proteins, particularly chimeric and humanized antibodies, which are characterized by the binding specificity and/or neutralizing activity of an anti-RSV monoclonal antibody (mAb). Also provided is a novel humanized antibody containing bovine antibody variable sequences in association with human immunoglobulin framework and constant regions. Methods for producing these products, which further include therapeutic and pharmaceutical compositions for treating R&V are also disclosed.
Other aspects and advantages of the present invention are described in the following detailed description.
Brief Description of the Drawinga Fig. 1 is a graph illustrating the isolation of recombinant LF1/1298, which contains the RSV Long strain F glycoprotein cDNA with a single transversion C to A at nucleotide 1298, cloned in the polylinker of pGEM4, This recombinant permits expression of the F protein in selected host cells.
Fig. 2 is a diagram of the F glycoprotein primary structure denoting the hydrophobic regions the site of proteolytic processing the potential sites for Nglycosyl'tion the cysteine residues and the amino acid residues which are changed in neutralization escape yu ants The locations of the trypsin fragments recognized by different mAbs are shown below the diagram.
Figs. 3A and 3B compare partial B4 and B13/B14 antibody variable light (VL) chain amino acid sequences (SEQ ID NOS: 1 and The B4 sequence is reported above the B13/B14 sequence to more readily illustrate comparison between the sequences. In the sequences, the symbol represents a gap in the sequence introduced to improve the alignment SUBSTITUTE SHEET WO 93/20210 PC/GB93/00725 3 between the sequences. The CDRs are boxed. The underlined sequences correspond to the sequences of the polymerase chain reaction (PCR) oligonucleotide primers used in amplifying these antibody sequences.
Figs. 4A and 4B compare partial B4 and B13/B14 antibody variable heavy (VH) chain amino acid sequences [SEQ ID NOS: 3 and 4] with the B4 sequence reported above the B13/B14 sequence. The symbol CDRs and PCR oligonucleotide primers sequences are defined and illustrated as in Figs. 3A and 3B.
Fig. 5 is a bar diagram showing the competitive binding of 10 anti-F bovine mAbs, labelled with 1251, to the A2 strain of RSV in the presence of increasing amounts of unlabelled antibodies. "Neut" represents the ability of the mAb to neutralize the RSV in a plaque neutralization assay.
"FI" refers to the ability of the antibody to inhibit fusion of multinucleated giant cells in an assay. "Protection" refers to whether the mAb was able to protect mice against RSV infection in an in vivo assay. Symbols: less than 11 to 80% (cross-hatched box), or greater than 80% (D) remaining bound at the highest amount of competing antibody tested.
Fig. 6 is a bar diagram showing the competitive binding of anti-F murine mAbs. "Neut", "Protection" and the symbols are defined as in Fig. Fig. 7 is a bar diagram showing the binding of anti-F mAbs to the RSV A2 strain and antibody-escape mutant RSVs.
The antibodies were tested in an ELISA using the purified viruses indicated at the top of the figure to coat microtitre plates. Symbols: less than 20% 20 to (cross-hatched box), greater than 80% of the absorbance values obtained with the A2 strain.
Fig. 8 is a bar diagram showing the binding of anti-F mAbs to RSV Long strain and antibody-escape mutant RSVs.
SUBSTITUTE SHEET WO 93/20210 PCI'/G1B93/00725 4 The antibodies were tested as described in Fig. 7. Symbols: less than 25% (open box), 25 to 50% (cross-hatched box), greater than 50% of the absorbance values obtained with the Long strain.
Fig. 9 is a series of 8 bar diagrams showing the binding of mAb B4 to synthetic octomeric peptides, bound to polyethylene pins, where each amino acid in the sequence corresponding to amino acid #266 through 273 of the RSV F protein [SEQ ID NO: 19] has been replaced in turn with other amino acids (indicated on the abscissa). The sequence of amino acids beneath each bar diagram shows which amino acid has been replaced (indicated by a box around the letter).
The antibody binding was tested in an ELISA and the black bars represent the absorbance values obtained with the native sequence of the peptide and the grey bars represent the absorbance obtained with the peptides containing the substituted amino acids.
Fig. 10 is a predicted humanized VH region sequence B4HuVH wherein bovine mAb B4 is the donor antibody (SEQ ID NO: CDRs are boxed. Underlined residues in the framework regions are murine residues which have been retained.
Fig. 11 is a predicted humanized constant heavy region sequence B4HuVK for use in constructing an altered antibody, wherein B4 is the donor antibody [SEQ ID NO: CDRs are boxed.
_._Figs. 12A and 12B provide a contiguous predicted humanized VH region sequence B13/Bl4HuV (SEQ ID NO: 7] for use in constructing an altered antibody, wherein B13/B14 is the donor antibody. CDRs are boxed and retained murine residues are underlined.
Fig. 13 is a predicted humanized constant heavy region sequence B13/Bl4HuVK (SEQ ID NO: 8] wherein B13/B14 is the donor antibody. CDRs are boxed.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 Figs. 14A and 14B provide a contiguous DNA sequence and corresponding amino acid sequence [SEQ ID NOS: 9 and 10) for the VH region of RSV19. CDRs are boxed. Underlined sequences correspond to the primers used.
Figs. 15A and 15B provide a contiguous DNA sequence and corresponding amino acid sequence of the RSV19 VL region [SEQ ID NOS: 11 and 12]. CDRs are boxed. Primers are underlined.
Fig. 16 shows th, plasmid pHuRSV19VH comprising a human Ig VH region framework and CDRs from murine RSV19.
Fig. 17 shows the plasmid pHuRSV19VK comprising a human Ig VL framework and CDRs derived from RSV19.
Fig.
sequences Fig.
sequences Fig.
sequences Fig.
sequences Fig.
sequences Fig.
framework 18 shows the derived Ig variable region amino acid encoded by murine RSV19VH [SEQ ID NO: 13].
19 shows the derived Ig variable region amino acid encoded by pHuRSV19VH [SEQ ID NO: 14].
20 shows the derived Ig variable region amino acid encoded by pHuRSV19VHFNS [SEQ ID NO: 21 shows the derived Ig variable region amino acid encoded by pHuRSV19VHNIK [SEQ ID NO: 16].
22 shows the derived Ig variable region amino acid encoded by pHuRSV19VK [SEQ ID NO: 17].
23 is the DNA and amino acid encoding the HuVL 4, [SEQ ID NOS: 20 and 21} showing the potential silice site. The underlined bases were changed to provide the genuine J1 gene sequence [SEQ ID NO: 22].
Detaided Description of the Invention I. Definitions.
As used herein, the term "first fusion partner" refers to a nucleic acid sequence encoding an amino acid sequence, which can be all or part of a heavy chain variable region, light chain variable region, CDR, functional fragment or analog thereof, having the antigen binding specificity of a selected antibody, preferably an anti-RSV antibody.
SUBSTITUTE SHEET WO 93/20210 PCT/Ge B93/00725 6 As used herein the term "second fusion partner" refers to another nucleotide sequence encoding a protein or peptide tc which the first fusion partner is fused in frame or by means of an optional conventional linker sequence. Such second fusion partners may be heterologous to the first fusion partner. A second fusion partner may include a nucleic acid sequence encoding a second antibody region of interest, an appropriate human constant region or framework reion.
The term "fusion molecule" refers to the product of a first fusion partner operatively linked to a second fusion partner. "Operative linkage" of the fusion partners is defined as an association which permits expression of the antigen specificity of the anti-RSV sequence (the first fusion partner) from the donor antibody as well as the desired characteristics of the second fusion partner. For example, a nucleic acid sequence encoding an amino acid linker may be optionally used, or linkage may be via fusion in frame to the second fusion partner.
The term "fusion protein" refers to the result of the expression of a fusion molecule. Such fusion proteins may be altered antibodies, chimeric antibodies, humanized antibodies, or any of the antibody regions identified herein fused to immunoglobulin or non-immunoglobulin proteins and the like.
As used herein, the term "donor antibody" refers to an antibody (polyclonal, monoclonal, or recombinant) which contributes the nucleic acid sequences of its naturallyoccurring or modified variable light and/or heavy chains, CDRs thereof or other functional fragments or analogs thereof to a first fusion partner, so as to provide the fusion molecule and resulting expressed fusion protein with the antigenic specificity io neutralizing activity characteristic of the donor antibody. An example of a donor SUBSTITUTE SHEET WO 93/20210 PCIr/GB93/00725 7 antibody suitable for use in this invention is bovine mAb B4 or B13/14.
As used herein the term "acceptor antibody" refers to an antibody (polyclonal, monoclonal, or recombinant) heterologous to the donor antibody, but homologous to the patient (human or animal) to be treated, which contributes all or a substantial portion of the nucleic acid seque~nces encoding its variable heavy and/or light chain 1 ramework regions and/or its heavy and/or light chain constant regions to a second fusion partner. Preferably a human antibody is an 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 which 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 functional fragments and analogs of the naturally occurring CDRs, which fragments and analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived. See, the CDRs indicated by boxes in Figs. 3A, 3B, 4A, 4B, and 10 through 13. By 'sharing the antigen binding specificity or neutrali-ing ability' is meant, for example, that although mAb B13/B14 may be characterized by a certain level of antigen affinity, and a CDR encoded by a nucleic acid sequence of B13/B14 in an appropriate structural environment may have a lower affinity, it is expected that CDRs of B13/B14 in such environments will nevertheless recognize the same epitope(s) as B13/B14.
A "functional fragment" is a partial CDR sequence or partial heavy or light chain variable sequence which retains SUBSTITUTE SHEET WO 93/20210 P1/~9/02 PC*f/GH93/00725 8 the same antigen binding specificity and/or neutralizing ability as the antibody from which the fragment was derived.
An "analog" is an amino acid or peptide sequence modified by replacement of at least one amino acid, modification or chemical substitution of an amino acid, which modification permits the amino acid sequence to retain the biological characteristics, antigen specificity, of the unmodified sequence.
An "allelic variation or modification" is an alteration in the nucleic acid sequence encodin~g the amino acid or peptide sequence~s of the invention. Such variations or modifications may be due to degeneracies, 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.
As used herein, an "altered antibody" describes a type of fusion protein, a synthetic antibody a chimeric or humanized antibody) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts of CDRs from one or more donor mAbs whtich have specificity for the selected epitope. These altered an t t oodies may also b~t characterized by minimal alteration of the nucleic acid sequences envcid'ng the acceptor mAb light and/or heavy variable domain framework regions in order to retain dono3r niAb binding specificity, These antibodies can comprise immunoglobuilin (1g) constant regions and variable framework regions from the acceptor mAb, and one or more CDRs from the anti-RSV donor antibodies described herein.
A "chimeric antibody" refers to a type of altered antibody which contains naturally-occurring variable region light chain and heavy chains (both CDR and framework regions) derived from a non-human donor antibody in SUGTUTE SHEET WO 93/20210 PCr/B93/00725 9 association with light and heavy chain constant regions derived from a human acceptor antibody.
A "humanized antibody" refers to an altered antibody having its CDRs and/or other portions of its light and/or heavy variable domain framework regions derived from a nonhuman donor immunoglobulin, the remaining immunoglobulinderived parts of the molecule being derived from one or mure human immunoglobulins. Such antibodies can also include altered antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa, The term "effector agents" refers to non-protein carrier molecules to which the fusion proteins, and/or natural or synthetic light or heavy chain of the donor antibody or other fragments of the donor antibody may be associated by conventional means. Such non-protein carriers can include conventional carriers used in the diagnostic field, polystyrene or other plastic beads, 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 a toxin, such as ricin. Such effector agents are useful to increase the half-life of the anti-RSV derived amino acid sequences.
II. Anti-RSV Antibodies For use in construccing the antibodies, fragments and fusion proteins of this invention, a non-human snecies may be employed to generate a desirable immunoglobulin upon presentment with the respiratory syncytial virus (RSV) F protein or a peptide epitope therefrom. Conventioncl hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human mAb to the RSV peptide.
For example, several neutralizing, fusion-inhibiting (FI and highly protective bovine and murine anti-RSV SUBSTITUTE SHEET WvO 93/20210 PCTIGB93100725 monoc2onal antibodies (mAbs) are provided by this invention.
The production and characterization of the bovine antibodies capable of binding tco the F protein, B13 and B14, and other suitable bovine mAbs designated herein as B4, B7 through B10, and murine mAbs, designated herein as 16 through 21, are described in detail in Examples 1 and 2, and in Figs. and 6.
The resulting 513 and B14 anti-RSV antibodies are characterized by the ability to neutralize RSV in a plaque reduction neutralization test. Both B13 and B14 are potent in fusion inhibition assays and are protective in mice.
Competition studies, together with studies of antibodyescape mutants, binding to F protein fragments and synthetic peptides suggest that the epitope recognized by mAbs B13 and B14 may be similar to, but not identical to, the epitope rece'nized by mAb RSV19 (also known as mAb 19 or RSMU19), the IgG 2 murine mAb specific for F protein amino acid 417- 438 of and described in Example 11 below and in PCT patent.
application No. PCT/GB91/01554. After sequencing, B13 and B14 have been determined to be substantially identical are referred to as a single mAb called B13/B14 in certain instances. Where the mAbs were tested separately, reference is made to mAb B13 or B14.
The inventors have determined that a previously disclosed anti-RSV mAb, B4, is effective in protecting calves against infection with bovine RSV, as well as protecting mice against infection with human RSV. The ability of bovine mAb B4, administered to calves by the i.t.
route, to protect against lower respiratory tract infection with RSV and against the development of pneumonic lesions, indicates that bovine mAbs are potentially effective prophylactic and therapeutic agents in the control of calf respiratory disease. B4 is also potent in fusion inhibition and virus neutralization assays (Examples 16 and 17t, SUBSTITUTE SHEET WO 93/20210 PCT/GB93/0)0725 11 These three bovine mAbs B4, B13, and B14 have been identified as desirable antibodies which may be altered for pharmaceutical or prophylactic use. However, this invention is not limited to the use of these three mAbs or their hypervariable sequences. These mAbs illustrate the products and methods of this invention; wherever in the following description the donor mAb is identified as B4, B13 or B14, it should be understood that any other appropriate anti-RSV neutralizing antibodies and corresponding anti-RSV CDRs may be substituted therefor.
It is anticipated that other antibodies, bovine as well as other species, which are developed against the RSV F protein epitope spanning amino acid 266 through 273 as well as other RSV epitopes of interest described herein, may be useful in compositions of this invention for treating RSV in mice, cattle and humans. Other anti-RSV antibodies may be developed by screening an antibody library including hybridoma products or libraries derived from any species immunoglobulin repertoires in a conventional competition assay, such as described in the examples below, with one or more bovine antibodies or RSV epitopes described herein.
Particularly desirable for screening for additional antibodies are the neutralizing and protective mAbs, B4 and B13/B14.
Thus, the invention may provide an antibody, other than B4 or B13/14, which is capable of binding to the RSV peptide spanning amino acid #266 through #273, ITNDQKKL, of the F protein [SEQ ID NO: 19] or other relevant RSV epitopes, This antibody may be a mAb or an altered antibody, an analog of such antibodies, a Fab fragment thereof, or an F(ab'), fragment thereof. Such other mAbs generated against a desired RSV epitope and produced by conventional techniques, include without limitation, genes encoding murine mAbs, human mAbs, and combinatorial antibodies.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/0725 12 These anti-RSV antibodies may be useful in pharmaceutical and therapeutic compositions for treating RSV in humans and other animals.
III. Antibody Fragments The anti-RSV antibodies described above may be useful as donors of desirable functional fragments, including the antibody light and heavy chain variable sequences and CDR peptides.
The present invention also includes the use of Fab fragments or F(ab') 2 fragments derived from mAbs directed against an epitope of RSV as agents protective in vivo against RSV infection and disease. A Fab fragment is the amino terminal half of the heavy chain and one light chain, and an F(ab') 2 fragment is the fragment formed by two Fab fragments bound by disulfide bonds. MAb B13/14 or other suitable RSV binding antibodies, provide a source of these fragments, which can be obtained by conventional means, cleavage of the mAb with the appropriate proteolytic enzymes, papain and/or pepsin.
These Fab and fragments are also useful themselves as therapeutic, prophylactic or diagnostic agents for RSV in humans and other animals, and are also useful as donors of variable chain sequences, CDRs ad other functional fragments useful in this invention.
IV, RSV F Protein Epitopes of Interest The above-described mAbs recognize certain protective epitopes on the fusion protein of RSV which are recognized by a natural host of RSV. The nucleotide sequence of the F mRNA and the predicted protein sequence of the F protein [SEQ ID NO: 19] have been previously reported in Collins et al., Proc. at'l Acad. Si. USA, 81:7683-7687 (1984) The amino acid numbering referred to herein is identical to the numbering in this latt?' reference. The inventors identified an eight amino aci- saquence spanning SUBSTITUTE SHEET WO 93/20210 PCr/GB93/0725 13 amino acids 266 through 273 of the F protein [SEQ ID NO: 19], as a suitable target for screening for neutralizing antibodies, as an antigen useful in therapeutic agents against RSV, and in particular, for producing monoclonal antibodies against RSV. Other epitopes of interest include epitopes at around amino acid #429 which are recognized by neutralizing antibodies, B13 and B14.
The regions of the F protein [SEQ ID NO: 191 which react with the neutralizing, fusion-inhibiting, and highly protective bovine and murine mAbs of the invention were mapped by competitive binding assays (Example isolation and sequencing of antibody neutralization escape mutants (Examples 7 and and synthesis of peptides with sequences containing the amino acids changed in the escape mutants and the assessment of the -'eactivity of these peptides with the mAbs (Examples 9-11).
Sequence analysis of the F protein [SEQ ID NO: 19] of the antibody-escape mutants permits identification of the amino acid residues important in the binding of the highly protective mAbs. Similarly, information on the binding of the protective mAbs to synthetic peptides permits the location of the epitopes that they recognize.
Briefly described, most of the bovine mAbs recognized epitopes similar to those recognized by the murine mAbs, and one of the protective antigenic areas (site B; site II of Fig. 8) is recognized both by cattle, which are a natural host for RSV, and mice. The epitope(s) recognized by the protective bovine mAbs B13 and B14 do not appear to be identical to any recognized by murine mAbs. B13 and B14 bind to a region of the F protein around amino acid 429.
This epitope is similar, but distinct from the epitope recognized by murine mAb RSV19 (PCT patent application No.
PCT/GB91/01554 and Example 11). For example, mAb B13/B14 does not recognize the peptides spanning F protein amino SUBSTITUTE SHEET WO 93/20210 PPGF/GB3/00725 14 acids #417-438, #417-432, and #422-438 all of SEQ ID NO: 19, which are recognized by mAb RSV19. A second antigenic site (area C, Figs 5 and 6; area IV, Fig. 8) on the F protein identified by neutralizing, protective murine mAbs RSV19 and 20 has been located towards the carboxy end of the Fl subunit and has also been described in the above-referenced PCT patent application.
The RSV epitope recognized by B4 is reproduced by the RSV F peptide at the amino acid sequence spanning #255-275 of SEQ ID NO: 19. The inventors have determined using the Geysen pepscan technique, that B4 recognizes an epitope spanning amino acid 266 to 273 of the F protein (SEQ ID NO: 19). Altered antibodies directed against functional fragments or analogs of this epitope may be designed to elicit enhanced binding with the same antibody. mAbs which are directed against this epitope have been shown to p otect mice and/or bovines from in vivo RSV infection.
Replacement of each amino acid in the sequence has enabled the discovery that enhanced binding of B4 occurs in mutant epitopes. Changes in amino acids 266, 279 and 273 did not affect binding of mAb B4. Changes in amino acid 267 resulted in reduced binding of mAb B4. Changes in amino acids 268, 269, and 272 resulted in total loss of binding.
Substitution at amino acid 271 resulted in significantly enhanced binding (See Example The epitopes of these antibodies are useful in the screening and development of additional anti-RSV antibodies as described above. Knowledge of these epitopes enables one of skill in the art to define synthetic peptides and identify naturally-occurring peptides which would be suitable as vaccines against RSV and to produce mAbs useful in the treatment, therapeutic and/or prophylactic, of RSV infection in humans or other animals.
IV. Anti-RSV Nucleotide Sequences of Interest SUBSTITUTE SHEET WO 93/20210 PCr/GB93/0725 The mAbs B4 and B13/14 or other anti-RSV murine, human and bovine, antibodies described herein may donate desirable nucleic acid sequences encoding variable heavy and/or light chain amino acid sequences and CDRs, functional fragments, and analogs thereof useful in the development of the first fusion partners, fusion molecules and resulting expressed fusion proteins according to this invention, including chimeric and humanized antibodies.
The present invention provides isolated naturallyoccurring or synthetic variable light chain and variable heavy chain sequences derived from the anti-RSV antibodies, which are characterized by the antigen binding specificity of the donor antibody. Exemplary nucleotide sequences of interest include the heavy and light chain variable chain sequences of the mAbs B4, B13 and B14, as described below in the examples. Based on this variable region sequence data, B13 and B14 appear to be substantially identical.
The naturally occurring variable light chain of B13/14 is characterized by the amino acid sequence of Figs. 3A and 3B [SEQ ID NO: 2] labelled B13/B14VL. The naturallyoccurring variable heavy chain of B13/14 is characterized by the amino acid sequence illustrated in Figs. 4A and 4B (SEQ ID NO: 4] labelled B13VH. These heavy and light chains are described in Example 18.
As described above for B13/B14, the amino acid sequences of the B4VL and VH chains are reported in Figs. 4A Lnd 4B (SEQ ID NO: 4] and 3A and 3B [SEQ ID NO: 2], respectively, with the putative CDR peptides boxed. In both VH chains of B13/B14 and B4, the CDR3 peptides are unusually long, having 25 and 21 amino acids, respectively, in contrast to the vast majority of human and rodent CDR3s which have less than 20 amino acids.
The nucleic acid sequences encoding the variable heavy and/or light chains, CDRs or functional fragments thereof, SUBSTITUTE SHEET WO 93/20210 PCfl/GB93/00725 16 are used in unmodified form or are synthesized to introduce desirable modifications. These sequences may optionally contain restriction sites to facilitate their insertion or ligation to a second fusion partner, a suitable nucleic acid sequence encoding a suitable antibody framework region or the second fusion partners defined above.
Taking into account the degeneracy of the genetic code, various coding sequences may be constructed which encode the VH and VL chain amino acid sequences, and CDR sequences Figs. 3A, 3B, 4A, 4B, and 10 through 13) and functional fragments and analogs thereof which share the antigen specificity of the donor antibody.
Thus, these isolated or synthetic nucleic acid sequences, or fragments thereof are first fusion partners, which, when operatively combined with a second fusion partner, can be used to produce the fusion molecules and the expressed fusion proteins, including altered antibodies of this invention. These nucleotide sequences are also useful for mutagenic insertion of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incorporation of the resulting modified or nucleic acid sequence into a vector for expression.
VI. Fusion Molecules, Fusion Proteins and Other Proteins of this Invention A fusion molecule may contain as a first fusion partner a nucleotide sequence from an anti-RSV donor mAb, fragment or analog which sequence encodes an amino acid sequence for the naturally occurring or synthetic VH or VL chain sequences, a functional fragment or an analog thereof. When the first fusion partner is operatively linked to a second fusion partner, the resulting fusion molecule and expressed fusion protein is characterized by desirable therapeutic or prophylactic characteristics.
SUBSTITUTE SHEET WO 93/20210 PCrGB93/00725 17 The fusion molecule, upon expression, can produce a fusion protein which is an altered antibody, a chimeric, humanized or partially humanized antibody. Altered antibodies directed against functional fragments or analogs Df RSV may be designed to elicit enhanced binding in comparison to the donor antibody.
An exemplary fusion molecule may contain a synthetic VH and/or VL chain nucleotide sequence from the donor mAb encoding a peptide or protein having the antigen specificity of mAb B4 or B13/14. Still another desirable fusion molecule may contain a nucleotide sequence encoding the amino acid sequence containing at least one, and preferably all of the CDRs of the VH and/or VL chains of the bovine mAbs B4 or B13/14 or a functional fragment or analog thereof. The second fusion partners with which the anti-RSV sequences first fusion partners are associated in the fusion molecule are defined in detail above.
