WO1994005690A1 - Novel antibodies for conferring passive immunity against infection by a pathogen in humans - Google Patents

Novel antibodies for conferring passive immunity against infection by a pathogen in humans Download PDF

Info

Publication number
WO1994005690A1
WO1994005690A1 PCT/US1993/008435 US9308435W WO9405690A1 WO 1994005690 A1 WO1994005690 A1 WO 1994005690A1 US 9308435 W US9308435 W US 9308435W WO 9405690 A1 WO9405690 A1 WO 9405690A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
ser
sequence
amino acid
Prior art date
Application number
PCT/US1993/008435
Other languages
English (en)
French (fr)
Inventor
Mitchell Stuart Gross
Martin Rosenberg
Jerald Charles Sadoff
Stephen Hoffman
Daniel R. Sylvester
Yupin Charoenvit
Mark Hurle
Original Assignee
Smithkline Beecham Corporation
United States Of America As Represented By The Secretary Of The Navy
United States Of America As Represented By The Secretary Of The Army
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation, United States Of America As Represented By The Secretary Of The Navy, United States Of America As Represented By The Secretary Of The Army filed Critical Smithkline Beecham Corporation
Priority to JP6507519A priority Critical patent/JPH08500979A/ja
Priority to AU48511/93A priority patent/AU4851193A/en
Priority to EP93921412A priority patent/EP0659192A4/en
Publication of WO1994005690A1 publication Critical patent/WO1994005690A1/en
Priority to KR1019950700936A priority patent/KR950703573A/ko

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to the field of monoclonal and reco binant antibodies directed to epitopes on selected pathogens, e.g., a malaria parasite, methods for preparing and using, and compositions employing, these antibodies.
  • pathogens e.g., a malaria parasite
  • Malaria is a severe and widespread disease, caused by various species of the protozoan parasite genus Plasmodium, including four species that infect man, e.g., P. falciparum, P. vivax, P. ovale and P. malariae [See, e.g., V. Enea et aJL. , Science, 225:628-630 (1984)]. Malaria remains one of the most widespread and fatal diseases in the world today because of the lack of an effective vaccine and programs to control vector populations, as well as new drug-resistant strains. Generally, treatment of malaria relies heavily on prophylactic drugs, such as the 4-aminoguinolines.
  • Plasmodium parasite particularly the circumsporozoite (CS) protein [Clyde et al., Am. J. Trop. Med. Hyg.. 4:397 (1975); Rieckman et al. , Bull. WHO. 57(1) :261 (1979); and U. S. Patent 4,957,869].
  • the cloning and characterization of the CS protein genes or fragments thereof of a number of Plasmodium species and recombinant expression thereof in E. coli or yeast host cells have been reported.
  • the central repeat domain of the CS proteins is immunodominant, i.e., if one injects sporozoites into an animal, the animal produces anti- repeat antibodies.
  • the first anti-sporozoite candidate vaccine tested in man was based upon the repetitive epitopes found on the CS protein of P. falciparum consisting of (AsnAlaAsnPro) 37 (AsnValAspPro) 4 [SEQ ID NO: 1] , which is invariant in a number of strains examined to date.
  • mAbs monoclonal antibodies directed toward proteins from various stages of the Plasmodium life cycle have been identified and shown to be effective in passive transfer experiments in mice and monkeys [Y.
  • the present invention provides complementarity determining region (CDR) peptides from a monoclonal antibody directed against a selected epitope on a pathogen, as well as fragments and analogs of these peptides.
  • the antibody is capable of binding an epitope of Plasmodium , particularly the CS repeat region epitope or a fragment thereof, e.g., murine anti-P. falciparum mAb NFS2.
  • CDRs retain the antigen binding specificity of the mAb from which they were derived.
  • Another aspect provides an isolated, naturally occurring or synthetic, humanized immunoglobulin light or heavy chain variable region amino acid sequence comprising one or more CDR sequences originating from the light or heavy chain of such a selected antibody.
  • the invention provides a fusion protein comprising a first amino acid sequence derived from the variable light chain and/or heavy chain of an anti -PI asmodium antibody, an anti -PI asmodi urn CDR, a functional fragment or analog thereof.
  • the first selected amino acid sequence is operatively linked or fused to a second selected amino acid sequence.
  • a further aspect of the invention provides an engineered antibody with specificity for the selected Plasmodium epitope, e.g., P. falciparum repeat region.
  • the invention provides a P. falciparum antibody or fragment thereof produced by screening hybridoma products derived from any species immunoglobulin repertoires, or human or murine antibody combinatorial libraries, with the epitope of mAb NFS2.
  • the present invention provides F ab fragments of the above-described engineered antibodies or anti -PI asmodium mAbs.
  • the invention provides nucleic acid sequences which encode the proteins, peptides, antibodies and fragments described herein, as well as plasmids containing one or more of the sequences, host cells transformed therewith, and methods for producing the products of expression of these nucleotide sequences in host cells, e.g., mammalian cells.
  • compositions and prophylactic methods for conferring passive immunity to a human anticipating exposure to a malarial parasite comprising an effective amount of at least one protein, antibody, peptide or fragment described herein and a pharmaceutically acceptable carrier or diluent.
  • Fig. 1 illustrates the amino acid SEQ ID NO: 4 and nucleotide SEQ ID NO: 3 sequences of the naturally occurring light chain variable region of mAb NFS2.
  • Fig. 2 illustrates the amino acid SEQ ID NO: 6 and nucleotide SEQ ID NO: 5 sequences of a synthetic humanized light chain variable region Pfhzlcl-1 containing ant i-Plasmodium CDRs SEQ ID NOs: 21-26. The CDRs are underlined.
  • Fig. 3 illustrates the amino acid SEQ ID NO: 8 and nucleotide SEQ ID NO: 7 sequences of synthetic humanized light chain variable region Pfhzlcl-2.
  • Fig. 4 illustrates the amino acid SEQ ID NO: 10 and nucleotide SEQ ID NO: 9 sequences of the naturally occurring heavy chain variable region of mAb NFS2.
  • Fig. 5 illustrates the amino acid SEQ ID NO: 12 and nucleotide SEQ ID NO: 11 sequences of a synthetic humanized heavy chain variable region Pfhzhc2-4.
  • Fig. 6 illustrates the amino acid SEQ ID NO: 14 and nucleotide SEQ ID NO: 13 sequences of synthetic humanized heavy chain variable region Pfhzhc2-3.
  • Fig. 7 is a schematic drawing of plasmid Pfhzhc2-3-Pcd employed to express a synthetic anti- Plasmodium heavy chain in mammalian cells.
  • the plasmid contains a beta lactamase (Beta-lac) gene, an SV40 origin of replication (SV40) , a cytomegalovirus promoter sequence (CMV) , the synthetic heavy chain Pfhzhc2-3 SEQ ID NO: 13, a poly A signal from bovine growth hormone (BGH) , a betaglobin promoter (beta glopro) , a dihydrofolate reductase gene (DHFR) , and another BGH sequence poly A signal in a pUC19 background.
  • BGH bovine growth hormone
  • beta glopro betaglobin promoter
  • DHFR dihydrofolate reductase gene
  • Fig. 8 is a schematic drawing of plasmid Pfhzlcl-1-Pcn employed to express a synthetic light chain in mammalian cells.
  • the plasmid differs from that of Fig. 7, in that it contains the synthetic humanized light 90
  • Fig. 9 illustrates the nucleotide SEQ ID NO: 42 and amino acid SEQ ID NO: 43 sequences of a synthetic humanized heavy chain variable region Pfhzhc2-6.
  • the present invention provides prophylactic agents capable of conferring a short duration, protective immune state against infection of humans by selected pathogens in the immunized human, e.g., for epidemic control and for use by those anticipating exposure to the pathogen.
  • Recombinant or engineered antibodies preferably chimeric, humanized or human monoclonal antibodies, are capable of use as such passive protective proteins. These proteins in a prophylactic composition may be administered before anticipated exposure to the pathogen and would not require daily regimens of follow- up doses to mediate the short term protection. While the following description refers specifically to antibodies capable of conferring passive protection to the sporozoite form of the pathogen, P.
  • the invention described herein is not limited to any particular stage of that pathogen nor to that pathogen alone.
  • the teachings of the present invention permit one skilled in the art to construct other recombinant antibodies directed to other selected pathogens, e.g., other species of Plasmodium, including the blood stages, liver stages, or gametocyte stages.
  • Antibodies of the invention directed against the circumsporozoite CS gene of the other human infective parasites e.g., P. malar iae , P. vivax and P. ovale, may also be constructed according to this invention to provide passive transfer proteins useful against these parasitic infections.
  • passive therapy agents prepared according to the invention may involve other infective agents, viruses, bacteria and the like. Additionally, such antibodies may also be useful as therapeutic agents for the treatment of acute stages of infections.