Where the second fusion partner is a nucleic acid sequence encoding a peptide, protein or fragment thereof heterologous to the nucleic acid sequence having anti-RSV antigen specificity, the resulting fusion molecule may express both anti-RSV antigen specificity and the characteristic of the second fusion partner. Typical characteristics of second fusion partners can be, a functional characteristic such as secretion from a recombinant host, or a therapeutic characteristic if the fusion partner is itself a therapeutic protein, or additional antigenic characteristics, if the second fusion partner has its own antigen specificity.
If the second fusion partner is derived from another antibody, any isotype or class of immunoglobulin framework or constant region (preferably human), or the like, the resulting fusion molecule of this invention provides, upon expression, an altered antibody. Thus a SUBSTITUTE SHEET WO 93/20210 PCr/GB93/00725 18 fusion molecule which on expression produces an altered antibody can comprise a nucleotide sequence encoding a complete antibody molecule, having full length heavy and light chains, or any fragment thereof, such as the Fab or fragment, a light chain or heavy chain dimer, or any minimal recombinant fragment thereof such as an F, or a single-chain antibody (SCA) or any other molecule with the same specificity as the donor mAb.
As K ample, a fusion molecule which on expression produces an altered antibody may contain a nucleic acid sequence encoding an amino acid sequence having the antigen specificity of an anti-RSV antibody directed against the F protein amino acid sequence spanning amino acid #266 through #273 of SEQ ID NO: 19, ITNDQKKL and analogs thereof, operatively linked to a selected second fusion partner.
Analogs of that epitope include those identified in the examples, such as SEQ ID NO: 56 when amino acid #266 is replaced with Ala, Cys, Asp, Glu, Phe, Gly, His, Leu, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; or SEQ ID NO: 57 when amino acid #269 is replaced with Glu, Phe, Ile, Leu, Met, Arg, Ser, Thr, Val, Trp, and Tyr; or SEQ ID NO: 58 when amino acid #271 is replaced with Asp, Glu, Phe, Ile, Leu, Met, Arg, Ser, Thr, Val, Trp, Tyr and Gln; or SEQ ID NO: 59 when amino acid #273 is replaced with Ala, Cys, Asp and Glu.
Desirably the source of the nucleic acid sequences is mAb B4.
Another fusion molecule which on expression produces an altered antibody may contain a nucleic acid sequence encoding the variable heavy chain sequence of Figs. 4A and 4B, a functional fragment or analog thereof, the variable light chain sequence of Figs. 3A and 3B, a functional fragment or analog thereof, or one or more B4 CDR peptides.
Another exemplary fusion molecule may contain a nucleic acid sequence encoding an amino acid sequence having the SUBSTITUTE SHEET WO 93/20210 PCTGB93/00725 19 antigen specificity of the anti-RSV antibody B13/B14, operatively linked to a selected second fusion partner. For example, the nucleic acid sequence may encode the VH chain sequence of Figs. 4A and 4B [SEQ ID NO: a functional fragment or analog thereof, the VL chain sequence of Figs.
3A and 3B [SEQ ID NO; 21, a functional fragment or analog thereof, or one or more B13/B14 CDR peptides.
When the fusion protein which is obtained upon expression of the fusion molecule is an altered antibody, the antibody contains at least fragments of the VH and/or VL domains of an acceptor mAb which have been replaced by analogous parts of the variable light and/or heavy chains from one or more donor monoclonal antibodies. These altered antibodies can comprise immunoglobulin (Ig) constant regions and variable framework regions from one source, the acceptor antibody, and one or more CDRs from the donor antibody, the anti-RSV antibodies described herein, An altered antibody may be further modified by changes in variable domain amino acids without necessarily affecting the specificity of the donor antibody. It is anticipated that heavy and light chain amino acids as many as thereof) may be substituted by other amino acids either in the variable domain frameworks or CDRs or both. Such altered antibodies may or may not also include minimal alteration of the acceptor mAb VH and/or VL domain framework region in order to retain donor mAb binding specificity.
In addition, these altered antibodies may also be characterized by minimal alteration, deletions, substitutions, or additions, of the acceptor mAb VL and/or VH domain framework region at the nucleic acid or amino acid levels may be made in order to retain donor antibody antigen binding specificity.
Such altered antibodies are designed to employ one or both of the VH or VL chains of a selected anti-RSV mAb SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 (optionally modified as described) or one c more of the above identified heavy and/or light chain CDR amino acid and encoding nucleic acid sequences. As another example, an altered antibody may be produced by expression of a fusion molecule containing a synthetic nucleic acid sequence encoding three CDRs of the VL chain region of the selected anti-RSV antibody or a functional fragment thereof in place of at least a part of the nucleic acid sequence encoding the VL region of an acceptor BAb, and a nucleic acid sequence encoding three CDRs of the VH chain region of a selected anti-RSV antibody, the bovine mAb B13/14, or a functional fragment thereof in place of at least a part of the nucleic acid sequence encoding the VH region of an acceptor mAb, such as a human antibody.
The altered antibodies can be directed against a specific protein epitope of RSV spanning amino acid #266-273 of SEQ ID NO: 19. It has been demonstrated that monoclonal antibodies which axe directed against this epitope protect mice and/or bovines from in vivo RSV infection.
A suitable acceptor antibody, for supplying nucleic acid sequences as second fusion partners, may be a human (or other animal) antibody selected from a conventional database, the Kabat database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. Desirably the acceptor antibody is selected from human IgG subtypes, such as IgG, or IgG,, although other Ig types may also be employed, IgM and IgA. For example, 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 the insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be SUBSTITUTE
SHEET
WO 93/20210 PCF/GBE93/00725 21 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.
The acceptor antibody need not contribute only human immunoglobulin nucleotide sequences to the desired fusion molecule, and resulting expressed fusion protein. For instance a fu.,ion molecule may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding the amino acid sequence of a polypeptide effector or reporter molecule.
Similarly rather than a human immunoglobin, a bovine or another species' immunoglobulin may be used, to create a 'bovinized' or other species' altered antibody.
One example of a particularly desirable fusion protein is a humanized antibody. As used herein, the term "humanized antibody" refers to a molecule having its CDR regions and/or other portions of its VL and/or VH domain framework regions derived from an immunoglobulin from a nonhuman species, the remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin.
Suitably, in these humanized antibodies one, two or preferably three CDRs from the anti-RSV antibody VH and/or VL regions are inserted into the framework regions of a selected human antibody, replacing the native CDRs of that latter antibody. Preferably, the variable domains in both human heavy and light chains have been altered by one or more CDR replacements. However, it is possible to replace the CDRs only in the human heavy chain, using as the light chain an unmodified light chain from the bovine donor antibody. Alternatively, a compatible light chain may be selected from a human acceptor antibody as described above.
A chimeric light chain may also be employed. The remainder of the altered antibody may be derived from any suitable acceptor human immunoglobulin.
SUBSTTTUTE SHEET WO 93/20210 PTG9/02 PCr/GB93/00725 22 Such altered antibodies according to this invention include a humanized antibody containing the framework regions of a humar IgG subtype into which are inserted one or more of the CDR regions of a bovine antibody. Such a humanized antibody can contain the VI CDR peptides of the bovine mAb inserted into the heavy chain framework region of a human antibody and in asscciation with the bovine light chain, or a bovine/human chimeric light chain. Such an exemplary humanized antibody is described in Example Alternatively, such an altered antibodjy may be associated with a desired human light chain. similarly, a chimeric antibody can contain the human heavy chain constant regions (preferably IgG) fused to the anti-RSV antibody, preferably bovine mAb, Fab regions. An exemplary chimeric antibody iz described in Example 19.
The altered antibody preferably has the structure of a natural antibody or a fragment thereof and possesses the combination of properties required for effective prevention and treatment of a desired condition in animals or man depending on the antigenicity supplied by the donor antibody, The altered humanized antibody thus preferably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required tor effective therapeutic use. Such "lhumanized" antibodies are effective in the prevention and treatment of RSV infection in an appropriate animal model for RSV infection in numans, and recognize~ a large variety of human clinical isolates of RlSV. Because of their above-denoted characteristics, nucleic acids encoding the bovine mAbs B4, B13 and B14 provide desirable RSV epitope specific donor sequences (first fusion partners) for the construction of a fusion molecule, which upon expression produces a humanized antibody according to this invention which can elicit a minimal immune response in humans. See, for exaniple, the SUBSTITUTE SHEERT WO 93/20210 PCT/GB93/00725 23 variable heavy and light chain sequences of Figs, 4A, 4B, 3A, 3B and 10 through 13, A fusion protein which is a chimeric antibody, as defined above, differs from the humanized antibodies by providing the entire non-human donor antibody heavy chain and light chain variable regions, including framework regions in association with human (or other heterologous animal, where desired) IgG constant regions for both chains.
It is anticipated that chimeric antibodies which retain additional non-human sequence in comparison to humanized antibodies of this invention, may also prove likely to elicit some desirable immuxte response in the human.
A preferred altered antibody is one directed against respiratory syncytial virus (RSV), preferably one specific for the fusion protein of RSV. A particularly preferred antibody of this kind has all or a portion of the variable domain amino acid sequences of B4 oz B13/B14 reported in Figs. 3A, 3B, 4A and 4B in its light and heavy chains, respectively. Figs. 10 through 13 illustrate predicted amino acid regions suitable for use in a "humanized" antibody and are described in the Brief Description of the Dray:ings section above. Additionally, an altered antibody of this invention may be characterized by the presence of one or more of the peptides identified in the above figures.
As one example, an alterad untibo may contain a the VL chain region of Fig. 11 or a functional fragment thereof in place oL at least a part of the VI. region of an acceptor mAb, and a VH chain region of Fig. 10 or a functional fragmerit thereof in place of at least a part of the VH region of an acceptor mAb, such as a human antibody. The resulting humanized antibody is characterized by the antigen binding specificity of mAb B4.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 24 Still another preferred altered antibody may contain a VL chain regioni of Fig. 13 or a functional fragment thereof in place of at least a part of the VL region of an acceptor mAb, and the VH chain region of Tigs. 12A and 12B or a functional fragment thereof in place of at least a part of the VH region of the acceptor mAb. The altered antibody is thus characterized by ,he antigen binding specificity of mAb B13/B14.
Alternatively, functional fragments of the variable sequences, such as the B4 CDR peptides, including: SYSVS (amino acids 31-35 of SEQ ID NO: 3); DASNGGIIYYNPALKS (amino acids 50-65 of SEQ ID NO: 3); CSVGDSGSYACTXaaGXaaRKGEYVDA, wherein Xaa is any or no aiiino acid (amino acids 100-122 of SEQ ID NO: 3); SGSS(S or D)NIG(R or I) (W or F) G or A)V(N or G) (alino acids 22-34 of SEQ ID NO: 1); YESSRPS (amino acids 50-56 of SEQ ID NO: 1); ATGDYNIA (amino acids 89-96 of SEQ ID NO: 1); ATGDYNIAV (amino acids 89-97 of SEQ ID NO: 1); Z0 or the B13/B14 CDR peptides, including GNTKRPS (amino acids 50-56 of SEQ ID NO: 2); VCGESKSATPV (amino acids 89-99 of SEQ ID NO: 2); DANVG (amino acids 31-35 of SEQ ID NO: 4); VIYKEGDKDYNPALKS (amino acids 50-65 of SEQ ID NO: 4); LGCYPVEGVGYDCTYGLQHTTFXaaDA, wherein Xaa is any amino acid (amino acids 98-122 of SEQ ID NO: 4), may be used in place of the larger variable region sequences of the figures.
Such altered antibodies can be effective in prevention and treatment of respiratory syncytial virus (RSV) infection in animals and man.
Another species of therapeutic, diagnostic or pharmaceutical protein of this invention is provided by the proteins or peptides encoded by the first fusion partner SUBSTITUTE SHEET WO 93/20210 1PCrGB9311,1072 23 which are associated with above-described effector agents.
One example of such a protein provides an anti-RSV amino acid sequence of the invention associated with a nonprotein carrier molecule. Another example contains a desired anti-RSV sequence of the invention to which is attached an non-protein reporter molecule. Additionally, the entire fusion proteins described above may be associated with an effector agent.
The procedure of recombinant DNA technology may be used to produce a protein of the invention in which the F, fragment or CH3 domain of a complete anti-RSV antibody molecule has been replaced by an enzyme or toxin molecule.
Another example of a protein of this invention contains an anti-RSV amino acid sequence of the invention with a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure.
In general, fusion or linkage between the anti-RSV antibody nucleotide sequences sequences and the second fusion partner in the fusion molecule or association of the peptides encoded by the first fusion partner and an effector agent, may be by way of any suitable conventional means.
Such conventional means can include conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional crosslinkers, carbodiimide, glutaraldehyde, and the like.
For association of the non-proteinaceous effector agents, conventional chemical linking agents may be used to fuse or join to the anti-RSV amino acid sequences.
Additionally, conventional inert linker sequences which simply provide for a desired amount of space between the first and second fusion partners in the fusion molecule may also be constructed into the molecule. The design of such linkers is well known. Such techniques and products are SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 26 known and readily described in conventional chemistry and biochemistry texts.
VII. Production of Fusion Proteins and Altered Antibodies Preferably the fusion proteins and altered antibodies of the invention will be produced by recombinant DNA technology using genetic engineering techniques. The same or similar techniques may also be employed to generate other embodiments of this invention, to construct the chimeric or humanized antibodies, the synthetic light and heavy chains, the CDRs, and the nucleic acid sequences encoding them, as above mentioned.
Briefly described, a hybridoma producing the anti-RSV antibody, the bovine mAb B4, is conventionally cloned, and the cDNA of its heavy and light chain variable regions obtained by techniques known to one of skill in the art, the techniques described in Sambrook et al., Molcular Clonina (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory (1989). The variable regions of the mAb B4 are Dbtained using PCR primers, and the CDRs identified using a known computer database, e.g, Kabat, for comparison to other antibodies.
Homologous framework regions of a heavy chain variable region from a human antibody are identified using the same databases, Kabat, and a human (or other desired animal) antibody having homology to the anti-RSV donor antibody is selected as the acceptor antibody. The sequences of synthetic VH regions containing the CDRs within the human antibody frameworks are defined in writing with optional nucleotide replacements in the framework regions for restriction sites. This pldtted sequence is then synthesized by overlapping oligonucleotides, amplified by polymerase chain reaction (PCR), and corrected for errors.
A suitable light chain variable framework region may be designed in a similar manner or selected from the donor or SUBSTITUTE SHEET WO 93/20210PC/G9072 PCr/GB93/00725 27 acceptor antibodies. As stated above, the source of the light chain is not a limiting factor of this invention, These synthetic VL and/or VH chain sequences and the CDRs of Ithe anti-RSV mAbs and their encoding nucleic acid sequences, are employed in the construction of fusion proteins and altered antibodies, preferably humanized antibodies, of thiis inventi.on, by the following process. By conventional techniques, a DNA sequence is obtained which encodes the non-human donor antibody B4, B13/B,4) yE or VL chain regions. In such a donor antibody at least the CDRs and those minimal portions of the acceptor mAb light and/or heavy variable domain framework region required in order to rttain donor mAb binding specificity as well as the remaining immunoglobulin-derived parts of the antibody chain are derived from a human immuzoglobulin.
A first conventional expression vector is produced by placing these sequences in operative association with conventional regulatory control sequences capable of controlling the replication and expression thereof in a host cell. Similarly, a second expression vector is produced having a DNA sequence which encodes the compl.ementary antibody light or heavy chain, wherein at least the CDRs (and those minimal portions of the acceptor monoclonal antibody light and/or heavy variable domain framework region required in order to retain donor monoclonal antibody binding specificity) of the variable domain are derived from a non-human ixnmunoglobulin. Preferably this second vector expression vector is identical to the first except in so far as the coding sequences and selectable markers are concerned so to ensure as far as possible that each polypeptide chain is equally expressed. Alternatively, a single vector of the invention may be used, the vector including the sequence encoding both light chain and heavy chain-derived SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 28 polypeptides. The DNA in the coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both.
A selected host cell is co-transfected by conventional techniques with both the first and second vectors 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 altered or humanized antibody of the invention. The humanized antibody which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as an ELISA assay. Similar conventional techniques may be employed to construct other fusion molecules of this invention.
Thus, the invention also includes a recombinant plasmid containing a fusion molecule, which upon expression produces an altered antibody of the invention. Such a vector is prepared by conventional techniques and suitably comprises the above described DNA sequences encoding the altered antibody and a suitable promoter operatively linked thereto.
The invention includes a recombinant plasmid containing the coding sequence of a mAb generated against the F protein 266-273 epitope.
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. For example, the conventional pUC series of cloning vectors commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden;, may be used. Additionally, any vector which is capable of replicating readily, has an abundance of cloning sites and marker genes, and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 29 Similarly, the vectors employed for expression of the altered antibodies according to this invention may be selected by one of skill in the art from any conventional vector. The expression vectors also contain selected regulatory sequences which are in operative association with the DNA coding sequences of the immunoglobulin regions and capable of directing the replication and expression of heterologous DNA sequences in selected host cells, such as CMV promoters. These vectori contain the above described DNA sequences which code for the altered antibody or fusion protein. Alternatively, 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 marker genes suitable for amplifying expression of the heterologous DNA sequences, the mammalian dihydrofolate reductase gene (DHFR) or neomycin resistance gene (neon). Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglupro).
The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art, The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, and the like, may be obtained from natural sources or synthesized by known procedures for use in directing the expression of the recombinant DNA in a selected host. 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.
Such a vector is transfected into a mammalian cell or other suitable cell lines via conventional techniques. The present invention also encompasses a cell-line transfected with these described recombinant plasmids. The host cell SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 used to express the altered antibody or molecule is preferably a eukaryotic cell, most preferably a mammalian cell, such as a CHO cell or a myeloid cell. Other primate cells may be used as host cells, including human cells which enable the molecule to be modified with human glycosylation patterns. 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, Sambrook et al., cited above.
Bacterial cells may prove useful as host cells suitable for the expression of the recombinant mAbs of the present invention. However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant mAb produced in a bacterial cell would have to be screened for retention of antigen binding ability. For example, various strains of E. coli, B. subtilis, Streptomyces, other bacilli and the like may also be employed in this method.
Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells and viral expression systems. See, e.g.
Miller et al., Genetic Engineerinag, .:277-298, Plenum Press (1986) and references cited therein.
The general methods by which the vectors of the invention may be constructed, transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the fusion protein or altered antibody of the invention from such host cell are all conventional. Likewise, once produced, the fusion proteins or 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 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/0/0725j 31 and the like. Such techniques are within the skill of the art and do not limit this invention.
Yet another method of expression of the humanized antibodies may utilize expression in a transgenic animal.
For example, a method of expression of the humanized antibodies of the invention may be by expression in the milk of a female transgenic animal, such as described in U.S.
Patent No. 4,873,316, which is incorporated herein by reference. For example, a DNA sequence for a selected humanized antibody of the invention may be operatively linked in an expression system to a milk-specific protein promoter, or any promoter sequence specifically activated in mammary tissue, through a DNA sequence coding for a signal peptide that permits secretion (and maturation, if necessary) of the desired protein in the mammary tissue.
Suitable promoters and signal peptides may be readily selected by one of skill in the art.
The expression system is transgenically introduced into a host genome using standard transgenic techniques, for example by microinjection into the pronuclei of fertilized mammalian eggs. See, e.g. B. Hogan et al, "Manipulating The Mouse Embryo: A Laboratory Manual" Cold Spring Harbor Laboratory (1986); R.L. Brinster et al, Cell, 21:223-231 (1991).] As a result, one or more copies of the construct or system are incorporated into the genome of the transgenic mammal. The presence of the expression system permits the female of the mammalian species to produce and secrete the recombinant humanized antibody into its milk. This system allows for high level production of the humanized antibodies of the invention.
This latter method of expression may be particularly suitable for a humanized antibody containing bovine CDRs, and especially suitable for the oral administration of this SUBSTITUTE SHEET WO 93/20210 PCr/GB93/00725 32 antibody to bovines as well as human infants. Other transgenic systems may also be employed.
Once expressed by the desired method, the altered antibody is then examined for in vitro activity by use of an appropriate assay. Presently, conventional enzyme linked immunosorbent assay (ELISA) formats are employed to assess qualitative and quantitative binding of the altered antibody to the RSV epitope (see Example Other assays may also be used to verify efficacy prior to subsequent human clinical stlidies performed to evaluate the persistence of the altered ant-body in the body despite the usual clearance mechanisms.
Example 11 below demonstrates the method of constructing the altered hunianited antibodies derived from the murine monoclonal antibody RSV19, such as HuRSVl9VH/VK and HuRSV19VHFNS/HuRSV19VK which are described in copending PCT patent application No. PCT/GB91/01554. Following the procedures described for humanized antibodies prepared from the murine RSV19, one of skill in the art may also construct humanized antibodies from the bovine antibodies, variable region sequences and CDR peptides described herein (see Examples 19 and 20). Altered antibodies can be produced with variable region frameworks potentially recognized as "self" by recipients of the altered antibody. Minor modifications to the variable region frameworks can be implemented to effect large increases in antigen binding without appreciable increased immunogenicity for the recipient. Such altered antibodies can effectively prevent and eradicate infection. Of particular interest for such humanized antibodies are the antibodies B4, B13 and B14 described herein. Such antibodies are useful in treating, therapeutically or prophylactically, a human against human RSV infection. Such antibodies may also be useful as diagnostic agents.
SUBSTITUTE SHEET WO 93/20210 PCr/G983/007255 33 VII. Therapeutic/Prophylactic Uses of the Invention This invention also relates to a method of treating, therapeutically or prophylactically, human RSV infection in a human in need thereof which comprises administering an effective, human RSV infection-treating dose of antibodies including one or more of the mAbs described herein, or fragments thereof, or an altered antibody as described herein, or another fusion protein, to such human. This invention also relates to a method of treating, therapeutically or prophylactically, bovine or other species' RSV infection in a bovine or other animal in need thereof which comprises administering an effective, RSV infection-treating dose of antibodies or molecules including one or more of the mAbs described herein, or fragments thereof, or an altered antibody as described herein, to such animal.
The fusion proteins, antibodies, altered antibodies or fragments thereof of this invention may also be used in conjunction with other antibodies, particularly human monoclonal antibodies reactive with other markers (epitopes) responsible for the disease against which the altered antibody of the invention is directed. Similarly monoclonal antibodies reactive with other markers (epitopes) responsible for the disease in a selected animal against which the antibody of the invention is directed may also be employed in veterinary compositions.
The fusion proteins or fragments thereof described by this invention may also be used as separately administered compositions given in conjunction with chemotherapeutic or immunosuppressive agents. The appropriate combination of agents to utilized can readily be determined by one of skill in the art using conventional techniques. As an example of one such combination, the altered antibody uRSVHuRSV1 /HuRSV19VK described in Example 11, or a SUBSTITUTE SHEET WO 93/20210 PCT/GB83/0725 34 similarly altered B4, B13 or B14 antibody, may be given in conjunction with the antiviral agent ribavirin in order to facilitate the treatment of RSV infection in a human.
One pharmaceutical composition of the present invention comprises the use of the antibodies of the subject invention in immunotoxins, molecules which are characterized by two components and are particularly useful for killing selected cells in vitro or in vivo. One component is a cytotoxic agent which is usually fatal to a cell when attached or absorbed. The second component, known as the "delivery vehicle" provides a means for delivering the toxic agent to a particular cell type, such as cells comprising a carcinoma. The two components are commonly chemically bonded together by any of a variety of well-known chemical procedures. For example, when the cytotoxic agent is a protein and the second component is an intact immunoglobulin, the linkage may be by way of heterobifunctional cross-linkers, carbodiimide, glutaraldehyde, and the like. Production of various immunotoxins is well-known in the art.
A variety of cytotoxic agents are suitable for use in immunotoxins, and may include, among others, radionuclides, chemotherapeutic drugs such as methotrexate, and cytotoxic proteins such as ribosomal inhibiting proteins ricin).