  • First fusion partner refers to a nucleic acid sequence encoding an amino acid sequence, which can be all or part of an immunoglobulin heavy chain, a light chain, functional fragment thereof including the variable region from one or both chains and CDRs therefor, or an analog thereof, having the antigen binding specificity of a selected high titer antibody, preferably the murine antibody, NFS2.
  • Strecond fusion partner refers to another nucleotide sequence encoding a protein or peptide to which the first fusion partner is fused in frame or by means of an optional conventional linker sequence. Such second fusion partner is preferably heterologous to the first fusion partner.
  • a second fusion partner may include a nucleic acid sequence encoding a second antibody region of interest, e.g., all or part of an appropriate human constant region or framework region.
  • Fusion molecule refers to the product of a first fusion partner operatively linked to a second fusion partner.
  • Oxperative linkage of the fusion partners is defined as an association which permits expression of the antigen specificity of the anti-P. falciparum sequence (the first fusion partner) from the donor antibody as well as the desired characteristics of the second fusion partner.
  • 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.
  • Fusion protein refers to the protein encoded by the fusion molecule, which may be obtained by expression of the fusion molecule in a selected host cell.
  • fusion proteins may be engineered antibodies, e.g., chimeric or humanized antibodies, or any of the antibody regions identified herein fused to immunoglobulin or non-immunoglobulin proteins and the like.
  • Donor antibody refers to an antibody (polyclonal, monoclonal or recombinant) which contributes its naturally-occurring or modified variable light and/or heavy chains, variable regions thereof, CDRs thereof or other functional fragments thereof to a first fusion partner, so as to provide the fusion molecule and fusion protein, with the antigenic specificity characteristic of the donor antibody.
  • One donor antibody suitable for use in this invention is murine mAb NFS2 and others are described below.
  • “Acceptor antibody” refers to an antibody (polyclonal, monoclonal or recombinant) heterologous to the donor antibody, but homologous to the patient (human or other mammal) to be treated, which contributes all or any portion of the sequences of its variable heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to a second fusion partner.
  • a human antibody is the acceptor antibody.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of the heavy and light chains. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include functional fragments and analogs of the naturally occurring CDRs, which share or retain the same antigen binding specificity as the donor antibody from which they were derived.
  • sharing the antigen binding specificity it is meant, for example, that although mAb NFS2 may be characterized by a certain level of antigen affinity, and a CDR encoded by a nucleic acid sequence of NFS2 in an appropriate structural environment may have a lower affinity, it is expected that CDRs of NFS2 in such environments will nevertheless recognize the same epitope(s) as NFS2.
  • a “functional fragment” is a partial CDR sequence or partial heavy or light chain variable sequence which retains the same antigen binding specificity 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, e.g., antigen specificity, of the unmodified sequence.
  • An "allelic variation or modification” is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention. Such variations or modifications may be due to 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.
  • An “engineered antibody” is a type of fusion protein, i.e., a synthetic antibody (e.g., a chimeric or humanized antibody) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody is replaced by analogous parts of CDRs from one or more donor antibodies which have specificity for the selected epitope.
  • engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain framework regions in order to retain donor antibody binding specificity.
  • These antibodies can comprise immunoglobulin constant regions and variable framework regions from the acceptor antibody, and one or more CDRs from the Plasmodium donor antibodies described herein.
  • the engineered antibodies of the invention will be produced by recombinant DNA technology.
  • Chimeric antibody refers to a type of engineered antibody which contains naturally-occurring variable region light chain and heavy chains (both CDR and framework regions) derived from a non-human donor mAb in association with light and heavy chain constant regions derived from a human (or other heterologous animal) acceptor mAb.
  • Humanized antibody refers to an engineered antibody having its CDRs and/or other portions of its light and/or heavy chain variable domain framework regions derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins.
  • Such antibodies can also include engineered antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa.
  • “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, e.g., 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 -Plasmodium derived amino acid sequences or to add to its properties.
  • non-human species may be employed to generate a desirable donor antibody upon presentment with an antigen from a Plasmodium strain capable of infecting humans.
  • Conventional hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human mAb to the selected antigen.
  • the murine mAb, NFS2 has been identified as a desirable antibody which may be employed for use in developing a chimeric or humanized antibody of this invention.
  • Murine IgG mAb NFS2 is characterized by an antigen binding specificity to the repeat region of the P. falciparum CS protein.