The delivery component of the immunotoxin may include one or more of the humanized immunoglobulins or bovine immunoglobulins of the present invention. Intact immunoglobulins or their binding fragments, such as Fab, are preferably used. Typically, the antibodies in the immunotoxins will be of the human IgM or IgG isotype, but other mammalian constant regions may be utilized if desired.
The mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the SUBSTITUTE SHEET WO 93/20210PC/G9075 PCr/GB93/00725 agent to the host. The fusion proteins, antibodies, altered .,itibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, subcutaneously, intramuscularly or intravenously. The compositions for parenteraJ. administration will commonly comprise a solution of the altered antibody of the invention or a cocktail thereof dissolved in an acceptable carriprf preferably an aqueous carrier. A variety of aqueous carriers may be employed, water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generalivy free of particulate matter. These solutions may be sterilized ty conventional, well known sterilization techniques. !Me 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, from less than about 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 admninistration selected.
Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 niL sterile buffered iater, a.-d 50 mg of an altered1 antibody of the invention. Similarly, iA pharmaceutical composition of the invention for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solutiLon, and 150 mg of an altered antibody of the invention. Actual methods for preparing parientev:ally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Rpmingtn.La SUBSTITUTE SHEET WO 93/20210 PCT/GB93/Q0725 36 Pharmace1tical Science, 15th ed., Macs Publishing Company, Easton, Fennsylvania, To effectively prevent RSV infection in a human or other animal, one dose of approximately 1 mg/kg to approximately 20 mg/kg of a molecule or an antibody of this invention should be administered parenterally, preferably i.v, (intravenously) or i.m. (intramuscularly); or one dose of approximately 20 ug/kg to approximately 2 mg/kg of such antibody should be adminiscered i.n. (intranasally).
Preferably, such dose should be repeated every six weeks starting at the beginning of the RSV season (October- November) until the end of the RSV season (March-April).
Alternatively, at the beginning of the RSV season, one dose of approximately 5 mg/kg to approximately 100 mg/kg of an antibody of this invention should be administered i.v, or i.m. or one dose of approximately 0.5 mg/kg to approximately mg/kg of such antibody should be administered i.n.
To effectively therapeutically treat RSV infection in a human or other animal, one dose of approximately 2 mg/kg to approximately 20 mg/kg of an antibody of this invention should be administered parenterally., preferably i.v. or or approximately 200 ug/kg to approximately 2 mg/kg of such antibody should be administered i.n. Such dose may, if necessary, be repeated at appropriate time intervals until the RSV infection has been eradicated.
For example. in Example 16, the dose of B4 required to protect calves when administered by the i.t. route was 300 A g/kg body weight. This is 300 to 1000-fold less than the amount of human IgG, containing high titres of RSVneutralizing antibody, required to reduce RSV infection in cotton-rats and owl monkeys, passively immunized by the it.
route (Hemming and Prince, Review of Infectious Diasge., 12:S470-S475 (1990)1. It has been shown that about less ar.ibody is required to reduce virus shedding when SUBTIrTUtE SHEET WO 93/20210 PCT/B93/00725 37 given by the topical route when compared with intravenous administration (Prince Hemming, (1990)]. Therefore, it is estimated that a dose of approximately 3 mg/kg of mAb B4 given i.v. wou±d be needed to significantly reduce RSV shedding in calves. This Is similar to the amount of murine or "humanized" mAb required to protect mice against P.SV infection (Tempest et al., (1991)].
The compositions of the invention may also be administered by inhalation. By "inhalation" is meant intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques. For example, to prepare a composition for administration by inhalation, for an aerosol container with a capacity of 15-20 ml: Mix 10 mg of an antibody of this invention with 0,2-0.2% of a lubricating agent, such as polysorbate 85 or oleic acid, and disperse such mixture in a propellant, such as freon, preferably in a combination of (1,2 dichlorotetrafluoroethane) and difluorochloromethane and put into an appropriate aerosol container adapted for either intranasal or oral inhalation administration. As a further example, for a composition fcr administration by inhalation, for an aerosol container with a capacity of 15-20 ml: Dissolve 10 mg of an antibody of this invention in ethanol (6-8 ml), add 0.1-0.2% of a lubricating agent, such as polysorbate 85 or oleic acid; and disperse such in a ',ropellant, such as freon, preferably a combination of (1.2 dichlorotetrafluoroethane) and difluorochloromethane, and put into an appropriate aerosol container adapted for either intranasal or oral inhalation administration.
The antibodies, altered antibodies or fragments thereof described herein can be 1yophilized for storage and reconstituted in a suitable carrier prior to use. This SUBSTITUTE SHEET WO 93/20210CV~l3/02 PCr,/G'093/00725 38 tec! nlue has been shown to be effective with conventional immune globulin6 and art-known lyophi2.izatiorn and reconstitution techniques can be employed.
Depending on t,-e intended result, the pharmaceutical composition of the invention can be administered for prophylactic and/or therapeutic treatments, In therapeutic application, compositionls are adzninisvtered tcq a patient already sufferi.ng from a disease, in an amount sufficieni to cure or at least partially arrest the disease and its -:omplications., In prophylactic applications, compositions containing the present antibodies or a cocitail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
single or multiple administrations of the pharmaceutical composit,4,ona can be car~ried out with dose levels and pa-.r-ejn beirm selected by the treating physician.
In any event, the pharmaceutical composition of the invention should provide A quantity oZT the altered antibodies of the invention sufficient to effectively treat t'ie patienL, It should alfso be noted that thie fusion proteins, antibodies, varl'able sequences, CDR~ peptides and epitopes of this inventioxi M~ay be used for the design and synthesis Of either peptide or non-pkeptids.: compounds (mimetias) wW~ch would be useful in the same therapy as the antibody. See, Sagragov-i et Sr Z,'-j*792-795 (1991).
il4atural RSV infections have also been reported in cattle, ioatst sheep aid chimpanzees, Thus, for example, utilizing th~e methodology descr{kbed above, an appropriate7 mouse antibody rzould be "bovinized", and appropriate I framework region residue alterations could bi effected, it' necessary, to restore spec'fic binding affini~ty. Once t-he appropriate mouse antibody has been created one of skill in 4,he art,. usLng conventional dosage determination techniques, SUBSTiTUTE SHEET WO 93 '0 PCT/GB93/00725 39 can readily determine the appropriate dose levels and regimens required to effectively treat, prophylactically or therapeutically, RSV infection in the selected animal.
The following examples illustrate various aspects of this invention and are not to be construed as limiting the sciope of this in-ention. All amino acids are identified by conventional three letter codes, single letter codes or by full name, unless otherwise indicated. All necessary restriction enzymes, plasmids, and other reagents and materials were obtained from commercial sources unless otherwise indicated. All general cloning ligation and other recombinant DNA methodology were as described in "Molecular Cloning, A Laboratory Manual." (1982), eds. T. Maniatis et al., published by Cold Spring Harbor Laboratory, Cold Spring Harbo, New York, ("Maniatis et al") or the second edition thereof (1989), eds. Sambrook et al., by the same publisher ("Sambrook etr The following examples illustrate the construction of exemplary altered antibodies and expression thereof in suitable vectors and host cells.
Example 1 PreaatJjon of Monoclonal Antibodiej Murine monoclonal antibodies 1 to 14 were described in Taylor et al., (1984) cited above and incorporated herein by reference. Several of these antibodies were produced by immunizing BALB/c mice with bovine RSV, strain 127. The bovine RSV, strain 127 was isolated at Compton in I )73 from a calf with respiratory disease. Others of these antibodies were produced with cells persistently infected with the Long strain of human RSV [Fernie et al., Proc. Soc. Exp.i.BL.
Medc, .fi:83-86 (1981)1. Murine monoclonal antibcdies 16 to 21 were produced from BALB/c mice inoculated intranasally on two occasions, three weeks apart, with 1X10 4 pfu of the hurr-n RSV strain A2, grown in Hep-2 cells. Human RSV, strain A2, subtype A was isolated from a child in SUBSTITUTE SHEET WO 93/20210 P1r/GD93/007'.15 Australia [Lewis et al., Med. J. Austr., Aa&:932-933 (1961)].
After an interval of four months, the mice were inoculated intraperitoneally with 2X10 7 pfu of the bovine 127 strain. Three days after the booster inoculation, the immune splenocytes were fused with NS-1 myeloma cells (American Type Culture Collection, designation TIB18]. The resulting hybridomas were screened for antibody to RSV by radioimmunoassay and immunofluorescence, cloned twice on soft agar and cloned cells inoculated into BALB/c mice to produce ascitic fluid as described in Taylor et al., cited above.
Bovine monoclonal antibodies B1 to B6 were produced as described in Kennedy et al., J. Gen. Virol., .9:3023-3032 (1988), incorporated herein by reference. At the same time, bovine mAbs B7 to B10, B13 and B14 were produced from bovine lymphocytes obtained from the same calf, but the lymphocytes were stored in liquid nitrogen and fused with NS1 cells at later dates. The resulting heterohybridomas were screened for bovine antibody to RSV by ELISA and in some cases also by the fusion inhibition assay [essentially as described in Kennedy et al. (1988), cited above], but adapted to microtitre plates. Cloned heterohybridoma cells secreting bovine mAbs to RSV were inoculated into 1.,stane-primed nude BALB/c mice to produce ascitic fluid or grown in serum-free, DCCM-1 medium (Biological Industries, Ltd., Glasgow, Antibody was purified from cell culture supernatant using Protein G Sepharose 4 Fast Flow (Pharmacia LKB]. Bound antibody was eluted with 0.1M glycine, pH2.7, neutralized with 1M Tris-HCl (pH9.0) and dialyzed against phosphate buffered saline (PBS).
The antibody AK13A2 raised against the Long F protein was a generous gift of Dr. P. Coppe, Centre d'Economie Rurale, Marloie, Belgium. The mAbs 1BC11 (a negative control antibody), 47F and 49F have been described by SUBSTITUTE SHEET WO 93/20210 PCr/GB393/00725 41 Garcia-Barreno et al., J. Virnl., .:925-932 (1989). MAb 7C2 is described in Trudel et al., (1987), cited above. The antibodies, 47F, AK13A2 and 49F, were purified from ascitic fluids by protein A-Sepharose chromatography and peroxidase labelled [Garcia-Barreno et sl., (1989), cited above] All of the murine and bovine mAbs and hybridoma cell lines producing them described herein, except mAbs 1BC11, 47F, 49F, AK13A2 and 7C2, are available from the laboratory of Dr. Geraldine Taylor, Institute for Animal Health, Compton Laboratory, Compton, Near Newbury, Berks, RG16ONN, England.
Example 2 Characterization of Monoclonal Antibodies The specificities of the mAbs for F protein viral polypeptides were determined by radioimmune precipitation of 35 S)-methionine or 3 H)-glucosamine labelled RSV infected cell lysates performed as described by Kennedy et al., 4L Gen. Virol., £2:3023-3032 (1988). The specificity was confirmed by Western blots (immunoblotting) of non-reduced and reduced RSV-infected cell lysates performed as described by Taketa et al., Electrophor., £:492-497 (1985). The antigens used in immunoblotting were either Hep-2 cells infected with the human RSV A2 strain or calf kidney (CK) cells infected with the bovine RSV strain 127. Uninfected Hep-2 or CK cells' were used as control antigens.
Only mAbs Bl, B4, B5 [Kennedy et al., cited above] and mAbs RSV19, 813 and 814 reacted with F protein denatured by boiling in dithiothreitol. Whereas mAbs B1, B4 and recognized 46K and 22K fragments of denatured F1 protein in Western blotting, mAbs RSV19, B13 and 814 only recognized 46K fragments. The properties of mAbs 16 to 18, 20 and 21, RSV19, B7 to 810, B13 and B14, not previously described, for the assays described below are shown in Table 1 below. The properties of all the other mAbs in these assays are summarized in Figs. 5 and 6.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 42 The ability of the mAbs to inhibit multinucleated giant cell formation was assayed in MA104 cells [American Type Culture Collection, Rockville, MD] 24 hours after infection with the RSV A2 strain [Kennedy et al., cited above, incorporated herein by reference] The results of this assay are reported in Table 1 under the column "Fusion Inhibition", and in Figs. 5 and 6 as A indication means that the mAb did not inhibit the giant cell formation.
A indication means that the mAb inhibited the formation of multinucleated giant cells.
Four murine mAbs (11, 13, RSV19 and 20) ana four bovine mAbs (B4, B5, B13 and B14) inhibited the formation of multinucleated giant cells.
The ability of mAbs to neutralize RSV was assayed by a plaque reduction neutralization test performed as described in Kennedy et al., cited above. The results of this assay are reported in Table 1 under the column "Neut. titre", and in Figs. 5 and 6 as "Neut". In the figures, a indication means no neutralization occurred; a indication means that the antibody was neutralizing, Seven of the murine mAbs and four of the twelve bovine mAbs, i.e., B4, B5, B13 and B14, neutralized RSV.
The ability of mAbs to protect against RSV infectioP was studied in BALB/c mice as follows. 100 jil of ascitic fluid containing the mAbs was injected intra-peritoneally into groups of five mice. One day later, the mice were inoculated i.n. with 104 pfu of the A2 RSV strain. On day of the infection, the mice were killed and their lungs assayed for RSV on secondary CK monolayers, according to the procedure described in Taylor et al., Infect. irnmun., a:649-655 (1984).
The results of this assay al- alsc 2eported in Table 1 under the column "Prot. of Mice", and in Figs. 5 and 6 under "Protection". In the figures, a indication means that SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 43 the mAb did not protect the immunized mice against RSV infection. A or indication means that the mAb did protect the animals to a lesser or gzeater degree, respectively. The eight mAbs thac were effective in the fusion inhibition assay murine mAbs 11, 13, RSV19 and and bovine mAbs B4, B5, B13, and B14) were highly effective in preventing RSV infection in BALB/c mice when administered i.p. 24 hours prio. to i.n. challenge with the A2 strain of RSV.
All antibodies, except murine mAbs 9 and 10 (Taylor et al., (1984)] and bovine mAb B8, which were specific for bovine RSV, reacted with both the A2 and the human B subtype (8/60) [Common Cold Unit, Salisbury, England] strains of human RSV (both grown in Hep-2 cells) and with bovine strains of RSV (Taylor et al., (1984), cited above; Kennedy et al., cited above]. These results indicate that the epitopes recognized by the highly protective, fusioninhibiting mAbs were highly conserved among strains of RSV.
Table 1 Properties of mAbs to the F protein of RSV Ig ELISA titre flogji Neut. Fusion %C Prot mAb class A2 8/60 BRSV titre 1 Inhib. lysis 2 of micfe 16 Gl 6.8 6.6 6.8 2.0 0 0.6 17 G2b 6.1 6.21 6.1 <1.0 59 0.6 18 G2a 7.0 6.2 3.4 43 1.6 RSV19 G2a 6.4 6.7 6.7 3.4 2 >3.8 20 G2a >6.0 8.6 7.5 4.3 76 >3.8 21 G2a 8.9 7.4 6.8 <1.0 68 B7 G1 3.0 4.9 4.9 <1.0 6 0 B8 G1 <2.0 <2.0 4.0 <1.0 8 0.2 B9 Gl 5.1 5.4 4.9 <1.0 2 0.4 B10 Gl 5.1 5.4 6.0 <1.0 9 B13 GI 6.0 5.1 5.4 5.8 0 2.2 BlI' GI 5.6 5.2 5.6 5.4 0 >2.2 50% plaque reduction titre expressed as logl 0 SUBSTITUTE SHEET WO 93/20210 PCT/GB3930725 44 2 Percent specific release with 1/100 dilution of mAb and rabbit complement 3 Log,, reduction in titre of RSV in the lungs of passively immunized mice compared with control animals Example 3 Enzyme Linked Immunosorbent Assay (ELTSA) RSV antigens to be tested in the ELISA were each prepared from Hep-2 cells, 3 to 4 days after infection.
Cells were scraped into medium, spun at 500 g for 5 minutes, resuspended in distilled water and treated with 0.5% (w/v) detergent to yield a cell lysate. Control antigen was made in a similar way using uninfected Hep-2 cells.
The ELISA was performed as follows: Microtitre plates were coated with RSV or control antigen, diluted in distilled water, overnight at 37 0 C, incubated with blocking buffer consisting of 5% normal pig serum in PBS and 0.05% Tween 20 for 1 hour at room temperature and washed 5x with PBS/Tween. Serial 3-fold dilutions of mAb were added to the wells and the plates incubated for 1 hour at room temperature. After washing 5x with PBS/Tween, HRPconjugated rabbit anti-bovine IgG (Sigma) diluted 1:4000 or HRP-conjugated goat anti-mouse IgG (Kpl, Maryland, USA) diluted 1:2000, was added to each well. After a final wash, bound conjugate was detected using the substrate tetramethylbenzidine (TMB, ICN, Immunobiologicals, Illinois).
Example 4 Purification of the F qlycoprotein and trypsin treatment The F protein was purified by immunoaffinity chromatography from extracts of Hep-2 cells infected with the Long strain (See, Walsh et al., J. Gen. Virol., 1£:409- 415 (1985); and Garcia-Barreno et al., (1989), cited above).
Several aliquots of the purified protein (15 pg each) were incubated and digested with either 2pg, 4gg, 84g or 16pg of trypsin for 4 hours at 37*C. The digestion was terminated SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 by the addition of electrophoresis sample buffer [Studier, J. Mol. Biol., 2:237-248 (1972)] and boiling of the samples for 3 minutes. SDS-PAGE separated the samples. The samples were electrotransferred to Immobilon membranes.
Example 5 Epitoes on F protein.
As an initial step to locate the epitopes recognized by the antibodies, AK13A2, 47F, 7C2, RSV19, 20 and B4, used in the selection of mutant viruses in Example 7, below, tae binding of mAbs to the trypsin fragments of purifiod F protein were tested by Western blot (Towbin et al., Proc.
Nat'l. Acad. Sci. USA, 26:4350-4354 (1979)). The protein fragments were either stained with Coomassie blue or developed with antibodies AK13A2 or 19.
Increasing amounts of trypsin generated smaller fragments of the Fl subunit which were stained by Coomassie blue. Four Fl fragments of 30, 20.5, 19 and 15 K were recognized by mAb AK13A2. The 20.5 and 19 K fragments had been mapped previously (Lopez et al., J. Gen. Virol., .E4A:927-930 (1990)) at the NH2 terminal end of the Fl subunit. Antibodies B4, 47F, and 7C2 recognized the same set of fragments as AK13A2. Thus, the epitope recognized by these mAbs can be ascribed to amino acid sequences included within the NH 2 terminal third of the F1 subunit.
In contrast, RSV19 reacts with a different set of F1 fragments. Only large size fragments (26 and 22 K), generated with low trypsin amounts, reacted with RSV19 (mAb reacted less efficiently with the same set of fragments).
Thus, epitopes 19 and 20 contain trypsin sensitive amino acid sequences which were tentatively located within the carboxy terminal two thirds of the Fl subunit (Fig. 2), outside the region covered by the fragments recognized by antibody B4. The NH 2 terminal end of the 26 and 22 K fragments could not be determined by direct protein sequencing because their low yield after trypsin treatment.
SUBSTITUTE SHEET WO 93/20210 PL-T/GB93/0072 46 The diagram of Fig. 2 shows the F gly oprotein primary structure denoting the hydrophobic regions, the site of proteolytic processing, the potential sites for Nglycosylation, the cysteine residues and the amino acid residues which are changed in the neutralization escape mutants (see Table 3A-3C below). The locations of the trypsin fragments recognized by different mAbs are shown below Fig. 2.
The region on the F protein recognized by mAbs B13 and B14 were identified by examining their binding to F protein fragments, expressed in E. coli. Recombinant C protein (rC,
F
3 77-5 24 of SEQ ID NO: 19 and recombinant D protein (rD, F 3 7t.
of SEQ ID NO: 19 were used as antigens in ELISA as described in Example 3. These peptide sequences of the F protein were fused to an influenza non-structural protein fragment containing amino acids 1 81 of the influenza nonstructural protein 1 (NS-1) at their amino termini, inserted into an expression plasmid and expressed in E.
coli. The production of these fusion peptides involved conventional procedures. MAbs B13, B14 and RSV19, but not B4, bound to these protein fragments. Table 2 below illustrates the binding of anti-F mAbs to recombinant F protein fragments in ELISA. These findings suggest that the region of the F protein recognized by B13 and B14 is similar to that recognized by RSV19 and is within the carboxy terminal third of the Fl subunit.
Table-2 mAh rC rD RSV B13 5.7' >3.0 5.6 B14 6.2 >3.0 5.6 RSV19 5.3 >3.0 7.4 B4 <1.5 <1.5 5.9 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 47 'Log, 1 titer by ELISA, rC at 2 u.g/well and rD at 1 ig/well used as antigens.
Example 6 Identification of antigenic areas in the F protein.
The epitope specificity of the 16 murine and 12 bovine mAbs to the F protein were analyzed by a competitive binding assay using purified and labelled mAbs. In summary, these competitive Linding assays identified twelve antigenic sites on the F protein, many of which overlapped extensively.
Three epitopic sites were recognized by both the neutralizing mAbs and the highly protective FI mAbs, e.g., B4, B5, 813 and B14. These findings are similar to those of others who have identified three antigenic sites on the F protein involved in neutralization using murine mAbs, two of which are invoved in Fl activity (Walsh et al., J. Gen.
Yirol., .:505-513 (1986) and Beeler et al., Virnl., .£:2941-2950 (1989)]. These findings suggest that virus neutralization can occur by a mechanism independent of preventing the fusion of the virus with the cell membrane, e.g. steric hindrance of virus attachment.
A. Purification and labelling of mAbs The IgG from ascitic fluid containing either murine or bovine mAbs was purified on either Protein Asepharose or Protein G-sepharose Fast-Flow (Pharmacia LKB].
The ascitic fluids were mixed with equal volumes of 0.1M phosphate buffer (pH and passed through a Protein Asepharose column with the same buffer. Bound antibodies were eluted with 0,1 M citrate buffer (pH 6.0 to Fractions eluted with low pH buffers were collected in 1M Tris-HCl (pH IgG from tissue culture supernatants was purified on Protein G-sepharose Fast-Flow and eluted with 0.1M glycine as described above. Purified IgG was dialyzed SUBSTITUTE SHEET WO 93/20210 P~r/GB93/00725 48 against PBS and labelled with using chloramine T or coupled to biotin.
B. Competitive Binding Assay A dilution of 12 "I-labelled, or biotinylated, mAbs, determined to give approximately 10,000 cpm at 90% of maximum binding to RSV antigen in a radioimmunoassay, was allowed to react with RSV antigen in the presence of increasing amounts of various unlabelled mAbs to the F protein. For mAbs B13 and B14, a dilution of biotinylated mAbs, determined to give 90% of maximum binding to RSVinfected cell lysate, was allowed to bind to RSV antigen in the presence of increasing amounts of unlabelled antibody.
An unlabelled mAb to the nucleoprotein was used as a control.
The results of this assay are illustrated in Figs.
and 6. Some mAbs inhibited the binding in a dosedependant manner; other mAbs, however, did not interfere with the binding of the test antibody. Unlabelled mAb to the N protein of RSV did not interfere with the binding of any of the mAbs to the F protein. These studies identified groups of mAbs that competed for simultaneous binding to antigen. Epitopes recognized by competing mAbs were considered to be operationally within the same antigenic area of the F protein. The competition profiles of the mAbs overlapped extensively (Figs. 5 and 6).
Therefore the clustering of epitopes was done on the basis of partial similarities and was analyzed using the Leucocyte typing database IV (Gilks, "Leukocyte typing database TV" Oxford UniverSity Press (1990)1, These studies showed that the 16 murine mAbs recognized 7 antigenic areas on the F protein [SEQ ID NO: 19) (Fig. mAbs 2 and competed with nearly all of the other murine mAbs. Two highly protective mAbs, 1I and 13, appeared to recognize the same epitope (site whereas two other mAbs, RSV19 and SUBSTITUTE SHEET WO 93/20210 PC/GB93/00725 49 which were also highly protective, were similar to each other but different from mAbs 11 and 13, and mapped to site
C.
Most of the 12 bovine mAbs mapped to the same sites as the murine mAbs (Figs. 5 and Murine mAbs 2 and competed with only 4 of the bovine mAbs (B2, B3, B4 and B6). A neutralizing murine mAb, 14, which mapped to site G in competition studies with the murine mAbs (Fig. showed a competition profile that was similar to the bovine mAbs B2, B3 and B6 and was therefore placed in group H (Fig. The binding of bovine mAbs B1 and B7 were not inhibited by any of the murine mAbs and, indeed, B7 appeared to recognize a distinct epitope. The epitopes recognized by 2 highly protective bovine mAbs B4 and B5, were similar to each other and to 2 of the highly protective murine mAbs, 11 and 13, mAb 18, which is partially protective in mice, and which is not protective, also map in this area (site B).
The binding of the protective bovine mAbs B13 and B14 was inhibited to various degrees by protective murine mAbs, RSV19 and 20, and the protective bovine mAbs B4 and B5. However, the competition profiles of mAbs B13 and B14 were different from those antibodies mapping to sites B and C, suggesting that they recognize a different site on the F protein.
Taken together, the murine and bovine mAbs recognized 12 antigenic areas, most of which overlapped extensively. The highly protective, fusion-inhibiting (FI), neutralizing mAbs mapped to 2 or possibly 3 sites (areas B, C and L in Fig. 5) on the F protein. mAbs that neutralized virus but did not have Fl activity mapped to 3 sites (areas B, D and However, mAbs which have neither neutralizing nor FI activity also map to these sites.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 Example 7 Antibody esc_e mutants The pattern of reactivity of antibody-escape mutants with the mAbs confirmed the mapping of the protective epitopes deduced from competitive binding assays. In summary, two regions of the F primary structure were identified where the epitopes recognized by neutralizing mAbs were located. The first region mapped within the trypsin resistant amino terminal third of the large Fl subunit. This region contained the overlapping epitopes recognized by mAbs 47F, 49F, 7C2, AK13A2, 11 and B4, included in antigenic area I (Fig. 8) and area B (Figs. and Antigenic areas II and B are identical. Most amino acid changes found in mutants selected with these antibodies were clustered around amino acids 262-272 of SEQ ID NO: 19.
Since these antibodies reacted in Western blots with proteolytic fragments of the Fl subunit, 4 was originally thought that they recognized "linear" epitopes determined by sequences of conecutive amino acids.
However, it seems that some conformations are needed for the integrity of certain epitopes, because only some of them were reproduced by synthetic peptides and amino acid substitutions located at a distant site influenced the binding of some antibodies. For example, the change at amino acid 216 (Asn to Asp), in the mutant 4/4 that conferred resistance to mAb AK13A2, also eliminated the reactivity with antibodies 7C2 and B4 (resistance to which are also conferred by selected changes at position 272).
The change at 216 is distantly located from the peptide 255- 275, which faithfully reproduced the epitope B4.
Consequently, some long range effect of amino acid 216 in the structure adopted by epitope B4 in the Fl subunit is likely to occur, Although the competition profiles in Figs. 5 and 6 of the mAbs overlapped extensively, protective mAbs 11, 13, B4 SUBSTITUTE SHEET WO 93/20210 PCr/GB93/00725 51 and B5 mapped to the same area 'site B in Figs. 5 and 7; site II in Fig. 8) and mutants resistant to these antibodies failed to bind only thopq mAbs recognizing site B. MAbs 7C2 and 47F also mapped to this area. Although there was inhibition of binding of mAbs RSV19 and 20 to RSV antigen by antibodies mapping to site B (site II), and vice versa, cluster analysis suggested that they recognized a different site (site C) This was confirmed by the finding that mutants selected for resistance to mAbs RSV19 and 20 still reacted with mAbs recognizing site B. Similarly, the binding of mAbs B13 and 814 to RSV was inhibited by mAbs mapping to sites B (II) and C. However, B13 and B14 appeared to map to a different region (site L) and this was confirmed by the observation that B13 and B14 bound to all the mutants selected with mAbs mapping to sites B (II) and
C.
The neutralization of RSV by the mAbs used to select the escape mutants is theorized to be related to their capacity to inhibit the membrane fusion of the F glycoprotein (Garcia-Barreno et al, (1989), cited above; Taylor et al., (1984), cited above]. By analogy with other paramyxoviruses [see, Morrison, Virus 1l:113-136 (1988)], it is assumed that the fusion activity of RSV depends upon the proteolytic processing of the F protein precursor. This modification generates the new NHW-terminal end of the Fl subunit, proposed to inteiai-t with lipid membranes through a short hydrophobic peptide, The antigenic areas of the F glycoprotein identified herein are distantly located from the fusion peptide in linear map; however, it is possible that other regions of the F protein influence the activity of the fusion peptide In this respect, mutants altered in the fusorenic activity of the influenza virus hemagglutinin (Daniels eot al., C.l, 4JI:431- SUBSTITUTE SHEET WO 93/20210 PCLT/G93/00725 52 439 (1985)] have been mapped outside the fusion peptide of the HA2 subunit.
The escape mutants were developed and evaluated as follows. The wild type and neutralization escape mutant viruses were grown in Hep-2 cells and purified from culture supernatants as previously described [Garcia-Barreno et al., viruss, 2:307-322 (1988)]. The Long and A2 strains of human RSV were plaque purified before being used to select viruses which escaped neutralization (mAb resistant mutants) by mAbs 47F, AK13A2, 7C2, 11, B4, B5, 19 or 20, and other mAbs directed against the F glycoprotein as described herein. These were selected in two different ways: A. A2 Strain Escape .Mtants Antibody escape mutant viruses of the RSV A2 strain, which are refractory to neutralizatior by one of the highly protective mAbs, 11, B4, B5, RSV19 and 20, were produced using plaque reduction techniques. For mAbs RSV19, B4 and B5, confluent monolayers of primary CK calls were infected with the A2 strain at a multiplicity of infection (MOI) of 0.2. Starting 24 hours after infection and continuing for 3 to 5 days, the culture medium was replaced daily with fresh medium containing 10% mAb. Virus was harvested when a cytopathic effect (CPE) was apparent.
Virus prepared in this way was mixed with an equal volume of the mAb under test for 1 hour at room temperature and inoculated onto CK monolayers in 35 mm multi-well plates (Nunc]. After 1 hour incubation at 370C, the plates were overlaid with medium containing 0.25% agarose incorporating a I in 10 dilution of the same mAb. Plates were then incubated at 370C in 5% CO, in air for 7 days before adding the vital stati, 0.3% 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide in 0.15M NaCI, to the overlay to visualize virus plaques.
SUBSTITUTE SHEET WO 93/20210 PC/GB93/00725 53 Putative mutant viruses were removed from plates in agar plugs containing single plaques, diluted in medium, mixed with an equal volume of mAb and inoculated onto CK monrolayers as before. Mutant viruses were plaque picked again and inoculated into tubes containing coverslips of calf testes cells or Hep-2 cells. After 4 to 6 days incubation, the coverslips were removed and stained with the mAb under test followed by FITC-labelled rabbit anti-mouse IgG [Sigma] or FITC-labelled rabbit anti-bovine IgG [Sigma].
A polyclonal bovine antibody to RSV followed by FITC- 4.abelled rabbit anti-bovine IgG was used as a positive control. RSVs that failed to react by immunofluorescence to the mAb under test were classed as mutants and were used to produce antigen for the ELISA described in Example 3 above.
Mutent viruses refractory to mAb 11 were selected essentially as described above, but without prior culture of the virus in cells containing 10% mAb in the supernatant.
Five mutant viruses were independently isolated from the A2 strain of RSV after plaquing in the presence of mAb 11. Eight mutants were independently isolated after culture in the presence of RSV19, 3 mutants after culture in the presence on mAb 20, 6 after culture in the presence oZ and 10 after culture in the presence of B4.
After cloning, each escape mutant was used as antigen in the ELISA described in Example 3 to test its reactivity with a panel of anti-F mAbs (Figs. 7 and 8).
Mutant viruses selected for resistance to mAb 11 lost the capacity to bind not only mAb 11 but also mAbs 13, B4, and B5, and had reduced binding to mAb 7C2, when compared with the parent A2 strain of RSV (Fig. All mutant viruses selected for resistance to either B4 or lost the capacity to bind not only B4 or B5 but also 11 and 13. However, some mutants selected with B4 C4947/5) SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 54 still bound to B5 but at a greatly reduced level when compared with the A2 strain.
As seen fcr mutants selected for resistance to mAb 11, some B4 and B5 mutants showed reduced binding to 7C2, however others failed to react with 7C2 C4947/5) In contrast to mutants selected with mAb 11, some mutants selected with B4 or B5 still reacted with mAb 18 (e.g.
C4947/5, 61:19, 61:16, 63:27 and C5014/7). B4 and mutants showed either the same, reduced or no binding to mAb B10 when compared with the parent A2 strain.
All mutant viruses selected with mAbs RSV19 or failed to react only with mAbs RSV19 and 20 (Fig. The binding of mAbs B13 and B14 (Fig. 7) and all other mAbs, described in Fig. 5, to all the mutants was the same as to the parent A2 strain of RSV, the mutant viruses retained the binding ot mAbs from other antigenic areas.
B. Long Strain Escape Mutants Escape mutants of the Long strain were isolated as previously described (Garcia-Barreno et al. (1989), cited above]. Briefly, virus stocks were enriched in mutant viruses by 4-5 consecutive passages in the presence of the selecting antibody, 47F, 7C2 or AK13A2.
Then, the viruses were plaque purified in antibody containing agar piates. Several viral plaques were isolated, and their resistance to antibody neutralization was confirmed. A single plaque originated from each aliquot of the virus stock was chosen for further analysis.
The epitopes recognized by the mAbs B4, 7C2 and AK13A2 were included in antigenic area II previously described by Garcia-Barreno et al. (1989), cited above, based solely on their reactivity with antibody-escape mutants. Similarly the epitopes recognized by mAbs RSV19 and 20 were included in antigenic area IV by the same criteria (Fig. 8).
SUBSTITUTE SHEET WO 93/20210 IPCTGB93/00725 The mutations selected in the escape viruses affected only epitopes from the antigenic area which included the selective antibody. For instance, mutant 4/4 did not react with any of the antibodies grouped in area II, whereas other mutants selected with the same antibody (11/3, 4, 5 and 7) reacted with mAbs 7C2 and B4 but not with 47F, 49F or AK13A2. Similarly, the mutants selected with mAbs 19 or 20 did not bind the antibodies grouped in the antigenic area IV, except mAb 52F. However, in all cases the mutant viruses retained the binding of mAbs from other antigenic areas.
The different reactivities of the antibodies from antigenic area II with the escape mutants indicated that their epitopes might overlap on the F molecule but were not identical. To further differentiate these epitopes, it was determined whether or not the corresponding mAbs would compete for simultaneous binding to the virus using a peroxidase labelled antibody in the 2LISA of Example 3 mixed with increasing, non-saturating amounts of each unlabeled antibody previously titrated against the Long strain.
The capacity of an anti-idiotype rabbit antiserum raised against mAb 47F to inhibit the binding of mAbs to RSV was also tested by ELISA (Palomo et al., J. Virol. _:4199- 4206 (1990)].
The results obtained indicated extensive competition between these antibodies for virus binding; however, antibody AK13A2 inhibited the binding of mAbs 47F and 49F in a non-reciprocal manner. In addition, the antiidiotype antiserum inhibited only the virus binding of mAbs 47F and 49F but not AK13A2, 7C2 and B4.
Thus, the epitopes included in antigenic area II could be distinguished by at least one of the following criteria: i) the reactivity of mAbs with escape mutants, ii) the competition of mAbs for virus binding and iii) the SUBSTITUTE SHEET WO 93/20210 PCT/B93/0725 56 inhibition of virus binding by an anti-Id antiserum. Only the epitopes 47F and 49F could not be distinguished by the above criteria, but they differ in both neutralizing capacity and susceptibility to denaturing agents.
Example 8 Location of amino,acid changes selected in neutralization escape mutants In order to identify the amino acid changes selected in the escape mutants, the F protein mRNAs obtained from cells infected with the different viruses were sequenced as follows. Hep-2 cells were infected with the different viruses and harvested 30-40 hours post-infection, when cytopathic effect was evident by the formation of syncytia.
Total RNA was isolated by the isothiocyanate-CsCl method [Chirgwin et al., Biohp, 18m:5294-5299 (1979)] and poly A+ RNA was selected by oligo dT-cellulose chromatography.
These mRNA preparations were used for sequencing by the dideoxy method [Sanger et al., 'Proc. Nat'l. Acad. Sci.. USA, 74:5463-5467 (1977)] using reverse transcriptase and 5'-"32p labelled oligonucleotides followed by a chase with terminal deoxynucleotidyl transferase [DeBorde et al., Anal.
Biochem, 1-52:275-282 (1986)]. The primers used for sequencing were synthesized according to the reported sequence of the Long F protein gene (Lopez et al., Zirus Res. J:249-262 (1988)].
The oligonucleotide primers used *or sequencing mutants selected with mAbs RSV19 and 20 were, in anti-RNA sense: SEQ ID NO: 23 F1216: SEQ ID NO: 24 F1300: SEQ ID NO: 25 F1339: SEQ ID NO: 26 F1478: SEQ ID NO: 27 F1548: SEQ ID NO: 28 F1707: The F mRNA of viruses selected with mAbs AK13A2, AK13A2, 7C2 and B4 were sequenced between nucleotid-4 420 SUBSTITUTE SHEET WO 93/20210 PCT/GIB93/0025 57 and 920, which encode the trypsin resistant fragments recognized by those antibodies (Fig. The F mRNA of viruses selected with mAb 11 were sequenced only between nucleotides 893 and 906, Similarly, the F mRNAs of viruses selected with mAbs 19 and 20 were sequenced between nucleotides 1100 and 1680, which encode the region of the tentatively located 26 kDa trypsin resistant fragment recognized by those antibodies.
Table 3 illustrates sequence changes selected in different neutralization escape mutants, including two previously reported mutants selected with mAb 47F [Lopez et al., (1990), cited above]. Only nucleotide (mRNA sense) and amino acid changes at the indicated positions, as compared to the Long and A2 strain sequences, are shown. Nb means not done.
This table, parts 3A, 3B, and 3C should be read across for each antibody. For example, for antibody 47F, virus 4, note that a nucleotide change Irom A to U at position 797 (Table 3A) results in an amino acid change from Asn to Tyr at position 262 (Table 3B), and a loss of antibody binding at 47F, 49F and AK13A2 (Table 3C).
SUJS1TiTUTE SPI.CT PCr/GB93/00725 WO 93/20210 ui 9/02 58 TABLE 3 Ab used for Specrtion ViruSes Long and A2 Nucleotide at position C A U A A A A C
U
U 47F AK13A2 4/4 11/3 4 7 41 7C2 A C 61: 16/7 61: 16/8 C4 84 f C4 90 C4909/6 C4 902 Wa C4 902Wb C4 902Wc SUBSTITUTE SHEET WO 93/20210 PTG9/02 PCT/GB93/00725 59 Ab used for Se ect ion mino Acid at position viruses 22Q 2iU 2U 2-EZ2 a .i217Z A2a 4 7F Long and A2 Ser Asn Leu Asn Asn Lys Arg Ile Tnyr Ile ~AK13A2 4/4 11/3 4 7 41 Asp Tyr Tyr Tyr Tyr Glu 7C2 Arg Ser Glu Thr Thr Thr B4 19 61: 16/7' 61:16/8 C4 84 f C4 90 9/5 C4909/6 C4 902 Wa C4 902Wb Ser Ser Ser Ser Ser Set C4902Wc SUBSITUTE SHEET WO 93/20210 WO 9320210PCr/GB93/00725 Ab used for Seeto Viruses Long arnd A2 TABLE 3 c Loss of binding with Antibodies 11 4 7F Not determined AK13A2 4/4 11/3 4 7 41 47F', 47F, 47F, 4 7F, 4 7F, 47F, 4 7F, 47F, 4 7F, 7C2 47, 49F, AK13A2 49F, AK13A2, 49F, AK13A2, 4 9F, AK13A2 49F, AK13A2 49F, AK13A2 49F, AK13A2 49F, AK13A2, 49F, AK13A2, 49F, AK13A2, 7C2, B4 7C2, B4 7C2, B4 -1C2, r54 7C2, B4 7C2 47F, 49F, AK13A2, 7C2, 61:16/7 61: 16/8 C484f C4909/5 C4 90 9/6 C4 902 Wa C49O2Wb C4902Wc 4 7F, 4 7F, 5 6F', 5 6F, 5 6F, 5 6F, 5 6F', 5 6F, 49F, AK13A2, 49F, AK13A2, 57F, 19, 57F, 19, 57F, 19, 57F, 19, 57F, 19, 57F, 19, 7C2, 7C2, SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 61 MAb 11 selected mitants wlhich had a single transversion (A to U) at nucleotide 816, which changed Asn-262 to Ile.
This change also led to the loss of the epitopes recognized by mAbs 13, B4 and B5, which are included in antigenic area B in Fig. 5 and is identical to that found in mutant 7 selected with mAb 47F which led to the loss of all the epitopes included in antigenic area II of Fig. 8.
Four viruses selected with mAb AK13A2 (11/3, 4, 5 and 7) has a single transversion (A to U) at nucleotide 797 which changed Asn-262 to Tyr. This change eliminated the .ainding sites for antibodies 47F, 49F and AK13A2 (see also Fig. 8) and it is identical to the change ob-' rved in mutant 4 selected with mAb 47F. A fifth virus selected with mAb AK13A2 had, in addition, a transition (2 to G) at nucleotide 659 which Asn-216 to Asp. This second amino acid change led to the loss of all the epitopes from antigenic area II (Fig. The last mutant selected with mAb AK13A2 had a single transition A to G at nucleotide 827, leading to the replacement of Lys-272 by Glu and the loss of all the epitopes from area II.
All mutants selected with mAb 7C2, except mutant 4, contained single nucleotide changes (A to G or A to C) at positions 827 or 828 which changed Lys-272 to Glu or Thr, respectively. These changes eliminated the reactivity with all the mAbs from antigenic area II. Mutant 4 had two nucleotide substitutions at position 583 (C to A) and 786 (U to C) which changed amino acids 190 (Ser to Arg) and 258 (Leu to Ser). The last mutant had only lost the binding site for mAb 7C2 but retained its reactivity with all the other anti-F antibodies (Fig. 8).
The two mutants selected with mAb B4 had a single nucleotide transversion at position 828 (A to C) which changed Lys-272 to Thr. Thus, all the amino acid changes SUBSTITUTE SHEEr WO 93/20210 PCT/GB93/00725 62 selected with mAbs from antigenic area II were clustered in a small segment of the F protein, between amino acids 262 and 272, except the changes at amino acids 258, 216 and 190 which were detected only in viruses with two amino acid substitutions.
All mutants selected with antibodies RSV19 or contained a single C to A transversion at nucleotide 1298 which changed Arg-429 to Ser. This amino acid change, located towards the carboxy terminal end of the cysteine rich region of the Fl subunit (Fig. eliminated the reactivity of all the mAbs grouped in the antigenic area IV, except antibody 52F. Amino acid 429 (Ser) is therefore important for the binding of mAbs RSV19 and 20 to the F protein. The synthetic peptides 417-432 and 422-438 of the F protein [SEQ ID NO: 191 reproduce at least part of the epitope recognized by mAb RSV19. The sequence results confirm the findings shown in Figs. 7 and 8 that antigenic areas II and IV do not overlap.
SUBSITrUTE SHEET WO 93/20210 PCT/GB93/00725 63 Example 9 Reactivity of antibodies with synthetjic peptid-s Since the antibodies used to select th\ escape mutants reacted in Western blot with trypsin fragments of the Fl subunit, whether or not synthetic peptides could reproduce the epitopes recognized by these antibodies was determined.
In summary, the results obtained with the synthetic peptides were also indicative of conformational constraints in the epitopes of antigenic area Tl. Epitope B4 was reproduced by the peptide 255-275 of SEQ ID NO: 55; however, other closely related peptides failed to react with that antibody. In addition, none of the peptides tested reproduced other epitopes of antigenic area II The region of the Fl subunit containing these epitopes is resistant to high doses of trypsin, indicative of a particular three-dimensional conformation which might be preserved in Western blots but not in synthetic peptides.
The peptides shown in Table 4 were synthesized in an Applied Biosystem 430 instrument, using the solid phase technology and t-Boc chemistry (Merrifield, Science, 212:341-347 (1986)]. The peptides were cleaved off the resin with trifluoromethyl sulfonic acid and purified from protecting groups and scavengers by Sephadex chromatography. The amino acid sequence of each peptide was confirmed by automated Edman degradation in an Applied Biosystem 477 protein sequencer.
Three peptides were synthesized with sequences corresponding to amino acids 250-273, 255-275 or 258-271 of the F1 subunit (SEQ ID NO: 55], which surrounded the positions changed in the mutants selected with mAbs from antigenic area II.
The binding of mAbs to synthetic peptides was tested by 3LISA of Example 3 in polyvinylchloride microtitre plates SUBSTITUTE SHEET WO 93/202:10 PCT/GB93/00725 64 coated overnight with 1-2 Ag of peptide. PBS containing pig serum was used as blocking reagent to eliminate spurious cross-reactions. The results are reported in Table 4 below.
Only antibody B4 and another bovine antibody, reacted with the peptide 255-275 of SEQ ID NO: 55. The B4 titre with this peptide was similar to that obtained against purified virus. However, this antibody did not react with peptides 250-273 nor 258-271 of SEQ ID NO: 55, which contained aimost the entire amino acid sequence included in peptide 255-275 of SEQ ID NO: 55. All other antibodies from area II failed to react with any of the peptides.
Three other peptidws, corresponding to the sequences 417-432, 422-438 and 435-450 of the F1 subunit [SEQ ID NO: which surrounded the position 429 changed in the escape mutanits selected with mAbs RSV19 or 20, were also tested by ELISA (Table Only antibodies RSV19, B13 and B14 reacted with the first two peptides (417-32 and 422-438 of SEQ ZD NO: Thus, two antigenic sites recognized by neutralizing, protective mAbs directed against the F protein have been identified. The first site contains several overlapping epitopes located within the trypsin resistant amino terminal third of ,*he Fl subunit, clustered around amino acids 262- 272 of SEQ ID NO: 55. Only one of these epitopes, that recognized by B4, was faithfully reproduced by a short synthetic peptide corresponding to amino acids 255-270 of the F protein (SEQ ID NO: 19]. The second antigenic site was located within the carboxy terminal third of the Fl subunit and the epitope recognized by mAb RSV19 and that recognized by B13 and B14 was reproduced by synthetic peptides corresponding to amino acids 417 to 432 and 422 to 438 of SEQ ID NO: 55. However, the epitopes recognized by SUBSTITUTE SHEET WO 93/20210 WO 9320210PCT/GB93/00725 MAbs RSV19, B13, and B14 do not appear to be identical since rnAbs 513 and B14 react with antibody-escape mutants selected with mAb RSV19 which have a substitution at amino acid 429- Ara (Fig. 7) indicating that an~ino acid 429-Arg is not essential for the binding of inAbs B13 and B14 to the F protein. The peptide fragmnents of the following Table 4 are taken from SE~Q ID NO: 55, the F1 subunit.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 66 Table 4 Reactivity of monoclonal antibodies with synthetic peptides Monoclonal antibody Peptide 7C2 47F AK13A2 11 B4 RSV19 20 B13 B14 )-273 <2.0 <2.0 <2.0 255-275 <2.0 <2.0 <2.0 <2.0 258-271 <2.0 <2.0 <2.0 <2.0 417-432 <2.0 <2.0 <2.0 <2.0 422-438 <2.0 <2.0 <2.0 <2,0 435-450 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 ND ND 6.7 <2.0 <2.0 o'2.0 <2.0 <2.0 <2.0 0 0 2.8 <2.0 6.0 <2.0 2.3 <2,0 <2.0 ND ND 4.5 3.2 5.0 4.3 ND ND
RSV
strain A2 8.4 9.1 4.9 6.4 5.3 6.4 6.3 5.6 5.6 s0 titre of antibody binding to synthetic peptides dried onto wells or RSV antigen tested in an ELISA.