  • NFS2 non-semiconductor styrene-maleic anhydride copolymer
  • mAb 2A10 which is directed against the CS repeat protein or other mAbs described in R. A. Wirtz et al, Bull WHO. 65:39-45 (1987) .
  • Antibodies produced in other animals protected by immunization with sporozoites or a protective epitope of a selected Plasmodium species may be similarly employed in this invention as a source of protective anti -PI asmodium sequences.
  • the P. falciparum CS protein repeat region protein R32tet32 NH 2 -Met-Asp-Pro-[ (Asn-Ala- Asn-Pro) 15 (Asn-Val-Asp-Pro) 2 ] 2 -Leu-Arg-Arg-Thr-His-Arg-
  • SEQ ID NO: 2 may be employed to elicit both human and murine mAbs with binding specificity therefor.
  • This repeat region protein is a suitable target for screening for neutralizing antibodies useful in prophylactic agents against malarial infection.
  • epitope to which NFS2 is responsive may be useful in the screening and development of additional P. falciparum antibodies, for use in the development of prophylactic compositions for short-term protection of humans against malaria.
  • Other epitopes of interest include non- repetitive flanking region epitopes, other repeat domains or various liver, and blood and sexual stage epitopes of Plasmodium species. Knowledge of these epitopes enables one of skill in the art to define synthetic, and to identify naturally-occurring, peptides which would be suitable to confer passive or active immunity against P. falciparum or other Plasmodium species. This knowledge also permits the production of mAbs useful in the prophylaxis of malarial infection in humans.
  • P. falciparum antibodies may be developed by screening hybridomas or other combinatorial libraries, or antibody phage displays [W. D. Huse et al.. Science. 246:1275-1281 (1988)] using the murine mAb epitope described herein.
  • a collection of antibodies, including hybridoma products or antibodies derived from any species immunoglobulin repertoire may be screened in a conventional competition assay, such as described in the examples below, with one or more epitopes described herein.
  • Antibodies such as those described above, including those generated against a desired epitope and produced by conventional techniques, including without limitation, genes encoding murine mAbs, human mAbs, and combinatorial antibodies, may be useful as donor antibodies, as sources of antibody fragments, as well as in prophylactic compositions against P. falciparum in humans.
  • the antibodies developed in response to Plasmodium, particularly P. falciparum, epitopes may be useful as donors of desirable variable heavy and/or light chain amino acid sequences, or functional fragments thereof (e.g., CDRs) useful in the development of fusion proteins, including engineered antibodies.
  • the invention may utilize a donor antibody, other than NFS2, which is capable of binding to the P.
  • falciparum peptide consisting essentially of the amino acid sequence of the repeat protein and analogs thereof. Additionally, the mAbs identified herein, other mAbs which are developed and are responsive to the use of the sporozoites, R32tet32 [SEQ ID NO: 2] or the repeat epitopes identified herein may be further altered or manipulated to impart additional desirable prophylactic characteristics.
  • the present invention provides isolated naturally-occurring or synthetic variable light chain and variable heavy chain sequences derived from mAb NFS2, as well as CDRs and fragments therefrom, which may be employed in the design of fusion proteins (including engineered antibodies) which are characterized by the antigen binding specificity of this mAb.
  • NFS2 is characterized by the amino acid and encoding nucleic acid sequences illustrated in Fig. 4 [SEQ ID NOS:
  • CDR 1 is characterized by the sequence:
  • CDR 2 nucleic acid and amino acid sequences are SEQ ID NOS: 17 and 18, respectively: GAAATTAGTGATGGTGGTAGTTACACCTACTATCCAGACACTGTGACGGGC GlulleSerAspGlyGlySerTyrThrTyrTyrProAspThrValThrGly.
  • the naturally-occurring CDR 3 has the nucleic acid and amino acid sequences SEQ ID NOS: 19 and 20, respectively:
  • NFS2 are characterized by the amino acid and encoding nucleic acid sequences illustrated in Fig. 5 [SEQ ID NOS:
  • CDRs have the following nucleotide and predicted amino acid sequences. Nucleotide changes were made in CDR 1 from the naturally occurring CDRs, and are indicated by underlining. Synthetic CDR 1 is characterized by the sequence: AGCTATGCCATGAGC SEQ ID NO: 15 SerTyrAlaMetSer SEQ ID NO: 16.
  • the synthetic CDR 3 has the same nucleic acid and amino acid sequences as does the naturally occurring CDR 3 SEQ ID NOS: 19 and 20, respectively.