Example 10 Pepscan Analysis of Epitope Rncognized by mAb Overlapping peptides corresponding to amino acids 255 to 275 of the F protein [SEQ ID NO: 191 were synthesized in duplicate as a series of octamers overlapping by seven amino acids and offset by one amino acid, bound to polyethylene pins using F-Moc chemistry following the method of Geyson et al, J. Immunol. Meth., .2Z:259-274 (1987). The software package, polyethylene pins and amino acids used to produce the peptides were obtained from Cambridge Research Biochemicals, Cheshire, England.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 67 The pins to which the peptides are bound were incubated with blocking buffer in 96 well microtitre plates (PBS containing 0.05% Tween 20 and 2% Marvel) on a rotary shaker.
After one hour incubation at room temperature, the pins were incubated with mAb B4, and diluted 1:600 in blocking buffer at 4 0 C with shaking. After being washed 10 times for minutes in PBS containing 0.05% Tween 20 (PBS/Tw), the pins were incubated with horseradish peroxidase (HRP)-rabbit anti-bovine IgG [Sigma], diluted 1:4000 in blocking buffer.
After one hour and 45 minutes, the pins were washed ten times for 5 minutes and incubated, in the dark with agitation, in microtiter plates containing 150pl/well of mg of azino-di-3-ethyl-benzthiazodisulpho-nate (Sigma] dissolved in 100 ml of substrate buffer (0.1M disodium hydrogen orthophosphate; 0,08M citric acid) containing 0.3 i/ml of 120 volume hydrogen peroxide. When sufficient color had developed, the O.D. was read at 405 nm on a Titertek Multiscan MCC 340 plate reader. MAb B4 recognized a single peptide extending from amino acid #266-273 of SEQ ID NO: 19 and havinig the sequence I T N D Q K K L bound to the pins.
The binding of B4 to this octomer was studied further using peptides, bound to pins, which represented the above sequence, but where every amino acid in this sequence was replaced in turn with each of the 20 naturally occurring amino acids. Duplicate peptides were synthesized as described above and the binding of mAb B4 to the peptides was determined by ELISA and is shown in Fig. 9. B4 bound to all peptides where amino acid 266-Ile was replaced in turn with all other amino acids, indicating that dmino acid 266- Ile was not essential for the binding of B4 to the peptide 266-273 of SEQ ID NO: 19. Similarly, replacement of amino acids 270-Glu and 273-Lys did not affect the binding of B4 SUBSTITUTE SHEET WO 93/20210 PCT/GB53/00725 68 to a significant extent. In contrast, substitution of amino acids 268-Asn, 269-Asp and 272-Lys resulted in the total loss of hinding of B4, indicating that these amino acids are essential for the binding of B4 to peptide 266-273 of SEQ ID NO: 19, These studies confirm the findings from the sequence analysis of antibody escape mutants (Example 8) which also showed that amino acids 268-Asn and 272-Lvs were critical for the binding of B4 to the F protein.
Substitution of amino acid 267-Thr resulted in reduced binding of B4 and replacement of amino acid 271-Lys resulted in significantly enhanced binding to the peptide. Maximum binding to the peptide 266-273 of SEQ ID NO: 19 was detected when amino acid 271-Lys was replaced by Ile.
Example 11 A Humanized Anti-RSV Antihbdy 1' The following example describes the preparation of an exemplary altered antibody utilizing the murine IgG, mAb called RSV!9 or RSMU19, described in co-pending PCT application No. PCT/GB91/01554 as the source of the donor var4 aole chain sequences and CDRs. Similar procedures may be allowed for the development of altered antibodies, using otner anti-RSV antibodies described herein.
RSV19 is specific for the fusion protein of RSV.
The RSV19 hybridoma cell line was obtained from Dr.
Geraldine Taylor. Methodology for the isolation of hybridoma cell lines secretin monoclonal antibodies specific for RSV is described by Taylor et al., Immunonogy, 52:137-142 (1984).
As described in the preceding example, cytoplasmic RNA was prepared by the method of Favaloro et al., (1980) cited above from the RSV19 hybridoma cell line, and cDNA was synthesized using Ig variable region primers as follows.
For the Ig heavy chain variable region, RSV19VH (see Figs.
SUBSTITUTE SHEET WO 93/20210 WO 9320210PCT/GB93/00725 69 15B and 19), the primer [SEQ ID NO: 33] VH1FOR 'TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG31 was used, and for the Ig light chain variable region, RSV19VK( (see Figs.
16A and 16B), the primer [SEQ ID NO: 34] VK1FOR 5'GTTAGATCTCCAGCTTGGTCCC3' was used.
cDNA synthesis reactions consisted of 20pgg RNA, 0.4p.M VHIFOR or VKlFOR, 250gAM each of dATP, dCTP, dGTP and dTTP, Tris-IICl pH{ 7.5, 75mM KCl, 10mM DTT, 3mM MgCl 2 and 27 units RNase inhibitor in a total volume off 5041l. Samples were heated at '700C for 10 minutes and slowly cooled to 42 0
C
over a period of 30 minutes. Then, 1.00g !M;MLV reverse transcriptase was added and incubation at 420C continued for 1 hour.
VII and VK, cDNAs were then amplified using PCR. For PCR, the primers uised were: VHIFOR; VK1FCR; V~lBACK (described in Example 318), and (SEQ ID NO: 351 VK1BACK 5'GACATTCAGCTGACCCAG7'CTCCA 3'.
Primers VH1FQR, VI(1FOR# VHIBACK and VIBACK, and their use for PCR-amplification of. mouse Ig DNA, are described by orlandi et al., (19G9), cit d above.
For PCR amplification of VII, DNA/primer mixtures consisted of 51.U RNA/cDNA hybr-id, and 0.5gVM VH1FOR. and VH1BACK primers. F'or PCR amplifications of VK, DNA/primer mixtuires consiste'd of 5gl RNA/cDNA hybrid, and 0.5pM VKFOR and VK1BACK pzimers. To these mixtures was added 200 gM each of dATP, dCTP, dGTP and dTTP, 10mM Tris-HCl pH 8.3, KC1, 1.5mM MgCl 2 0.01% gelatin, 0.01% (v/v) Tween 20, 0.01% (vlv) Nonidet P40 and 2 units Taq DNA polyrnerase (United State-s Biochemicals -Cleveland, Ohio, USA). Samples were subjectedi to 25 thermal cycles of PCR at 940C, 1 minute; 60'C, 1 miinute; 721C, 2 minutes; ending with minutes at 720C. For cloning and sequencing, amplified VH SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 DNA was purified on a low melting point agarose gel and by Elutip-d column chromatography and cloned into phage M13.
The general cloning and ligation methodology was as described in Maniatis et al., cited above.
VH DNA was either directly ligated into the SmaI site of M13 mp 18/19 or, following digestion with Pstl, into the PstI site of M13tgl31 [Amersham International-Little Chalfont, UK]. Amplified VK was similarly gel purified and cloned by the following alternatives: PvuII digest into M13mpl9 (Smal site); PvuII and BglII digest into M13mpl8/19 (Smal-BamHI site); PvuII and BglII digest into M13tgl31 (EcoRV-BglII site); BglII digest into M13tgl31 (SmaI-BglII site). The resultant collections of overlapping clones were sequenced by the dideoxy method [Sanger et al., cited above] using Sequenase [United States Biochemicals-Cleveland, Ohio, USA].
From the sequence of RSV19 VH and VK domains, as shown in Figs. 14A and 14B, and 15A, and 15B, respectively, the CDR sequences were elucidated in accordance with the methodology of Kabat et al., in "Sequences of Proteins of Immunological Interest", US Dept of Health and Human Services, US Government Printing Office, (1987) utilizing computer assisted alignment with other VH and VK sequences.
The murine RSV19 CDRs were transferred to human frameworks by site directed mutagenesis. The primers used were: (SEQ ID NO: 36] VHCDR1 CTGAAGGTGAAGCCAGACACGGT 3' (SEQ ID NO: 37] VRCDR' 5' CATTGTCACTCTGCCCTGGAACTTCGGGG CATATGGAACATCATCATTCTCAbGATCAATCCA 3' [SEQ ID NO: 38] VHCDR3 5' CCCTTGGCCCCAGTGGTCAAAGTCACTCCC CCATCTTGCACAATA 3' SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 71 [SEQ ID NO: 39] VKCDR1 5' CTGCTGGTACCATTCTAAATAGGTGTTTCCA TCAGTATGTACAAGGGTCTGACTAGATCTACAGGTGATGGTCA 3' [SEQ ID NO: 40] VKCDR2 5' GCTTGGCACACCAGAAAATCGGTTGGAAACTC TGTAGATCAGCAG 3' [SEQ ID NO: 41] VKCDR3 5' CCCTTGGCCGAACGTCCGAGGAAGATGT GAACCTTGAAAGCAGTAGTAGGT 3' The DNA templates for mutagenesis comprised human framework regions derived from the crystallographically solved proteins, NEW [Saul, et al., J. Biol..Chem., 53:585- 597 (1978)] with a substitution of amino acid 27 from serine to phenylalanine [See, Riechmann et al., DLc.cit and REI [Epp et al., Eur J. Biochem. 45:513-524 (1974)] for VH and VK domains, respectively. M13 based templates comprising human frameworks with irrelevant CDRs were prepared as described by Riechmann et al., Nature. 332 (1988).
Oligonucleotide site directed mutagenesis of the human VH and VK genes was based on the method of Nakamaye et al., Nucl. Acid Res., LA:9679-9698 (1986). To 54g of VH or VK single-stranded DNA in M13 was added a two-fold molar excess of each of the three VH or VK phosphorylated oligonucleotides encoding the three mouse CDR (complementarity determining region) sequences. Primers were annealed to the template by heating to 70 0 C and slowly cooled to 37 0 C. To the annealed DNA was added 6 units T4DNA ligase [Life Technologies, Paisley, UK]; 0.5 mM of each of the following nucleoside triphosphates (dATP, dGTP, dTTP and 2'-deoxycytidine 5'-0-)l-thiotriphosphate) (thiodCTP); Tris-HCl (pH 6mM MgCl 2 5mM DTT [Sigma, Poole, UK]; and 10mM ATP in a reaction volume of 50pl. This mixture was incubated at 16 0 C for 15 hours. The DNA was then ethanol precipitated and digested with 5 units Ncil [Life Technc.- gles, Paisley, UK] which nicks the parental strand SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 72 but leaves the newly synthesized strand containing thiodCTP intact. The parental strar-i was then removed by digesting for 30 minutes with 100 units exonuclease III [Pharmacia, Milton Keynes, United Kingdom] in 50 p.1 of 60mM Tris-HCl (pH 0.66mM MgCl 2 and 1mM DTT. The DNA was then repaired through addition of 3 units of DNA polymerase I (Life Technologies, Paisley, UK], 2 units T4 DNA ligase in 5J3 .1 of 60 mM Tris-HCl (pH 6mM MgC1 2 5mM DTT, 10mM ATP and mM each of dATP, dCTP, dGTP and dTTP. The DNA was transformed into competent E. coli TG1 cells (Amersham International, Little Chalfont, UK] by the method of Maniatis et al., cited above.
Single-stranded DNA was prepared from individual plaques and sequenced by the method of Messing, Methods in Enzymoloav, 11:20-78 (1983) If only single or double mutants were obtained, then these were subjected to further rounds of mutagenesis (utilizing the methodology described above) by using the appropriate oligonucleotides until the triple CDR mutants were obtained.
The CDR replaced VH and VK genes were cloned in expression vectors (by the method of Maniatis et al.) tc yield the plasmids pHuRSV19VH and pHuRSV19VK. The plasmids are shown in Figs. 16 and 17, respectively. For pHuRSV19VH, the CDR replaced VH gene together with the Ig heavy chain promoter, appropriate splice sites and signal peptide sequences were excised from M13 by digestion with HindIIl and BamHI, and cloned into an expression vector containing the murine Ig heavy chain enhancer, the SV40 promoter, the gpt gene for selection in mammalian cells and genes for replication and selection in E. col.. The variable region amino acid sequence is shown in Fig. 19. A human IgGI constant region was then added as a BamHI fragment.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 73 The construction of the pHuRSV19VK plasmid was essentially the same except that the gpt gene was replaced by the hygromvcin resistance gene and a human kappa chain constant region was added (see Figs. 17 and 22).
10g of pHuRSV19VH and 20Lg of pHuRSV19VK were digested with P .I utilizing conventional techniques. The DNAs were mixed together, ethanol precipitated and dissolved in water. Approximately 10 7 YB2/0 cells [American Type Culture Collection, Rockville, Maryland, USA] were grown to semiconfluency, harvested by centrifugation and resuspended in DMEM [Gibco, Paisley, UK] together with the digested DNA in a cuvette. After 5 minutes on ice, the cells were given a single pulse of 170V at 960uF (Gene-Pulser, Bio-Rad- Richmond, California, USA) and left in :lce for a further minute. The cells were then put into 20 ml DMEM plus foetal calf serum and allowed to recover for 48 hours.
After this time, the cells were distributed into a 24-well plate and selective medium applied (DMEM, 10% foetal calf serum, 0.8.g/ml mycophenolic acid, and 250pg/ml xanthine).
After 3-4 days, the medium and dead cells were removed and replaced with fresh selective medium. Transfected clones were visible with the naked eye 10-12 days later.
The presence of human antibody in the medium of wells containing transfected clones was measured by conventional ELISA techniques. Micro-titre plates were coated overnight at 40C with goat anti-human IgG (gamma chain specific) antibodies [Sera-Lab-Ltd., Crawley Down, UK) at 1 gg per well. After washing with PBST (phosphate buffered saline containing 0.02% Tween 20x 100l of culture medium from the wells containing transfectants was added to each microtitre well for 1 hour at 37 0 C. The wells were then emptied, washed with PBST and either peroxidase-conjugated SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 74 goat anti-human IgG or peroxidase-conjugated goat anti-human kappa constant region antibodies [both obtained from Sera- Lab Ltd., Crawley Down, UK] were added at 100 ng per well, Plates were then incubated at 37 0 C for 1 hour. The wells were then emptied and washed with PBST. 340 pg/ml qphenylenediamine in 50mM sodium citrate, 50mM sodium phosphate (pH 5.0) and 0.003% H 2 0 were added at 200( 1 per well. Reactions were stopped after 1 to 5 minutes by the addition of 12.5% sulphuric acid at 50Ll per well. The absorbance at 492 nm was then measured spectrophotometrically.
The resulting humanized antibody HuRSV19VH/VK (also called RSH200), secreted from cell lines cotransfected with pHuRSV19VH and pHuRSV19VK, was purified on Protein-A agarose columns [Boehringer Mannheim, Lewes, UK] and tested for binding to RSV virus in an ELISA assay. Antigen consisted of calf kidney (CK) cells infected with RSV A2 strain (Lewis et al., Med. J. Australia, AU:932-933 (1961)] and treated with 0.5% NP40 detergent to yield a cell lysate. A control cell lysate was similarly prepared using uninfected CK cells. Microtitre plate wells were coated wita either infected or control cell lysate. Antigen coated platen were blocked with PBST for 1 hour at 37 0 C, washed with PBST, and thereafter humanized antibody was applied HuRSV19VH/VK) After 1 hour at 37 0 C, the wells were emptied, washed with PBST and 200 ng goat anti-human IgG antibodies [Sera Lab-Ltd., Crawley Down, UK] added per well.
After 1 hour at 37 0 C, the wells were emptied, washed with PBST and 200L1 of a 1:1000 dilution of HRP-conjugated rabbit anti-goat IgG antibodies [Sigma-Poole, UK] were added.
After 1 hour at 37 0 C, the wells were emptied and washed with PBST. To each well was added 200g1 substrate buffer SUBSTITUTE SHEET WO093/20210 PCT/GB93/00725 (3404g/ml q-phenylenediamine in 50mM sodium citrate, sodium phosphate (pH 5.0) and 0.003% H 2 0 2 Reactions were stopped by the addition of 50gl 12.5% sulphuric acid.
The absorbance at 492 nm was then measured.
This humanized antibody HuRSV19VH/VK (RSHZO0), generate, by the straight replacement of the RSV19 heavy and light chain CDRs into the human heavy chain framework regions (variable and constant regions REI and kappa, respectively) bound to whole RSV preparations, although with an affinity less than the donor murine RSV19 antibody.
p 2 Production of High Affinity Anti-RSV Antibodies High affinity antibodies specific for RSV were developed by a method designed to achieve minimal variable region framework modifications giving rise to high affinity binding. The method involves the following order of steps of alteration and testing: 1. Individual framework amino acid residues which are known to be critical for interaction with CDRs are compared in the primary antibody and the altered CDR-replacement antibody. For example, heavy chain amino acid residue 94 (Kabat numbering-see Kabat et al., cited above) is compared in the primary (donor) and altered antibodies. An ,rg residue at this position is thought to interact with the invariant heavy chain CDR Asp residue at position 101.
If amino acid 94 comprises Arg in the framework of the primary antibody but not in the framework of the altered antibody, then an alternative heavy chain gene comprising Arg 94 in the altered antibody is produced. In the reverse situation whereby the altered antibody framework comprises an Arg residue at position 94 but the primary antibody does not, then an alternative heavy chain gene comprising the original amino acid at position 94 is produced. Prior to SUBSTITUTE SHEET WO093/20210 PCT/GB93/00725 76 any further analysis, alternative plasmids produced on this basis are tested for production of high affinity altered antibodies.
2. Framework amino acids within 4 residues of the CDRs as defined according to Kabat (see Kabat et al., cited above) are compared in the primary aw ibody and altered CDRreplacement antibody. Where differences are present, then for each region upstream of VHCDRI) the specific amino acids of that region are substituted for those in the corresponding region of the altered antibody to provide a emall number of altered genes. Alternative plasmids produced on this basis are then tested for production of high affinity antibodies.
3. Framework residues in the primary and altered CDRreplacement antibodies are compared and residues with major differences in charge, size or hydrophobicity are highlighted. Alternative plasmids are produced on this basis with tha individual highlighted amino acids represented by the corresponding amino acids of the primary antibody and such alternative plasmids are tested for production of high affinity antibndies.
The method is exemplified by the production of a high affinity altered antibody derivative of HuRSV19VH/VK specific for RSV. Comparison of VH gene sequences between RSV19VH and pHURSV19VH (Figs. 18-22) indicates that 3 out of 4 amino acid differences occur between amino acids 91 to 94 of the F protein of SEQ ID NO: 19, which defines a framework sequence adjacent to heavy chain CDR3.
Thus, plasmid pHuRSV19VHFNS (Fig. 20) was produced by inserting the RSVI9 heavy chain CDRs and the four amino acid framework sequence amino acids 91 to 94 into the human framework described in the preceding example. Using SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 77 oligonucleotide site directed mutagenesis, the following oligonucleotide was used for mutagenesis of the HuRSV19VH gene in M13: [SEQ ID NO: 421 HuRSV19VHFNS ACCG 3'.
The cell line cotransfected with pHuRSV19VHFNS and pHuRSV19VK (Fig. 22) produced a second humanized antibody, HuRSV19VHFNS/HuRSV19VK (abbreviated hereafter as RSHZ19).
This antibody was tested in an ELISA assay for analysis of binding to RSV antigen prepared from detergent-extracted, virus-infected cells. The substitution of VH residues 91 to 94 in HuRSV19VH/VK with VH residues from mouse RSV19VH partially restored antigen binding levels. Additional analysis of HuFNS binding properties was performed using an ELISA assay in which intact Type A RSV (Long strain) was used as the antigen. The data from such additional analysis show that there is little if any difference between the ability of the murine mAb RSV19 and the humanized RSHZ19 antibodies to bind to intact, non-denatured RSV. This additional analysis also showed detectable binding of HuRSV19VH/VK to intact virus, although of a much lower magnitude than was seen with either RSV19 or RSHZ19.
Thus, the data from this additional analysis suggests that the affinity for the native antigen was restored in the RSHZ19 mAb. Specificity of RSHZ19 for RSV F protein was shown by conventional Western blot analysis using a truncated soluble F protein construct expressed in CHO cells.
Example 13 immunofluorescence Analysis; Qo Humanized Antibodies In order to ascertain the potential clinical usefulness of a humanized antibody specific for RSV, an immuno- SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 '78 fluorescence analysis of binding to 24 RSV clinical isolates was undertaken. The isolates were obtained from children during the winter of 1983-84 by the Bristol Public Health Laboratory (Bristol, England) and represented both of the major subgroups of FSV. Thirteen isolates were serotyped as subgroup A and 11 isolates as subgroup B. HeLa or MA 104 cells infected with RSV isolates were grown in tissue culture. When the cells showed evidence of cytopathic effect, 20 ml of 0,02% disodium ethylenediaminetetraacetic acid (EDTA) [BDH Chemicals Ltd., Poole, UK) in PBS and 3ml of 0.25% trypsin in PBS were added and the cell suspension spotted into wells of PTFE-coated slides (polytetrafluoroethylene coated slides) (Hendley, Essex, UK]. After 3 hours at 37 0 C, the slides were dried and fixed in 80% acetone. Cells were overlaid with monoclonal antibody either humanized antibody, RSHZ19 or the murine antibody RSV19) for 1 hour at room temperature.
After extensive washing, either fluorescein-conjugated rabbit anti-mouse IgG (Nordic Laboratories-Tilburg, The Nett.rlands] or fluorescein-conjugated goat anti-human IgGI [Southern Biotechnology, Birmingham, Alabama, USA] was added, and the incubation was repeated. After further washing, cells were mounted in glycerol and examined under UV light.
The results of comparative immunofluorescance for the humanized antibody, RSHZ19, and the murine antibody RSV19 indicated that 100% of clinical isolates are recognized by both the humanized and murine antibodies. Such data demonstrated that the humanized antibody has the potential for recognition of most clinical isolates comprising both of the major RSV subgroups.
SUBSTITUTE SHEET WO 93/20210 PCT/IGB9300725 79 The humanized antibody, RSHZ19, was next tested for biological activity in vitro in a fusion inhibition assay.
A suspension of MA104 cells was infected with RSV at an m.o.i. (multiplicity of infection) of 0.01 PFU (plaque forming units) per cell. After 1 hour at 37 0 C, 2 ml of cells at 10 5 /ml were distributed to glass coverslips in tubes. After a further 24 hours at 37 0 C, the culture medium was replaced by medium containing dilutions "f humanized antibody, RSHZ19. Twenty-four hours later, coverslip .0 cultures were fixed in methanol for 10 minutes and stained with May Grunwald stain [BDH Chemicals Ltd., Poole, UK], The effect of increasing concentrations of RSHZ19 in inhibiting the frequency of giant cells demonstrates the biological activity of the humanized antibody RSHZ19 in inhibiting Type A RSV induced cell fusion. Additional studies showed that the fusion inhibition titres for RSV19 versus RSHZ19 were comparable, providing additional evidence that affinity for the native viral ai.cigen was fully restored in the humanized RSHZ19. The humanized antibody RSHZ19 has also been shown, using methodology analogous to that utilized above for showing inhibition of Type A RSV induced cell fusion, to exhibit a dose dependent inhibition of Type B RSV (strain 8/60) induced giant cell fusion.
The humanized antibody, RSHZ19, was next tested for biological activity in vitro in an RSV-mouse infection model. BALB/c mice [Charles Rivers: specific pathogen free category 4 standard] were challenged ,ntranasally with 104 PFU of the A2 strain of human RSV (Taylor et al., Infct.
Immun., A3:649-655 (1984)]. Groups of mice were administered with 25gg of humanized antibody either one day prior to virus infection or 4 days following infection.
SUBSTITUTE SHEET WO 93/20210 PCr/GB93/00725 Administration of antibody was either by the intranasal or intraperitoneal routes. 5 days after RSV infection, mice were sacrificed and lungs were assayed for RSV PFU [see, Taylor et al., c:,ted above]. The data showed that RSHZ19 at a single dose of 25 Ig per mouse is extremely effective in prevention and treatment of RSV infection.
RSHZ19 was also shown to be active in vivo when administered prophylactically to mice challenged with Type B RSV (strain 8/60) using methodology similar to that described above. In addition, the humanized antibody HuRSV19VH/VK was also shown to be active in vivo when administered prophylactically to mice challenged with "ype B RSV (strain 8/60) using methodology similar to that described above.