  • NFS2 is characterized by the amino acid sequence and encoding nucleic acid sequence of Fig. 1 [SEQ ID NOS: 3 and 4], This chain is further characterized by CDRs having the following nucleotide and amino acid sequences.
  • CDR 1 is characterized by the nucleic acid and amino acid sequences SEQ ID NOS: 34 and 35, respectively: AAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAATTACTTGGCC LysSerSerGlnSerLeuLeuTyrSerSerAsnGlnLysAsnTyrLeuAla.
  • CDR 2 is characterized by the nucleic acid and amino acid sequences SEQ ID NOS: 36 and 37, respectively:
  • CDR 3 is characterized by the nucleic acid and amino acid sequences SEQ ID NOS: 38 and 39, respectively:
  • NFS2 is characterized by the amino acid and encoding nucleic acid sequences illustrated in Fig. 2 [SEQ ID NOS: 5 and 6]. This chain is characterized by CDRs having the following predicted amino acid sequences and encoded by the illustrated nucleotide sequences. Nucleotide changes were made in the three CDRs from the naturally occurring corresponding CDRs, and are indicated by underlining.
  • Synthetic CDR 1 is characterized by the nucleic acid and amino acid sequences SEQ ID NOS: 21 and 22, respectively: AAGAGCTCTCAGAGCCTTTTATACTCGAGCAATCAAAAGAATTACTTGGCC LysSerSerGlnSerLeuLeuTyrSerSerAsnGlnLysAsnTyrLeuAla.
  • Synthetic CDR 2 is characterized by the nucleic acid and amino acid sequences SEQ ID NOS: 23 and 24, respectively:
  • Synthetic CDR 3 is characterized by the amino acid sequences SEQ ID NO: 26 and encoding nucleotide sequences SEQ ID NO:25, respectively:
  • Another synthetic humanized variable light chain of NFS2 is characterized by the amino acid and encoding nucleic acid sequences illustrated in Fig. 3
  • the present invention also includes the use of F ab fragments or F, ab , )2 fragments.
  • a F ab fragment contains the entire light chain and amino terminal portion of the heavy chain; and a F, ab ,> 2 fragment is the fragment formed by two F ab fragments bound by disulfide bonds.
  • MAb NFS2 and engineered antibodies derived therefrom and described below provide sources of F ab fragments and F, ab , )2 fragments which can be obtained by conventional means, e.g. cleavage of the mAb with the appropriate proteolytic enzymes, papain and/or pepsin, or by recombinant methods.
  • the F ab fragments or F, ab , )2 fragments may be derived from any of the mAbs described above, as agents protective in vivo against infection by malarial pathogens, particularly P. falciparum .
  • variable chain peptide sequences of murine mAb NFS2 may be useful in obtaining various fusion molecules encoding desired fusion proteins, particularly engineered antibodies, and in methods for preparing and administering pharmaceutical compositions containing them.
  • nucleic acid sequences of the invention or fragments thereof, encoding the variable light chains and heavy chain peptide sequences or CDR peptides, or functional fragments thereof are used in unmodified form or are synthesized to introduce desirable modifications.
  • the isolated naturally-occurring or synthetic nucleic acid sequences which are derived from mAB NFS2 or from other desired anti-Plasmodium antibodies, may optionally contain restriction sites to facilitate insertion or ligation into a suitable nucleic acid sequence encoding a desired antibody framework region, ligation with mutagenized CDRs, or fusion with a nucleic acid sequence encoding a selected second fusion partner.
  • variable heavy and light chain amino acid sequences and CDR sequences of the invention, e.g., Figs. 1 - 6 [SEQ ID NOS: 3-26], and functional fragments and analogs thereof which share the antigen specificity of the donor antibody.
  • the isolated or synthetic nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs or functional fragments thereof can be used to produce fusion proteins, i.e. chimeric or humanized antibodies, or other engineered antibodies of this invention, when operatively combined with a second fusion partner.
  • 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 fusion nucleic acid sequence into a vector for expression.
  • silent nucleotide substitutions may be made in the nucleotide sequences encoding the CDRs to create restriction enzyme sites to facilitate insertion of the mutagenic frameworks, or to modify the selected frameworks at nucleotide positions analogous to those of the donor antibody.
  • Such mutations may include those inserted for the purpose of contributing to higher antigen binding affinity.
  • Fusion molecules of this invention can encode engineered antibodies, chimeric antibodies and humanized antibodies.
  • a desired fusion molecule may contain a first fusion partner encoding an amino acid sequence having the antigen specificity of a Plasmodium antibody directed against the amino acid sequence of the repeat protein and analogs thereof, operatively linked to a second fusion partner.