Example 14 Comparison of blood levels of RSHZI9 after i.v.
or i.p. Inoculation of Mice Five female BALB/c mice (weighing approximately were inoculated i.p. with 50 )g RSHZ19 (CHO) and another were inoculated intravenously with 50 ig RSHZ19 (CHO). Mice were bled from the tail 2 hours, 1, 4, 7, 14, 21 and 46 days later and the levels of RSHZ19 in the sera were determined using two different ELISAs as follows.
Plates were coated with a lysate of either RSV (strain) A2-infected or uninfected Hep-2 cells, followed by dilutions of mouse sera and HRP-anti human IgG.
(ii) Plates were coated with 200ng of anti-idiotypic mAb B12, followed by mouse sera and HRP-anti human IgG.
Both assays gave essentially the same results, although the B12 ELISA appeared to be more sensitive. Two hours after inoculation the serum level of RSHZ19 was SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 81 greater in mice inoculated i.v. compared with those inoculated However, titres of RSHZ19 were equivalent in both groups of mice by 24 hours after inoculation. The level of RSHZ19 remained constant for at least 4 days after inoculation and was beginning to decline at 7 days. After this time, there was a rapid decline in serum levels of RSHZ19 in mice inoculated whereas the level of RSHZ19 declined more slowly in mice inoculated i.p. These results are summarized in Table Table Comparison of Serum Levels of RSHZ19 (CHO) After IV or IP Inoculation of Mice 1 a- ELISA ttre in mice inoculated IV Tp Day R FLTA RS EIJ A FJ. TSA 0.1 3.5 0.2 4.6 _L 0.2 3.2 0.1 4.2 0.1 1 3.1 0.2 4.2 0.04 3.3 0.04 4.3 0.1 4 3.2 0.1 4.2 0.1 3.3 0.2 4 2 ±0.04 7 3.1 0.2 3.7 C.3 3.6 0.2 3.9 0.1 14 1.5 1.5 3.1 0.2 3.8 0.1 21 1.5 1.5 2.3 1.3 3.5 0.2 46 ND 1.5 ND 3.3 0.1 To investigate if the rapid decline in RSHZ19 between days 7 and 14 in mice inoculated i.v. was due to an immune response to RSHZ19, the sera were tested for antibody to RSHZ19 in an ELISA. Plates were coated with 50ng of RSHZ19, followed by D21 mouse sera and HRP-anti mouse IgG. As seen SUBSTITUTE SHEET WO) 93/20210 PCr/GB93/00725 82 in Table 6, mice inoculated i.v. developed antibody to RSHZ19 at day 21, whereas mice inoculated i.p. had no detectable antibody to RSHZ19. These results suggest that tolerance to RSHZ19 developed following but not i.v., inoculation of mice with this antibody. Mice are inoculated i.p. or i.v. with RSHZ19 produced from CHO or myeloma cells to further confirm these results.
Table 6 Antibody Response to RSHZ19 in Sera of Mice Inoculated i.v. or i.p. with 50 pg RSHZ19 (CHO) Mice log,, ELISA Inoculatd _titre* i.v. 2.5 0.2 i.p. Plates coated with 50ng RSHZ19 (CHO) Example 15 ReconiJtion of Clinical Isolates Preliminary experiments using biotin-labeled, RSHZ19 (B01, 2.5 pg/ml; 9/29/92 from SmithKline Beecham) and FITCstreptavidin (Sigma) on RSV-infected and uninfected calf testes cells showed that biotin-RSHZl9 at 1/40 with FITCstreptavidin at 1/80 gave spezific fluorescence of RSVinfected cells.
Nine slides of nasopharyngeal aspirates from children hospitalized with RSV infection were obtained from the WHO Collaborating Centre for Reference and Research on Rapid Laboratory Viral Diagnosis, the Royal Victoria Infirmary, SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 83 Newcastle-upon-Tyne, England. Each slide consisted of 3 replicate samples in separate chambers. One sample was stained with Imagen T RSV, (Novo Nordisk Diagnostics Ltd, Cambridge CB4 4WS, UK) as instructed in the technical data.
Another sample was stained with a 1:40 dilution of biotinylated RSHZ19 for lh at room temperature, washed 3x with PBS, and incubated with FITC-Streptavidin for lh at room temperature. The third sample was stained with FITC- Streptavidin only. After washing 3x with PBS, the samples stained with FITC-Streptavidin were counterstained with 0.01% Evans blue for 5 min. washed and mounted in glycerol. RSV-infected cells in the nasopharyngeal aspirate samples stained using IMAGEN T RSV showed discrete fluorescent intracellular cytoplasmic inclusions typical of infected cells stained with mAb to the N protein of RSV. In contrast, nasopharyngeal aspirate cells stained with biotinylated-RSHZ19 and FITC-Streptavidin showed more generalized granular cytoplasmic staining, typical for the F protein. There was no fluorescence of samples stained with FITC-Streptavidin alone.
The results are illustrated in Table 7. Biotinylated RSHZ19 recognized RSV in all the nasopharyngeal aspirates studied. The intensity of fluorescence in samples stained with biotinylated RSHZ19 was less than in those stained with 2o IMAGEN T RSV; however, the numbers of stained cells appeared to be similar in both samples.
SUBSTITUTE SHEET WO 93/20210 WO 9320210PCT/GB93/00725 Table 7 Binding of RSHZ19 to RSV in Nasopharyngeal Aspirates Date Spec. Specimen 6513 02/02/88 7430 15/03/88 9997 16/07/85 7920 22/03/85 8195 20/11/91 8818 13/12/91 Flimorescenne Sub- Imagen T m FITC- Biot. RSHZ19 typ _,RL Sten F'TTC-Strept.
A A B ND ND 8845 14/12/91 9495 16/01/92 9575 08/01/92 ND ND These studies indicate that RSHV19 recognizes all 310 clinical isolates of RSV examined so far.
E~xample 16 Prop~hvlactin effectf bovine rnAb B4 on Ray fnfection n alvesp~ Three 1 to 2 week old gnotobiotic calves, weighing 43 to 55 kg, were inoculated intratracheally with 215 mg of purified bovine mAb, B4, and three were inoculated i.t.
with PBS. Twenty-four hours later, all calves were challenged i.n. and i.t. with approximately 105 pfu of the Snook strain of bovine RSV. The Snook strain of bovine RSV was isolated from the lung of a calf which died of pmx.=oia (Thomas et al., Brit. J. Exp) Pathol., £5.:19-28 (1984)1, and grown in secondary CK cells. Nasopharyngeal swabs were SUBSTITUTE SHEET WO 93/20210 Pe/G93/00725 obtained daily after infection and calves were killed on day 7 of infection. Lung washings were obtained at post-mortem by filling the lungs with 800 ml of PBS. Lung washings were centrifuged at 1300 g and the cell pellet resuspended in ml of medium. All samples were assayed for RSV on secondary CK monolayers.
Treatment of calves with mAb B4 24 hours prior to challenge with the bovine strain of RSV had no effect on virus shedding from the nasopharynx throughout the 7 days of infection. However, as reported in Table 8 below, little or no virus was recovered from the lungs of the calves treated with B4, 7 days after RSV challenge. In contrast, between 103 and 104 pfu/ml was recovered from the lungs of the control calves. Calves given mAb B4 did not develop pneumonic lesions, whereas the lungs of the control animals were pneumonic.
Table 8 Prophylactic effects of bovine mAb B4 on RSV infection in calves D7 Virus Titre (logo PFU/ml) Treatment Calf No. Nose Lung Wash Pneumonic Lesions B4 d-l' 1097 2.4 <0.7 <1 1230 3.5 0.7 0 1242 3.6 <0.7 <1 i~ne 1098 2.2 3.2 9 1231 <0.7 3.2 6 1245 2.1 4.2 6 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 86 Example 17 Prohylactic Effects of Bovine mAbs on RSV infection in Calves Calves were also treated i.t. with 15 mg B13 or 15 mg Bl 24 hours prior to challenge with bovine RSV (BRSV). MAb B1 is an anti-F antibody that is non-neutralizing, nonprotective in mice but fixes complement (Kennedy et al, (1988)). Although there was a reduction in the titre of virus in the lungs of calves given B13, the difference in titre of virus compared with control calves given PBS was rot statistically significant (p 0.07) (Table 8).
However, there was a statistically significant reduction in the severity of pneumonic lesions in calves given B13 when compared with controls. There were no significant differences in either the level of virus in the lungs or the severity of pneumonia in calves given B1 when compared with controls (Table 9).
These studies indicate that E4 is more protective against BRSV infection in the calf than B13. Further, a non-protective, complement fixing mAb, whilst not protective in the calf, does not exacerbate pneumonic lesions.
SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 87 Table 9 Prophylactic Effects of Bovine mAbs on RSV infection in Calves Nasal Shedding Luna Virus Mean Lung Treatmt No, Duration peak No. Wash Pneumonic calves (days) titre p Infec. titreA lesions B4 d-I 3 5.0 0 3.9 0.7 1 <0.
7 b <Ic B13 d-1 4 4.5 0.6 2.9 0.2 2 1.3±1.5C 22.6c Bi d-1 4 4.8 0.5 3.2 0.7 4 2.6+1.8d 5.5+2.4 d PBS d-1 9 4.4 1.2 3.0 0.5 9 3.1±1.5 10.5±7.0 a logu PFU/ml b Probability that passively immunized animals are significantly different from controls. p<0.01; c p=0.07; d NS; p<0.05 Example 18 Cloning and Sequencina of B4. B13 and B14 Cytoplasmic RNA was prepared by the method of Favaloro et al., Meth. Enzymol., .6:718-749 (1980) from B4, B13 and B14 hybridoma cell lines. The primers BCG1FOR: 5'TTGAATTCAGACTTTCGGGGCTGTGGTGGAGG 3' [SEQ ID NO: 29], which is based on sequence complementary to the 5' end of bovine 7-1 and 7-2 constant region genes, and BCL1FOR: 5'CCGAATTCGACCGAGGGTGGGGACTTGGGCTG 3' [SEQ ID NO: 30], which is complementary to the 5' end of the bovine lambda constant region gene, were used in the synthesis of Ig heavy (VH) and light (VL) chain variable region cDNAs, respectively.
cDNA synthesis reactions consisted of 20Lg RNA, 0.4PM BCG1FOR or BCL1FOR, 250IM each of dATP, dCTP, dGTP and dTTP, SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 88 Tris-HCI pH 7.5, 75mM KC1, 10mM DTT, 3mM MgC1, and 27 units RNase inhibitor [Pharmacia, Milton Keynes, United Kingdom] in a total volume of 50l. Samples were heated at 0 C for 10 minutes and slowly cooled to 42 0 C over a period of 30 minutes. Then, 100 MMLV reverse transcriptase [Life Technologies, Ppisley, United Kingdom] was added and incubation at 42 0 C continued for 1 hour.
VH and VK cDNAs were then amplified using the polymerase chain reaction (PCR) as described by Saiki et al., 9cience, 291:487-491 (1988). For the PCR, the primers used were BCG1FOR, BCL1FOR, [SEQ ID NO: 31] VHIBACK: (R)CTGCAG(S)AGTC(W)GG 3' [SEQ ID NO: 32] VL2BACK: 5'TTGACGCTCAGTCTGTGGTGAC(K)CAG(S) (M)GCCCTC 3' VH1BACK is described by Orlandi et al., Proc. Nat'l.
Acad. Sci.. USA, 8£:3833-3937 (1989). The sequence of VL2BACK was based on nucleotide sequences listed for the end of human lambda variable regions (Kabat et al., (1987), cited above].
For PCR amplification of VH, DNA/primer mixtures consisted of 5p1 RNA/cDNA hybrid and 0.5lM BCG1FOR and VHlBACK primers. For PCR amplifications of VL, DNA/primer mixtures consisted of 5pl RNA/cDNA hybrid and 0.5.M BCL1FOR and VL2BACK primers. To these mixtures was added 250plM each of dATP, dCTP, dGTP and dTTP, 10mM Tris-HCl pH 8.3, KC1, 1.5mM MgCl, 0.01% gelatin, 0.01% Tween 0.01% Nonidet P40 and 5 units AmpliTaq (Cetus].
Samples were subjected to 25 thermal cycles of PCR at 94 0
C,
30 seconds; 55 0 C, 30 seconds; 72 0 C, 45 seconds; ending with minutes at 72 0 C. For cloning and sequencing, amplified VH DNA was purified on a low melting point agarose gel and by SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 89 Elutip-d column chromatography (Schleicher and Schuell- Dussel, Germany] and cloned into phage M13 [Pharmacia-Milton Keynez, United Kingdom]. The general cloning and ligation methodology was as described in Maniatis et al., cited above.
VH DNA was cloned as PstI-EcoRI fragments into similarly-digested M13mpl8/19 [Pharmacia-Milton Keynes, UK].
VL DNA wa. 'loned as SstI-EcoRI fragments into M13mpl8/19 digested the same enzymes. Representative clones were sequenced by the dideoxy method [Sanger et al., Proc. Nat'l.
Acad. Sci.. USA, 14:5463-5467 (1977)] using T7 DNA polymerase [Pharmacia].
The amino acid sequences obtained by translation of the variable region gene inserts were aligned with known VH and VL sequences to allow identification of the CDRs, The VL and VH amino acid sequences of B4 and the apparently substantially identical B13 and 814 antibodies are reported in Figs. 3A and 3B and 4A and 4B (VH).
The B4 sequences are reported above the B13/B14 sequences to demonstrate the homologies therebetween.
Example 19 Chimeric B4 Antibody To construct the B4 chimeric heavy chain expression vector, the B4VH gene was amplified from an M13 clone (Example 18) by PCR with oligonucleotides VH1BACK (doscribed in Example 18) and VHIFOR TGAGGAGACGGTGACCGTGGTCCCT TGGCCCCAG 3' [SEQ ID NO: 431 described by Orlandi et al, Proc. Nat'1. Acad. Sci. USA, i~:3833-3937 (1989)). The PCR mixture consisted of 0.5 .1 M13 phage supernatant 0.5 uM each of the above primers, 250 uM each of dATP, dCTP, dGTP and dTTP, 10 mM KC1, 20 mM Tris-HC1 pH 8.8, 10 mM (NH 4 )2SO 4 2 mM MgSO 4 0.1% Triton X-100 and 1 unit Vent DNA polymerase (New England Biolabs) in a volume of 50 ul. Samples were SUBSTITUTE SHEET WO 93/20210 P/GB3/00725 subjected to 15 rounds of amplification at 94 0 C, 30 seconds; 0 C, 30 seconds; 75 0 C, 1 minute; ending with 5 minutes at 0 C. Amplified DNA was purified on a low melting point agarose gel and by Elutip-d column chromatography (Schleicher and Schuell-Dussel, Germany). The DNA was cloned as PstI-BstEII fragments into similarly-digested M13VHPCR1 (Orlandi et la, 1989, cited above). The integrity of a chosen clone was confirmed by nucleotide sequencing.
The B4VH was cloned into an expression vector as described in Example 11 except that the human IgGl constant region was already present in the vector. The plasmid was termed pSVgptB4BoVHHulgG.1 To create B4 chimeric light chain expression vector, the vector M13VKPCR1 (Orlandi et al, 1989, cited above) was first modified to allow it to accept a lambda, rather than kappa, chain variable region. This was achieved by mutating the 5' end of the existing VK gene using the oligonucleotide TGGGCTCTGGGTTAACACGGACTGGGAGTGGACACC 3'[SEQ ID NO: 44] and the 3' end using the oligonucleotide ATTCTACTCACGACCCATGGCCACCACCTTGGT 3' (SEQ ID NO: introducing Hpal and Ncol restriction sites respectively.
The existing Ncol site in the vector was deleted using the oligonucleotide 5' CTCCATCCCATGCTGAGGTCCTGTG 3' [SEQ ID NO: 46].
M13VKPCR1 was grown in E. coli RZ1032 (dut'ung") to give single-stranded template DNA containing uracil in place of thymine. 0.5 ug DNA was mixed with 1 pmol each of the three phosphorylated oligonucleotides above and 1 pmol of an oligonucleotide VKPCRFOR GCGGGCCTCTTCGCTATTA(GC [SEQ ID NO: 471 which anneals to the M13 template downstream of the insert DNA. The oligonucl.eotides were annealed to the template in 20 ul of 50 mM Tris-HCl p1 7.5, 25 mM MgCl 2 63 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 91 mM NaCl by heating to 80 0 C for 5 minutes and cooling slowly to room temperature. dATP, dCTP, dGTP and dTTP were added to a 250 M final concentration, DTT to 7 mM, ATP to 1 mM with 0.5 unit T7 DNA polymerase (USB) and 0.5 unit T4 DNA ligase (BRL) in the same buffer. The 30 ll reaction was incubated at room temperature for one hour and the DNA ethanol precipitated.
In order to nick the parental strand the DNA was dissolved in 50 pl of 60 mM Tris.HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA containing 1 unit uracil DNA glycosylase and incubated at 37 0 C for one hour before NaOH was added to 0.2 M and incubation continued at room temperature for 5 minutes. The DhA was ethanol precipitated, dissolved in 20 .l TE and the insert fragment amplified by Pr"R. The reaction mixture contained 2 .1 mutant DNA, 0.5 JM each VKPCRFOR and VKPCRBACK CTGTCTCAGGGCCAGGCGGTGA [SEQ ID NO: 48], 250 JM each of dATP, dCTP, dGTP and dTTP, 10 mM Tris.HCl pH 8.3, 50 mM KC1, mM MgCl 2 0.01% Tween-20, 0.01% gelatin, 0.01% NP40 and 2 units Thermalase (IBI) in 50 ul. Amplification was achieved with 15 cycles of 94 0 C, 30 seconds; 50 0 C, seconds; 72 0 C, 1 minute; ending with 72 0 C, 5 minutes.
The product DNA was cloned int M13mpl9 as a Hindt'X- BamHI fragment. Representative clones were sequenced and a clone mutant in all three areas was chosen and named M13VLPCR1.
HpaI and N-ol restriction sites were introduced at the ends of the B4VL by amplifying the DNA from an M13 clone (Example 18) using oligonucleotides VL3BACK TCTGTGTTAACGCAGGCGCCCTCCGTG [SEQ ID NO: 49] and VL1FOR GGCTGACCCATGGCGATCAGTGTGGTC (SEQ ID NO: 501 and Vent DNA polymerase as described above for the B4VH above. The SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 92 product DNA was purified, digested with Hpal and NcoI and cloned into similarly-digested M13VLPCR1 RF DNA; Clones containing the B4VL were identified by sequencing and the HindIII-BamHI insert of one such clone used to construct an expression vector, pSVhygB4BoVLHuVK, as described in Example 11.
The expression vectors were co-transfected into myeloma cells, transfectomas secreting antibody identified and a chimeric antibody B4BoVH/BoVL purified as described in Example 11. The chimeric antibody was compared to the B4 bovine antibody for binding to RSV-infected cell lysate in an ELISA. The method was essentially as described in Example 11 except that RSV-infected and uninfected Hep2 cell lysates were used. The reporter antibodies were goat antihuman IgG antibodies, HRPO-conjugated (Sera-Lab Ltd, Crawley Down, UK) and rabbit anti-bovine IgG antibodies, HRPOconjugated (Sigma, Poole, UK), used as 1 in 1000 dilutions.
The bovine and chimeric (BoVH/BoVL) B4 antibodies bound to the infected cell lysate whereas an irrelevant humanized antibody did not. None of the antibodies reacted against the control lysate. It is not possible to draw a direct comparison between the bovine and chimaeric antibodies from this experiment as different reporter antibodies were used.
In a separate experiment comparing the conjugates, about 2.5 fold more bovine antibody than human antibody was required to obtain the same OD reading. Thus the bovine and chimeric antibodies are approximately equivalent in binding.
Example 20 Humanized 84 A. B4 Humanized Heavy Chain The B4VH was humanized by transferring the bovine CDRs onto human NEWM VH frameworks (Saul et al, 1978, cited above) using site-directed mutagenesis. The following SUBSTITUTE SHEET WO 93/20210 PC/GB93/00725 93 bovinn framework residues (numbering as Kabat et al, (1987), cited above) were incorporated into the humanized VH alongside the CDRs (see Figure Phe27, Ser28, Leu29 while not being part of the hypervariable region, these residues are part of the structural loop of CDRl (Chothia and Lesk, J. Mol. Biol., 12:901-917 (1987)).
Leu48 adjacent to CDR2, this residue has affected the binding of other reshaped antibodies.
Arg71 this residue has been shown to be important in other reshaped examples and is involved in the packing of CDRs 1 and 2 (Tramontano et al., Mol. Biol. 215:175-182 (1990)).
Lys94 the amino acid at this position can affect the conformation of CDR3 by formation of a salt bridge (Chothia and Lesk (1987), cited above).
The template DNA was M13mpl9-based and contained a VH gene comprising NEWM frameworks and irrelevant CDRs, similar to that described by Riechmann et al., Nature, 112:323-327 (1988). The mutagenesis was carried out as described above for the construction of M13VLPCI1. The oligonucleotides employed were: VHCDRl: 5' CTGTCTCACCCAGCTTACAGAATAGCTGCTCAATGAGAAG CCAGACAC 3' [SEQ ID NO: 51] VHCDR2: 5' CATTGTCACTCTGGATTTCAGGGCTGGGTTATAATATATGATT CCGCCATTGCTTGCGTCTCCAAGCCACTCAAGACC 3' [SEQ ID NO: 52] VHCDR3: 5' CAAGGACCCTTGGCCCCAGGCGTCGACATACTCGCCCTTGC
GTCCAGTACAAGCATAACTTCCACTATCACCAACAGAACACTTTGCACAATA
ATAGACCGC 3' (SEQ ID NO: 53] and the universal M13/pUC-20 primer, 5' GTAAAACGACGGCCAGT 3' [SEQ ID NO: 541.
SUBSTITUTE SHEET WO 93/20210 PP/GB93/00725 94 DNA encoding a VH containing all three B4 CDRs was subsequently excised from the M13 and cloned into the expression vector described for the chimeric VH in Examples 11 and 19 and resulting in pSVgptB4HuVHHuIgG1.
pSVgptB4HuVHHuIgGl was co-transfected with the chimeric light chain vector, pSVhygB4BoVLHuVK as described in Example 11. The resulting partially humanized antibody B4HuVH/BoVL therefore contains a humanized B4 heavy chain (B4HuVH) with a B4 light chain chimeric B4BoVLHuVK. Cells secreting 84HuVH/BoVL antibody were expanded and antibody purified from 400ml conditioned medium.
The B4HuVH/BoVL antibody was compared to the chimeric antibody B4BoVH/BoVL in binding to RSV strain A2-infected cell lydate in an ELISA. This allowed assessment of the relative binding abilities of the chimeric and humanized heavy chains.
The humanized heavy chain HuVH binds to RSV-infected cell lysate, but is 2-3 fold deficient in binding relative to the chimeric heavy chain BoVH.
Additional murine residues were included to attempt to increase binding. The HuVH gene was mutated to encode the following changes: T at position 73, N at position 76 and F at position 78 to NSV. These residues are part of a 1-turn which forms a fourth loop at the antigen binding surface.
A HuVHNSV/BoVL antibody was produced and tested for binding to a lysate of cells infected with the Snook strain of RSV by ELISA. Inclusion of the RSV residues gave no advantage over the original HuVH.
One other difference between the BoVH and HuVH which might affect binding is the region spanning amino acids #67- It is anticipated that the inclusion of the bovine B4 residues L at position 67 and I at position 69 are more SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 likely to influence the antigen interaction as their side chains pack inside the domain. Additionally the block change to L at position 67, G at position 68, I at position 69 and T at position 70 is also anticipated to be advantageous.
B. B4 Humanized Light Chain A humanized version of the B4 light chain B4HuVL was constructed by site-directed mutagenesis of the bovine B4VL frameworks to give frameworks of the human KOL lambda variable region (see Figure 11). Cells were selected for the presence of the gpt gene which is found on the heavy chain expression vector.
Northern blotting was used to determine if the HuVL RNA was of the correct size. Total RNA was prepared from BoVH/HuVL and BoVH/HuVL FR4 transfectomas and from BoVH/BoVL transfectomas and untransfected YB2/0 as positive and negative controls. Initial results using BoVL and HuVL probes show bands of approximately the same s4.ze for all three species of light chain. In a similar investigation cDNA was prepared from each cell line and PCRs carried out using a constant region primer and VL3BACK. Again the same sized product was obtained for all three species of light chain, indicating no major splicing problem.
Two more humanized light chain constructs a human REI kappa framework-based version of the light chain and a CDRgrafted light chain with frameworks of the human KIM46L lambda chain, may be made using the actual nucleotide sequence of the KIM46L VL gene (Cairns et al, J. Jmmual., 141:685-691 (1989)) This is believed to be the first example of a bovine antibody being humanized. The lack of bovine variable regiun sequences in the databases meant that it was SUBSTITUTE SHEET WO 93/20210 P~T//GB93/00725 96 difficult to design primers for PCR amplification and thus to isolate DNA for the initial cloning and sequencing.
Example 21 Effect of RSHZ19 and RSBV04 administered therapeutically to RSV infected mice Groups of five mice were inoculated intranasally with approximately 10 5 PFU of the A2 strain of RSV and were treated on day 4 of infection with different amounts of RSBV04 administered intraperitoneally either alone or with mg/kg RSHZ19, as shown in Table 10 below. Mice were killed five days after RSV challenge, and the level of virus in the lungs determined on CK cells. The results are shown in Table 10 and indicated that the effect of combined therapy with RSHZ19 and RSBV04 is additive rather than synergistic.
TABLE Dose (mg/kg)' RSV titer in lungs Group R(SHZ19 RSBV04 lo1gl PFU/q A 0.5 3.6 0.7 B 0.5 0.5 2.2 0.6 C 0.5 0.25 2.3 0.8 D 0.5 0.125 2.6 0.9 E 1.0 2.1 0.8 F 0.75 2.3 0.6 G 0.625 2.6 0.7 H 4.8 0.1 'mAbs administered IP on day 4 of infection.
SUBSTTUTE SHEET WO 93/20210 PCr/GB9/00725 97 Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art.
Such modification and alterations are believed to be encompassed in the scope of the claims appended hereto.
SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Taylor, Geraldine Stott, Edward J.
(ii) TITLE OF INVENTION: Novel Antibodies for Treatment and Prevention of Respiratory Syncytial Virus Infection in Animals and Man (iii) NUMBER OF SEQUENCES: 59 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: SmithKline Beecham Corporation Corporate Patents STREET: 709 Swedeland Road CITY: King of Prussia STATE: PA COUNTRY: USA ZIP: 19406-2799 COMPUTER READABLE FORM: MED-IUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO FILING DATE:
CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: GB 9207479.8 FILING DATE: 06-APR-1992 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 98 (viii) ATTORNEY/AGENT INFORMATION: NAME: Jervis, Herbert H.
REGISTRATION NUMBER: 31,171 REFERENCE/DOCKET NUMBER: P50153 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 215-270-5019 TELEFAX: 215-270-5090 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 112 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Ser Val Val Thr Gin Glu Pro Ser Val Ser Gly Ser Leu Gly Gin 1 5 10 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg 25 Trp Gly Val Asn Trp Tyr Gin Gin Val Pro Gly Ser Gly Leu Arg 40 Thr Ile Ile Tyr Tyr Glu Ser Ser Arg Pro Ser Gly Val Pro Asp 55 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile 65 70 Ser Ser Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Thr 85 Gly Asp Tyr Asn Ile Ala Val Phe Gly Ser Gly Thr Thr Leu Ile 100 105 Val Met Gly Gin Pro Lys Ser 110 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 116 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUEVZE DESCRIPTION: SEQ ID NO:2; SUBSTITUTE SHEET WO 93/20210 PFG9102 PCr/GB93/00725 Ser Arg Phe Thr Arg Asn Gly Leu Val Vai Val Ser Ala Val Ile Ile Phe Ser Ser Leu Glu Ser Thr Val.
Thr Ile Gly Tyr Giy Gin Lys Leu 99 Gin Gin Pro Ser Val. Ser Giy 10 Thr Cys Ser Gly Ser Ser Asp 25 Trp Tyr Gin Gin Vai Pro Gly 40 Giy Asn Thr Lys Arg Pro Ser 55 Ser Lys Ser Giy Asn Thr Ala 65 70 Ala Glu Asp Glu Ala Asp Tyr 85 Ser Ala Thr Pro Val. Phe Giy 100 Ser Gin Pro Lys Ser Pro Pro 110 115 Ser Leu Gly Gin Asn Ile Giy Ile Ser Giy Leu Arg Giy Val Pro Asp Thr Leu Thr Ile Phe Cys Val Cys Gly Giy Thr Thr 105 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 137 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gin Vai Xaa Leu Gin Giu Ser Gly Pro Ser Leu Val. Lys Pro 1. 5 10 Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Giy Phe Ser Leu 25 Ser Tyr Set Vai Ser Trp Val. Arg Gin Ala Pro Giy Lys Thr 35 40 Giu Trp Leu Gly Asp Ala Set Asn Gly Giy Ile le Tyr Tyr 55 Pro Ala Leu Lys Ser Arg Leu Gly Ile Thr Arg Asp Asn Ser 70 Ser Gin Val Ser Leu Ser Leu Asn Thr .Ie Thr Pro Giu Asp 85 Ala Thr Tyr Tyr Cys Ala Lys Cys Ser Val. Gly Asp Ser Gly 100 Tyr Ala Cys Thr Gly Arg Lys Gly Giu Tyr Val. Asp Ala Trp, 110 115 Gin Giy Leu Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala 125 130 ser Ser LeU Asn Ly s Thr Ser 105 Giy 120 Pro 135 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 Lys (2) 100 Val INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 141 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gin Val Xaa Leu Gin Gin Ser Gly Pro Ser Leu Val 1 G1n Asp Glu Pro Ser Ala Val Asp Thr Thr Leu His Asn Trp Leu Ala Leu Gln Val Thr Tyr Gly Tyr Ala Trp Thr Ala 5 Ser Leu Val Gly Gly Val Lys Ser Ser Leu 80 Tyr Cys Asp Cys 110 Gly Gin 125 Pro Lys 140 Thr Trp lie Arg Ser Ala Thr Gly Val Cys Thr Val Ile Arg Gln Tyr Lys Glu Leu Ser Ile Leu Ser Ser Thr Leu Gly Tyr Gly Leu Leu Leu Val 10 Ser Gly Leu 25 Ala Pro Gly 40 Gly Asp Lys 55 Thr Lys Asp 70 Val Thr Thr 85 Cys Tyr Phe 100 Gin His Thr 115 Thr Val Ser 130 Lys Pro Ser Ser Leu Ser Lys Ala Leu Asp Tyr Asn Asn Ser Lys Glu Asp Thr Val Glu Gly 105 Thr Phe Xaa 120 Ser Ala Ser 135 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 129 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Val Gin Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser 1 5 10 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 Gin Ser Glu Pro Asn Ala Tyr Gin Thr Tyr Trp Ala Gin Val Ala Gly Leu Ser Leu Thr Cys Ser Val Ser Trp Val Leu Gly Asp Ala Ser Leu Lys Ser Arg Val Phe Ser Leu Arg Leu 80 Tyr Tyr Cys Ala Lys Er Gly Arg Lys 110 Thr Thr Val Thr Val 125 101 Thr Arg Asn Thr Ser Cys Gly Ser Val Ser Gly 25 Gin Pro Pro 40 Gly Gly Ile 55 Met Leu Arg 70 Ser Val Thr 85 Ser Val Gly 100 Glu Tyr Val 115 Ser Phe Ser Leu Ser Gly Arg Gly Leu Ile Tyr Tyr Asn Asp Thr Ser Lys Ala Ala Asp Thr Asp Ser Gly Ser 105 Asp Ala Trp Gly 120 INFORMATICU FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 109 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Asp Ile Gln Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 Gly Asp Arg Val Thr Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile 25 Gly Arg Trp Gly Val Asn Trp Tyr Gin Gln Lys Pro Gly Lys Ala 35 40 Pro Lys Leu Leu Ile Tyr Tyr Glu Ser Ser Arg Pro Ser Gly Val 55 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe 70 Thr Ile Ser Ser Leu Gin Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 85 Ala Thr Gly Asp Tyr Asn Ile Ala Val Phe Gly Gin Gly Thr Lys 95 100 105 Leu Glu Ile Lys SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro 1 Gin Asp Glu Pro Asn Ala Val Asp 5 Thr Leu Ser Leu His Asn Val Gly Trp Leu Gly Val 50 Ala Leu Lys Ser Gin Phe Ser Leu Val Tyr Tyr Cys Gly Tyr Asp Cys 110 Ala Trp Gly Gin 125 Thr Trp Ile Arg Arg Ala Thr Gly Cys Val Tyr Val Leu Thr Tyr Thr 10 Thr Val Ser Gly Leu Ser Leu 25 Arg Gin Pro Pro Gly Arg Gly 40 Lys Glu Gly Asp Lys Asp Tyr 55 Thr Met Leu Lys Asp Thr Ser 70 Ser Ser Val Thr Ala Ala Asp 85 Leu Gly Cys Tyr Phe Val Glu 100 Gly Leu Gin His Thr Thr Phe 115 Thr Val Thr Val Ser Ser 130 Ser Ser Leu Asn Lys Thr Gly 105 Xaa 120 INFORMATION FOR SEQ ID NO:8: Asp 1 Gly Gly SEQUENCE CHARACTERISTICS: LENGTH: 111 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ile Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 5 10 Asp Arg Val Thr Ile Thr Cys Ser Gly Ser Ser Asp Asn Ile 25 Ile Phe Ala Val Gly Trp Tyr Gin Gin Lys Pro Gly Lys Ala 40 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/60725 103 Pro Lys Leu Leu Ile Tyr Gly Asn Thr Lys Arc Pro Ser Gly Val 55 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe 70 Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 85 Val Cys Gly Glu Ser Lys Ser Ala Thr Pro Val Phe Gly Gin Gly 100 105 Thr Lys Leu Glu Ile Lys 110 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 348 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CAGGTCCAGC TGCAGSAGTC WGGGACAGAG CTTGAGAGGT CAGGGGCCTC AGTCAAGTTG TCCTGCACAG CTTCTGGCTT CAACATTAAA GACTACTATA 100 TGCACTGGAT GAAGCAGAGG CCTGACCAGG GCCTGGAGTG GATTGGATGG 150 ATTGATCCTG AGAATGATGA TGTTCAATAT GCCCCGAAGT TCCAGGGCAA 200 GGCCACTATG ACTGCAGACA CGTCCTCCAA CACAGCCTAC CTGCAGCTCA 250 CCAGCCTGAC ATTTGAGGAC ACTGCCGTCT ATTTCTGTAA TTCATGGGGG 300 AGTGACTTTG ACCACTGGGG CCAAGGGACC ACGGTCACCG TCTCCTCA 348 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 116 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein SUBSTITUTE SHEET WO 93/20210 T/B3005 PCr/GB93/0072$ 104 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 6 OTHER INFORMATION: /note= "amino acid at position 6 can be either giu or gin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i0: Gin Val Gin Leu Gin Xaa Ser Giy Thr Giu Leu Giu Arg Ser Gly 1 5 10 Ala Ser Val. Lys Leu Ser Cys Thr Ala Ser Gly ?he Asn Ile Lys 25 Asp Tyr Tyr Met His Trp Met Lys Gin Atg Pro Asp Gin Gly Leu 40 Glu Trp Ile Gly Trp Ile Asp Pro Giu Asn Asp Asp Val, Gin Tyr 55 Ala Pro Lys Phe Gin Gly Lys Ala Thr Met Thr Ala Asp Thr Ser 70 Ser ?isn Thr Ala Tyr Leu Gin Leu Thr Ser Leu Thr Phe Glu Asp 80 85 Thr Ala Val T;'r Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His 100 105 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser ii(0 115 INFORMATION FOR9 SEQ ID NO:1i: SEQUENCE CHARACI ERISTICS: LENGTH: 337 base pairs TYPE: nucleic acid STIVNDEDNESS: double TOPOLOGY: unknown (ii) MOLICULE TYPE: DNA (genomic) (ix) FEATO'RE: NAME/KEY:- CDS LOCATION: 1..333 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GAC ATT CAG CTG ACC CAG TCT CCA CTC TCC CTG CC? GTC, ACT 4 Asp Ilie Gln Leu Thr Gin Ser Pro Leu Ser Leu Pro Vai Thr 1 5 CTT GGA GA? CAA GCC TCC ATC TCT TGC AGA TCT AG? CAG ACC 8 Leu Gly Asp Gin Ala Ser Ile Ser Cys Arg Ser Ser Gin Thr 20 SUBSTITUTE SHEET WO 93/202 10 PCTr/G B93/00725 105 CTT GTA CAT ACT GAT GGA AAC ACC TAT TTA GAA TGG TTT CTG 126 Leu Val His Thr Asp Gly Asn Thr Tyr Leu Giu Trp Phe Leu 35 CAG AAA CCA GGC CA, TCT CCA 4NAG CTC CTG ATC TAC AGA GTT 2,68 Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Tyr Arg Val 50 TCC AAC CGA TTT TCT GGG GTC CCA GAC AGG TTC AGT GGC AGT 210 Ser Asn Arg Phe Ser GJy Val Pro Asp Arg Phe Ser Giy Ser 65 GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC AGC AGA GTG GAG 252 Gly Ser Giy Thr Asp Phe Thr Leu Lys le Ser Arg Val Glu GCT GAG GAT CTG GGA GT TAT TTC TGC TTT CAA GGT TCA CAT 294 Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gin Gly $er His 90 CTT CCT CGG ACG TTC GGT GGA GGG ACC AAG CTG GAG ATC TAAC 337 Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: III amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Asp Lie Gin Leu Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Leu 1 5 10 Gly Asp Gin Ala Ser Ile Ser Cys Arg Ser Ser GIn Thr Leu Val 25 SUBSTITUTE SHEET WOP 93/20210 PC/GB93/00725 106 His Thr Asp Gly Asn Tbr Tyr Leu Giu Trp Phe Leu Gin Lys Pro 40 Gly Gin Ser Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe 55 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 70 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Giu Asp Leu Gly Val 85 Tyr Phe Cys Phe Gin GJy Ser His Leu Pro Arg Thr Phe Gly Gly 95 100 105 Gly Thr Lys Leu GIP Ile 110 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 116 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO;13: Gin 1 Ala Asp Glu Ala Ser Thr Trp Val Ser Tyr Trp ?ro Asn Al Gly Gin Leu Gin 5 Val Lys Leu Tyr Met Uis Ile Gly Trp Lys Phe Gin 65 Thr Ala Tyi: Val Tyr Phe Gin Gly Thr 110 Glu Ser Gly Thr Ser Cys Thr Ala Trp Met Lys Gjn Ile Asp Pro Glu Glyv 1Is Ala Thr Leu Gin Leu Thr Cys Asn Ser Trp Thr Val Thr Val Glu Leu Glu Arg 10 Ser Gly Phe Asn 25 Arg Pro Asp Gin 40 Asn Asp Asp Val 55 Met Thr Ala Asp 70 Ser Leu Thr ?he 85 Gly Ser Asp Phe 1Q0 Qer Ser 115 Ser Gly Ile Lys Gly Leu Gin Tyr Thr Ser Glu Asp ,Asp His 105 LNFORMATIQW FOR SEQ ID NO:14: SEQUlENCE CHARACTERISTICS: LWGTU: 116 amino acids SUBSTmrJTE SHEET WO 93/20210 P~r/B9300725 107 TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SFQ ID NO:14: Gin Val Gin Leu Gin Giu Ser Gly Pro Gly Leu 1 5 iO Gin Thr Leu Ser Leu Thr \ys Thr Val Ser Giy 25 Asp Tyr Tyr Met His Trp Val Arg Gin Pro Pro 40 Glu Trp Ile Gly Trp lie Asp Pro Giu Asn Asp 50 55 Ala Pro Lys Phe Gin Gly Arg Val Thr Asn Leu 70 LyS Asn Gin Phe Ser Leu Arg Leu Ser Ser Val 8$ Val Arg Pro Ser Phe Thr Phe Ser Gly Arg Gly Leu Asp Val Gin Tyr Vai Asp Thr Ser Thr Ala Ala Asp Asp Phe Asp Ifis irls Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gxy 100 Trp Gly Gin Gly Thr Thr Val Thr Val Ser 110 115 Ser See 44) INFORMATION FOR SEQ ID 2) SEQUENCE CHARACTERISTICS: ',ENGTH: 116 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEOUENCE DESCRIPTION: SEQ ID Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser 1 5 10 Gin Thr Leu Ser Leu Thr Cys Thr Val. Ser Gly Phe Thr Phe Ser 25 Asp Tyr Tyr Met His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 35 40 Glu Trp le Gly Trp Ile Asp Pro Giu Asn Asp Asp Val Gin Tyr 55 Ala Pro Lys Phe Gln Giy Arg Val Thr Met Ler Vai Asp Thr Ser 70 SUESrrrur SHC"ET WO 91/2010 PCT/GB93/0725 108 Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp 85 Thr Ala Val Tyr Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 116 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser 1 5 10 Gln Thr Leu Ser Leu Thr Cys Thr Val Set Gly Phe Asn Ile Lys 25 Asp Tyr Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu 35 40 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr 55 Ala Pro Lys Phe Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser 70 Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp 85 Thr Ala Val Tyr Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 INFORMATION FOR SEQ ID N0:17: SEQUENCE CHARACTERISTICS: LENGTH: 112 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: SUBSTITUTE SHEET
I
WO 93/20210 WO 93/02 10PCT'/GB93/00725 Asp 1 Gly His Giy Ser Phe Tyr Giy 109 Ile Gin Leu Thr Gin Ser Pro Ser Ser Leu 5 10 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser 20 25 Thr Asp Giy Asn Thr Tyr Leu Glu Trp Tyr 40 Lys Ala Pro Lys Leu Leu Ile Tyr Arg Val 55 Gly Vai Pro Ser Arg Phe Ser Giy Ser Giy 70 Thr Phe Thr Ile Ser Ser Leu Gin Pro CG1u 85 Tyr Cys Phe Gin Giy Ser His Leu Pro Arg 95 100 Thr Lys Val Giu Ile Lys 110 Ser Ala Ser Val Gin Thr Leu Val Gin Gin Lys Pro Ser Asn Arg Phe Ser Gly Thr Asp Asp Ile Aia Thr Thr Phe Giy Gin 105 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: ),899 base pairs TYPE: nucleic acid STRANDEDNIESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE; NAME/KEY: CDS LOCATION: 14..1735 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGGGCAAATA ACA ATG GAG TTG CTA ATC CTC AAA GCA AAT GCA ATT4 6 Met Glu Leu Leu Ile Leu Lys Aia Asn Ala Ile 1 5 ACC ACA ATC CTC ACT GCA GTC ACA TTT TGT TTT GCT TCT GGT 88 Thr Thr Ile Leu Thr Ala Vai Thr Phe Cys Phe Ala Ser Giy 1s 20 CAA AAC ATC ACT GAA GAA TTT TAT CAA TCA ACA TGC AGT GCA 130 Gin Asn Ile Thr Glu Giu Phe Tyr Gin Ser Thr Cys Ser Ala GTT AGC, AAA GGC TAT CTT AGT GCT CTG AGA ACT GGT TGG TAT 172 Val Ser Lys G2ly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr 45 SUBSTITUTE SHEET WO 93/20210 PCT/GB93/00725 GAA TTT AGT GTT Glu Phe Ser Val
AAT
Asn 240 GCA GGT GTA ACT Ala Gly Val Thr
'CA
Thr 245 CCT GTA AGC ACT Pro Val Ser Thr 760 802 TAC Tyr 250 ATG TTA ACT ATT Met Leu Thr Asn
AGT
Ser 255 GAA TTA TTG TCA Glu Leu Leu Ser
TTA
Leu 260 ATC AAT GAT Ile Asn Asp TCC AAC AAT Ser Asn Asn 275 ATG CCT Met Pro 265 ATA ACA AAT GAT Ile Thr Asn Asp
CAG
Gin 270 AAA AAG TTA ATG Lys Lys Leu Met GTT CAA ATA Val GltR Ile 280 GTT AGA GAG CAA Val Arg Gin Gin
AGT
Ser 285 TAC TCT ATC ATG Tyr Ser Ile Met TCC ATA Ser Ile 290 844 886 928 970 ATA AAA GAG I Ie Lys Glu
GAA
Glu 295 GTC TTA GCA TAT Val Leu Ala Tyr
GTA
Val 300 GTA CAA TTA CCA Val Gin Leu Pro
CTA
Leu 305 TAT GGT GTT ATA Tyr Gly Val, le
GAT
Asp 310 ACA CCC TG' TGG Thr Pro Cys Trp
AAA
Lys 315 CTA CAC ACA TCC Leu Hi1s Thr Ser
CCT
Pro 320 CTA TGT ACA Leu Cys Thr ACC AAC Thr Asn 325 ACA AAA G1IA GGG Thr Lys Giu Gly
TCC
Ser 330 TTA ACA AGA Leu Thr Arg 335 TCA GTA TCT Ser Vai Ser 350 ACT GAG AGA Thr Asp Arg
GGA
Gly 340
TGG
Trp
GCT
Ala 355 TAC TGT GAG Tyr Cys Asp GAA ACA TGT Glu Thr Cys AAC ATC TGT 1012 Asn lie Cys AAT GCA GGA 1054 Asn Ala Gly AAA GTT CAA 1096 Lys Val Gin 360 TTA ACA TTA 1038 Leu Thr Leu 375 TTC TTC CCA CAA Phe Phe Pro Gin TCA AAT CGA Ser Asn Arg
GTA
Val 365 TTT TGT GAC ACA Phe Cys Asp Thr
ATG
Met 370 AAC AGT Asn Ser CCA AGT GAA ATA Pro Ser Giu Ile
AAT
Asn 380 CTC TGC AAT GTT Leu Cys Asn Val
GAC
Asp 385 ATA TTC AAC CCC 1180 Ile Phe Asn Pro
AAA
Lys 390
AGC
Ser TAT GAT TGT AAA Tyr Asp Cys Lys
ATT
Tie 395
TCT
Ser ATG ACT TCA AAA Met Thr Ser Lys
ACA
Thr 400
GTG
Val GAT GTA AGC Asp Val Ser 1222
TCC
Ser 405 GTT ATC ACA Val Ile Thr
CTA
Leu 410 GGA GCC ATT Gly Ala Ile TCA TGC, TAT 1264 Ser Cys Tyr 415 SUBSTITUTE SHEET WO'93/20210 WO'9320210PCr/GB93/00725 113 ACTATCTGCT CATAGACAAC CCATCTGTCA TTGGATTTTC TTAAAATCTG 1825 AACTTCATCG AAACTCTCAT CTATAAACCA TCTCACTTAC ACTATTTAAG 1875 TAGATTCCTA GTTTATAGTT ATAT 1899 SUBSTITUTE SHEET WO 93/20210 w6 9320210PCT/GB93/00725 115 Giu Leu Leu Ser Leu 260 SLys Lys Leu Met Ser 275 Tyr Ser Ile Val Gin Leu Leu His Thr Asn Ile Cys Ala Gly Ser Gin Ser Asn Pro Ser Giu Tyr Asp Cys Val Ile Thr Lys Cys Thr Ser Asn Gly Ser Val Gly Ser Leu Tyr Leu Val Phe Met Ser 290 Pro Leu 305 Sex Pro 320 Leu Thr 335 Val Ser 350 Arg Val 365 Ile Asn 380 Lys Ile 395 Ser Leu 410 Ala Ser 425 Cys Asp 440 Asn Thr 455 Val. Lys 470 Pro Ser 485 Ile Asn 500 His Asn 515 Thr Ile 530 Ile Asn Asp Met Pro Ile Thr Asn 265 Asn Asn Val Gin Ile Val Arg Gin 280 Ile Ile Lys Giu Giu Val Leu Ala 295 Tyr Giy Val Ile Asp Thr Pro Cys 310 Leu Cys Thr Thr Asn Thr Lys Giu 325 Arg Thr Asp Arg Giy Trp Tyr Cys 340 Phe Phe Pro Gin Ala Giu Thr Cys 355 Phe Cys Asp Thr Met Asn Ser Leu 370 Leu Cys Asn VTal Asp Ile Phe Asn 385 Met Thr Ser Lys Thr Asp Vai Ser 400 Gly Ala Ile Val Ser Cys Tyr Gly 415 Asn Lys Asn Arg Giy Ile Ile Lys 430 Tyr Vai Ser Asn Lys Giy Met Asp 445 Leu Tyr Tyr Val Asn Lys Gin Giu 460 Giy Giu Pro Ile Ile Asn Phe Tyr 475 Asp Giu Phe Asp Ala Ser Ile Ser 490 Gin Ser Leu Ala Phe Ile Arg Lys 505 Val Asn Ala Giy Lys Ser Thr Thr 520 Ile Ile Val Ile Ile Val Ile Leu 535 Asp Gin 270 Gin Ser 285 Tyr Val 300 Trp Lys 315 Giy Ser 330 Asp Asn 345 Lys Val 360 Thr Leu 375 Pro Lys 390 Ser Ser 405 Lys Thr 420 Thr Phe 435 Thr Va2l 450 Gly Lys 465 Asp Pro 480 Gin Val 495 Ser Asp 510 Asn Ile 525 Leu Ser 540 Asn Glu Lys Giu Leu Leu Met Ile Thr SUBSTITUTE SHEET WO93/20210 PCT/GB93/00725 117 SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTCGGAACTG GGACCAAGGT CACCGTCCTA GGTAAGTGG 39 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATCTGTTTTT GAAGTCAT 18 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24: ACGATTTTAT TGGATGC 17 SUBSTITUTE SHEET WO 93/203210 PCT/GB93/00725 119 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CTCAGTTGAT CCTTGCTTAG INFORMATION FOR SEQ ID NOx29: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acd STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: TTGAATTCAG ACTTTCGGGG CTGTGGTGGA GG 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID SUBSTITUTE
SHECET
W6C 93/20)210 PCT/GB93/00725 121 TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG 34 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GTTAGATCTC CAGCTTGGTC CC 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GACATTCAGC TGACCCAGTC TCCA 24 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: SUBSTITUTE SHEET WOT 93/20210 PCT/GB93/00725 123 CTGCTGGTAC CATTCTAAAT AGGTGTTTCC ATCAGTATGT ACAAGGGTCT GACTAGATCT ACAGGTGATG GTCA 74 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TY-E: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GCTTGGCACA CCAGAAAATC GGTTGGAAAC TCTGTAGATC AGCAG INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CCCTTGGCCG AACGTCCGAG GAAGATGTGA ACCTTGAAAG CAGTAGTAGG T 51 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42: SUBSTITUTE SHEET IWO9320210 PCr/GB93/00725 125 (xi) SEQUENCE DESCRIPTION: SEQ ID ATTCTACTCA CGACCCATGG CCACCACCTT GGT 33 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CTCCATCCCA TGCTGAGGTC CTGTG INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GCGGGCCTCT TCGCTATTAC GC 22 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET WO 93/20210 W093/0210PCT/GB93/00725 127 CTGTCTCACC CAGCTTACAG AATAGCTGCT CAATGAGAAG CCAGACAC 4 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHA7RACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECUL3E TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CATTGTCACT CTGGATTTCA GGGCTGGGTT ATAATATATG ATTCCGCCAT TGCTTGCGTC TCCAAGCCAC TCAAGACC 78 INFORMATIONI F5OR SEQ ID NO:53: SEQUENiC-E CHACTERISTICS:- LIMNTH., 102 base pairs TVYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLFCULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CAAGGACCCT TGGCCCCAGG CGTCGACATA CTCGCCCTTG CGTCCAGTAC AAGCATAACT TCCACTATCA CCAACAGAAC ACTTTGCACA. ATAATAGACC GC102 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGT11: 17 bie pairs TYPE: nucleic a(,id STRANDEDNESS! sinqle TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET WO 93/202108 PCr/GB93/00725 129 Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp 200 205 Gly Ser VdI Ser Phe Phe Pro Gin Ala Glu 215 220 Ser Asn Arg Val Phe Cys Asp Thr Met Asn 230 235 Ser Asp le Cys Asn Val Leu Val Giu Leu Ile Glu Ile Cys Lys Thr Ser Thr Ala Gly Cys Gly Asn Tyr Val Phe Pro tys le Leu His Thr Thr Asn Leu Cys Asn Val 245 Ile Met Thr Ser Lys 260 Leu Gly Ala Iil Val 275 Ser Asn Lys Asn Arg 290 Asp Tyr Val Ser Asn 305 Thr Leu Tyr Tyr Val 320 Lys Qly Glu Pro Ile 335 Ser Asp Giu Phe Asp 350 Asn Gin Ser Leu Ala 365 Asn Val Asn Ala Gly 380 Ile lie tie Val Ile 395 Gjy Leu Leu Lou Tyr 410 Ser Lys Asp Gln Lou 425 Asp lie 250 Thr Asp 265 Ser Cys 280 Gly Ile 295 Lys Gly 310 Asn Lys 325 Ile Asn 340 Ala Ser 355 Phe Ile 370 Lys Ser 385 ile Val 400 Qys Lys 415 Ser Gly 430 Tyr Cys Asp Asn Ala 210 Thr Cys Lys Val Gin 225 Ser Leu Thr Leu Pro 240 Phe Asn Pro Lys Tyr 255 Val Ser Ser Ser Val 270 Tyr Gly Lys Thr Lys 285 Ile Lys Thr Phe Ser 300 Met Asp Thr Val Ser 315 Gin Glu Giy L~s Ser 330 Phe Tyr Asp Pro Leu 345 le Ser Gin Val Asn 360 Arg Lys Ser Asp Glu 375 Thr Thr Arsn le Met 390 Ile Leu Lou Set Leu 405 Ala Arg Set Thr Pro 420 i0 Asn Asn Ile Ala 435 Ile Ala Val Val Phe Thr eu Sez, Asn INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOG.: unknown (ii) MOLEC3LS TYPE: protein SUBSTITUTE
SHEET
WO 93/20210 PCr/GB93/00725 131 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 6 OTHER "'NFORM2.TION; /note= 11X can be Asp, Glu, Phe, Ilef Leu, Met, Arg, Serf Thr, Val, Trp, Tyr or Glfl" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Ile Thr Asti Asp G~.n Xaa Lys Leu 1 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHqARCTERISTICS; LENGTH: 8 amino acids TYPE; amino acid TOPOLOGY: u~nknown (ii) MOLECULJE TYPE: protein (ix) FEATURE: NP4/KEY: Modified-site LOCATION: 8 OTHER INFOP.hTION: /note- "X can be Ala, Cys, Asp or Glu" (xi) SEQUENCE DESC.RIPTION:o SEQ ID NO:,599; Ile Thr Asrn Asp Gin Lys Lys Xaa 1 SUBSTITUTE SHEET

Claims (18)

1. A fusion protzin comprising an amino acid sequence having antigen specificity directed against the amino acid sequence of the F protein SEQ ID NO;1_0 spanning amino ac'i~l #266 through #273 and analgr., 'hereof fusF,6 to a second peptide or' protein sequence.
2. The protein. according to claim 1 wherein said antibody is a bovine antibody, The pj.otein acco'rding, to claim 2 wherein, said antibody Is s~2lepted from the group consisting of bovine. monoclonal antibody Bl4 and thto bovine anti-RSV antibody 4 The pr.. tein according to claim 1 wherein oaid amino acid isequence 40 sallected from the gxoup consisting of the variable heosvy chain of said anLibody, the variable _h haix, of said antibody, alt least oije CDR from gaid 0 variable heavy chain, at least one DR froM isald vari able light chain, a funcvtio-.ial fragment or anal og 1 The proteirl according to claiv I wherein said Atino, acid sequenice hai* the formula oelected fro the qroup cotsioiting of a, SEQ ID NO,# 56: X-Thr-Asn-Asp-aln-Lys-Lys-Lou, whein X is selected from the amino acids consioting ot Ala, Cva,# Asp$ Glu, Phe, Gly, His, i II :i' 23 4 o7;i5 r, pj\oPrAAMM390Qo 93C4 23/419 -133- Leu, Pro, Gln, Arg, Sert Thr, Val, Trp, and Tyr, b. SEQ ID NO: 57: Ile-Thr-Asn-Asp-Y-Lys-Lys-Leu, wherein Y io selected from the amino acids consisting of Asp, Glu, Phe, Ile, Leu, Met, Arg, Ser, TZr, Val, Trp, and Tyr, c. SEQ ID NO: 58; Ile-Thr-Asn-Asp-Glf-Z-Lys-Leu, wherein Z is selected from the amino acids consisting of Asp, Glu, Phe, lie, Leu, Met, Arg, Ser, Thr, Val, Trp, Tyr and Cln, and S* SEQ ID NO: 59: Ile-Thr-Asn-Asp-Gln-Lys-Lys-W, $E .NO too* wherein w is selected from the amino acids g 4 consistiang of Ala, Cys, Asp and G1u. veto go6. The protein according to claim 1 wherein Asaid amino acid :000 sequence is selected from the group consisting of a sequence, comprising a variable heavy chain sequence of Fig. 4A and 4B SEQ ID NO: 3, a sequence, comprising V' a variable light chain sequence of Fig. 3A and 31' SEQ to oID NO: 4, and a functional fragment or analog of (a) or
7. The protein according to claim 1 wherein said amin acid 9 sequence comprises one or more CDR peptides selected r* *94 from the group consisting of .4 4 a. amino acids 31 through 35 of SEQ ID NO: 3t SerTyr- Ser-Val-Ser; b. amino acids 50 through 65 of SEQ ID NO. 3: Asp-Ala- Ser-Awn-Glyly-Gy-le-Ile-Tyr-Tyr-Asn-Pro-Ala-Leu- L o -eri; c. amino acids 100 through 122 of SEQ ID NO: 3: Cys- 4 97 1 :rN, 2 PA0OP9RUMS39LOO-93,CL2M 2314/97 -134- Ser-Val -Gly--Asp-Ser-Gly- Ser-Tyr-Ala- Cys -Thr-X-Gly- Xaa -Arg -Lys -Gly-Glu -Tyr -Val -Asp-Ala, wherein X is any or no amino acid. d- amino acids 22 through 34 of SEQ ID NO: 1 and 2: Ser-0Vly-Ser-Ser- (Ser or Asp) -Asn-Ile-Gly- (Arg or Ile) -(Trp or Phe) -(Gly or Ala) -Val- (Asn or Gly) ease *06. sees- S 4 e. amino acids 50 through 56 of SEQ ID NO: 1: Tyr-Glu- Ser-Ser-Arg- Pro-Ser; f. amino acids 89 through 96 of SEQ ID NO: 1: Ala-Thr- Gly-Asp-Tyr-Asn-Ile-Ala; g. amino acids 89 through 97 of SEQ ID NO: Ala-Thr- GJly-Asp-Tyr-Asn- Ile-Ala-Val; h. amino acids 50 through 56 of SEQ ID NO; 2: Gly-Asn- Thr-Lys-Arg-Pro-Ser; i. amino acids 89 through 99 of SEQ ID NO: 2: Val-Cys- Gly-Glu-Ser-Lys-Ser-Ala-Thr-Pro-Val; J. amino acids 31 through 35 of SEQ ID NO: Asn-Val.-Gly k. amino acids So through 65 of SEQ b a. a 950 *0O5 a ae a S a.. S east r. a a S. S SO 4: Asp-His- 4: Val-Ile- Tyr-Lys-Glu-Gly-Asp-Lys-Asp-Tyr-Asn-Pro-Ala-Leu- Lys-Ser; and 1. amino acids 98 through 122 of SEQ ID NOt 4: Leu- Gly-Cys-Tyr- Phe-Val-Glu-Gly-Val-Gly-Tyr-Asp-Cys Thr- Tyr-Gly- Leu-Gln -His -Thr-Thr- Phe -Y-As-D-Ala, wherein Y is any amino acid.
8. A fusion molecule comprising a first fusion partner nucleotide sequence encoding the, fusion protein of claim I. operatively linked to a second fusion partner nucleotide sequence. P:\OPFRUMS39000.93.CLM -23/497 -135-
9. An anti-RSV CDR peptide selected from through of claim 7 and m. a fragment thereof, or an analog thereof, characterized by the antigen specificity of any of the above peptides. An isolated bovine anti-RSV antibody variable light chain amino acid sequence, a fragment or analog thereof S"sharing the anti-RSV antigen specificity of said 0 S* sequence, *oe*
11. The sequence according to claim 10 wherein said light chain sequence is naturally occurring in said antibody 0* or modified, and is selected from the group consisting of the sequences of Figs. 3A and 3Z SEQ ID NOS: 2 and 2, Fig. 11 SEQ ID NO: 6, and Fig. 13 SEQ ID NO: 8. *eg
12. An isolated bovine anti-RSV antibody variable heavy chain amino acid sequence, a fragment or analog thereof sharing the anti-RSV antigen specificity of said sequence. S00 o *0 g
13. The sequence according to claim 12 wherein said heavy chain sequence is naturally occurring in said antibody or modified, and is selected from the group consisting of the sequences of Figs. 4A and 4B SEQ ID NOS: 3 and 4, Fig. 10 SEQ ID NO: 5, and Fig. 12 SEQ ID NO: 7,
14. An isolated nucleic acid sequence encoding the variable 1J6- heavy chain amino acid sequence or variable light chain amino acid sequence of an anti-RSV antibody selected from the group consisting of B4 and B13/B14, or a functional fragment or analog thereof, optionally containing restriction sites to fe zilitate insertion into a desired antibody framework region or fusion with a selected fusion partner. An altered antibody comprising an amino acid sequence in S.which at least part of the heavy chain variable region Soso of an acceptor antibody has been replaced by an eec analogous part of the heavy chain varipble region of at :C least one donor antibody having specificity for respiratory syncytial virus, and a suitable light chain *CV ,sequence, wherein the variable light chain region of the donor antibody is that of Fig. 3A and 3B SEQ ID NO: I or a functional fragment thereof and the variable heavy chain region of the donor antibody is that of Fig. 4A and 4B SEQ ID NO: 3, or a functional fragment thereof, a wherein the resulting altered antibody is characterized by the antigen binding specificity of mAb B4, and wherein said acceptor antibody is heterologous to said *donor antibody. CC
16. The antibody according to claim 15 wherein the variable heavy chain region of the donor antibody is intact and fused to the heavy chain const,. re5jon of the acceptor antibody.
17. The antibody according to claim 15 wherein the variable 4 1 :1 I r) :1 6 RjU .39~3CLM 23/4/97 -137- heavy chain CDR fragments of the donor antibody replace the heavy chain CU~R fragments of the acceptor antibody.
18. The antibody according to claim 15 wherein the light chain is selected from the group consisting of a. a variable light chain -egion of the donor antibody fused to the light chain constant region of the acceptor antibody; b. ax light chain comprising light chain CDR fragments a. ae of the donor antibody replacing the light chain CDR fragments of the acceptor antibody; C. the donor antibody light chain; and a heterologous acceptor antibody light chaiin, An altered antibody comprising an amino acid sequence in which at least part of the heavy chain variable region of an acceptor antibody has been replaced by an analogous part of the heavy chain variable region of at *least one donor antibody having specificity for respiratory syncyti~l viras, and a suitable light chain too@sequence, wherein thie variable light chain region of the donor antibody is that of Fig. 3A and 38 SEQ ID NOiq 2 or a functional fragmnent thereof and the variable heavy chain region of the donor antibody is that of Fig. 4A and 4B SEQ ID NO: 4 or a functional fragment thereof, wherein the resulting altered antibody is characterized by the antigen binding specificity of mAb B13/B14, and wherein said acceptor antibody is heterologous to said donor antibody. 4 #J 2I:j 2 22 PM? MUNS 92.CLM -23/4J9 -138- A humanized antibody comprising an amino acid sequence in which at least parts of the sequence of the heavy chain variable region of a human acceptor antibody have been replaced by analogous parts of the amino sequence of the heavy chain variable region of at least one bovine donor antibody, and a suitable light chain sequence, said humanized antibody ch~aracterized by the antigen specificity of the bovine donor antibody. *21. The antibody according to claim 20 wherein the antigen specificity is binding to an epitope of RSV, said antibody comprising a humanized heavy chain variable region sequence .elected from the group con~isting of the sequence of Fig. 10 SEQ ID NO: 5 and the seq~uence of Fig. 12 SEQ ID NO; 7.
22. The antibody according to claim 21 characterized by a light chain selected from the group consisting of the humanized light chain sequence of Fig. 11 SEQ ID NO: 6, the humanized sequence of Fig. 13 SEQ ID NO- 8, a naturally occurring bovine monoclonal c~ntibody light chain characterized by the light chain variable ~*sequences of Figs. 3A and 3B SEQ ID NO, 1$ and a chimeric bovine/human light chain characterized by the light chain variable sequences of Figs. 3A and 3B SEQ ID NO; I fused to the light chain constant regions of a human acceptor antibody.
23. An antibody, other than B4, which is capable of binding to the RSV peptide consisting essentially of the amino 0 ia/ a2 2; 1 4 91 l 1 6 t 6 8 I PAOPERUMS390X93.CLM 23/497 -139- acid sequence of the F protein spanning amino acid #266 through #273 of SEQ ID NO: 19 and analogs thereof, a Fab fragment thereof, or an F(ab') 2 fragment thereof, said antibody being a monoclonal antibody or an altered humanized antibody.
24. An anti-RSV antibody, a Fab fragment or a F(ab') 2 fragment thereof produced by screening an antibody library comprising hybridoma products and libraries e* derived from any species immunoglobulin repertoires, with one or more antibodies selected from the group *so consisting of B4 and B13/B14. a too* *og s o 25. A pharmaceutical composition comprising one or more of S. a fusion protein of claims 1-7, a CDR peptide of claim 9, the sequences of claim 10-13 or the antibodies of claim 15-24, and a pharmaceutically acceptable carrier or diluent. em
26. A method of preventing or treating human RSV infection :so. too$ in a human in need thereof which comprises administering to said human an effective dosage of a pharmaceutical composition of claim S
27. A recombinant plasmid comprising a nucleic acid sequence of claim 14 or a nucleic acid sequence of a fusion molecule of claim 8.
28. A mammalian cell line transfected with the recombinant plasmid of claim 27.
AU39000/93A 1992-04-06 1993-04-06 Antibodies for treatment and prevention of respiratory syncytial virus infection Ceased AU679440B2 (en)

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GB929207479A GB9207479D0 (en) 1992-04-06 1992-04-06 Novel antibodies for treatment and prevention of respiratory syncytial virus infection in animals and man
PCT/GB1993/000725 WO1993020210A1 (en) 1992-04-06 1993-04-06 Antibodies for treatment and prevention of respiratory syncytial virus infection

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US5824307A (en) 1991-12-23 1998-10-20 Medimmune, Inc. Human-murine chimeric antibodies against respiratory syncytial virus
US6258529B1 (en) * 1994-12-01 2001-07-10 Oravax, Inc. PCR amplification of rearranged genomic variable regions of immunoglobulin genes
US7153508B2 (en) 1995-06-07 2006-12-26 Biogen Idec Inc. Treatment of B cell lymphoma using anti-CD80 antibodies that do not inhibit the binding of CD80 to CTLA-4
US7175847B1 (en) 1995-06-07 2007-02-13 Biogen Idec Inc. Treating intestinal inflammation with anti-CD80 antibodies that do not inhibit CD80 binding to CTLA-4
US5811524A (en) * 1995-06-07 1998-09-22 Idec Pharmaceuticals Corporation Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
EP0853487B1 (en) * 1995-09-18 2000-07-05 Intracel Corporation Neutralizing monoclonal antibodies to respiratory syncytial virus
FR2758331B1 (en) * 1997-01-14 1999-03-05 Univ Bourgogne NEW MEANS FOR DIAGNOSIS, PREVENTION AND TREATMENT FOR CONTAMINATION OR INFECTIONS WITH MUCOUS TROPISM VIRUSES
US6160099A (en) * 1998-11-24 2000-12-12 Jonak; Zdenka Ludmila Anti-human αv β3 and αv β5 antibodies
WO2001082965A1 (en) * 2000-05-03 2001-11-08 Medimmune, Inc. Combination therapy of respiratory diseases using antibodies
AU2001263443A1 (en) * 2000-05-25 2001-12-03 Med Immune, Inc. F-protein epitope-based vaccine for respiratory syncytial virus infection
US7132100B2 (en) 2002-06-14 2006-11-07 Medimmune, Inc. Stabilized liquid anti-RSV antibody formulations
TW200636064A (en) * 2004-10-28 2006-10-16 Centocor Inc Anti-respiratory syncytial virus antibodies, antigens and uses thereof
EP1997830A1 (en) 2007-06-01 2008-12-03 AIMM Therapeutics B.V. RSV specific binding molecules and means for producing them
US8568726B2 (en) 2009-10-06 2013-10-29 Medimmune Limited RSV specific binding molecule
SG10201506906VA (en) 2010-09-09 2015-10-29 Pfizer 4-1bb binding molecules
FR2970968A1 (en) 2011-02-01 2012-08-03 Isp Investments Inc NOVEL PEPTIDES INVOLVED IN THE SCF C-KIT SIGNALING PATHWAY AND COMPOSITIONS COMPRISING THE SAME
US9364414B2 (en) 2011-02-01 2016-06-14 Isp Investments Inc. Method to protect skin from ultraviolet radiation using novel peptides involved in the improvement of microparasol organization in keratinocytes
JP2015509091A (en) * 2012-01-09 2015-03-26 ザ スクリプス リサーチ インスティテュート Humanized antibody
JP6684490B2 (en) 2012-01-09 2020-04-22 ザ・スクリップス・リサーチ・インスティテュート Ultralong complementarity determining regions and uses thereof
WO2014110368A1 (en) 2013-01-11 2014-07-17 The California Institute For Biomedical Research Bovine fusion antibodies
AU2014290361B2 (en) 2013-07-18 2019-04-18 Taurus Biosciences, Llc Humanized antibodies with ultralong complementarity determining regions
JO3555B1 (en) 2015-10-29 2020-07-05 Merck Sharp & Dohme Antibody neutralizing human respiratory syncytial virus
EP3848389A4 (en) * 2018-09-03 2022-04-13 Pontificia Universidad Católica De Chile Specific monoclonal antibody against the n antigen of human respiratory syncytial virus (hrsv) useful for treating infection, detection thereof and diagnosis
EP4265637A1 (en) * 2020-12-18 2023-10-25 Zhuhai Trinomab Pharmaceutical Co., Ltd. Respiratory syncytial virus-specific binding molecule

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2785089A (en) * 1987-12-23 1989-07-19 Pharmacia & Upjohn Company Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus
AU4927193A (en) * 1992-09-16 1994-04-12 Scripps Research Institute, The Human neutralizing monoclonal antibodies to respiratory syncytial virus
AU3660893A (en) * 1993-01-29 1994-08-15 Med Immune, Inc. Human-murine chimeric antibodies against respiratory syncytial virus

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69130708T2 (en) * 1990-07-19 1999-05-27 Us Commerce IMPROVED IMMUNOTHERAPEUTIC METHOD FOR PREVENTING OR TREATING VIRAL DISEASES OF THE respiratory tract
GB9019812D0 (en) * 1990-09-11 1990-10-24 Scotgen Ltd Novel antibodies for treatment and prevention of infection in animals and man

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2785089A (en) * 1987-12-23 1989-07-19 Pharmacia & Upjohn Company Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus
AU4927193A (en) * 1992-09-16 1994-04-12 Scripps Research Institute, The Human neutralizing monoclonal antibodies to respiratory syncytial virus
AU3660893A (en) * 1993-01-29 1994-08-15 Med Immune, Inc. Human-murine chimeric antibodies against respiratory syncytial virus

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ZA932445B (en) 1995-01-05
JPH07508401A (en) 1995-09-21
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