  • the source of the first fusion partner is a selected mAb, e.,g., mAb NFS2, the source of nucleic acid sequences of Figs. 1 [SEQ ID NO: 3] and 4 [SEQ ID NO: 9] .
  • a fusion molecule may encode an amino acid sequence for a naturally occurring variable heavy chain sequence of Fig. 4 [SEQ ID NOS: 9 and 10], a functional fragment or analog thereof, a naturally occurring variable light chain sequence of Fig. 1 [SEQ ID NO: 3 and 4], a functional fragment or analog thereof, or one or more NFS2 CDRs [SEQ ID NO: 15-26].
  • Another exemplary fusion molecule may encode a synthetic variable heavy and/or light chain from the donor mAb, such as those of
  • a desirable fusion molecule of this invention may be characterized by encoding an amino acid sequence containing at least one, and preferably all of the CDRs [SEQ ID NOS: 15-26] of the variable region of the heavy and/or light chains of the murine antibody NFS2, or a functional fragment or analog thereof.
  • the second fusion partners are defined above, and may include a sequence encoding a peptide, protein or fragment thereof heterologous to the CDR-containing sequence having the antigen specificity of NFS2.
  • One example is a sequence encoding a second antibody region of interest and may optionally include a linker sequence.
  • the resulting fusion molecule may encode both anti-P. falciparum antigen specificity and the characteristic of the second fusion partner, e.g., a functional characteristic such as secretion from a recombinant host, or a therapeutic characteristic if the fusion partner itself encodes a therapeutic protein, or additional antigenic characteristics, if the fusion partner encodes a protein having its own antigen specificity.
  • the second fusion partner is derived from another antibody, e.g., any isotype or class of immunoglobulin framework or constant region (preferably human) , or the like, an engineered antibody is provided.
  • a fusion molecule of this invention may comprise a complete antibody molecule, having full length heavy and light chains (Figs.
  • the invention includes isolated naturally-occurring or synthetic nucleic acid sequences, which may encode variable region sequences, CDR peptides, fragments thereof derived from desired Plasmodium mAbs, any fragment of an engineered antibody, such as the F ab or F ( a b')2 fragment, a heavy chain dimer, or any minimal recombinant fragment thereof such as an F v or a single- chain antibody (SCA) or any other sequence encoding a protein with the same specificity as the selected mAb, e.g., the Plasmodium mAb NFS2.
  • the first fusion partner may also be associated with effector agents as defined above, to which the first fusion partner may be operatively linked by conventional means, e.g., attached to the NFS2 encoding nucleic acids by a covalent bridging structure.
  • Fusion or linkage between the first fusion partners and the selected second fusion partner may be by way of any suitable means, e.g., by conventional covalent or ionic bonds, protein fusions, or heterobifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde, and the like.
  • first fusion partner is associated with an effector agent, non-proteinaceous
  • conventional chemical linking agents may be used to fuse or join the anti-P. falciparum amino acid sequences to the effector agent. Such techniques are known in the art and readily described in conventional chemistry and biochemistry texts.
  • One particularly desirable type of fusion protein includes the engineered antibody in which, at a minimum, fragments of the variable heavy and/or light domains of an acceptor mAb have been replaced by analogous parts of the variable light and/or heavy chains from one or more donor monoclonal antibodies, which include the Plasmodium mAbs described herein, such as NFS2.
  • One example of a particularly desirable engineered antibody is a humanized antibody, in which CDRs from a desired donor murine mAb are inserted into the framework regions of a human antibody.
  • a preferred donor antibody is one directed against a Plasmodium epitope, preferably one specific for the repeat region epitope of P. falciparum .
  • a particularly preferred donor antibody has all or a portion of the variable domain amino acid sequences of NFS2.
  • these humanized antibodies one, two or preferably three CDRs from the Plasmodium antibody heavy chain and/or light chain variable regions are inserted into the framework regions of a selected human antibody, replacing the native CDRs of that latter antibody.
  • the variable domains in both human heavy and light chains have been altered by CDR replacement.
  • This engineered humanized antibody thus preferably has the structure of a natural human antibody or a fragment thereof.
  • Such humanized antibodies may or may not also include minimal alteration of the acceptor mAb light and/or heavy variable domain framework region in order to retain donor mAb binding specificity.
  • the humanized antibody possesses the combination of properties required for effective prevention and treatment of infectious P. falciparum disease in animals or man.
  • the remainder of the engineered antibody may be derived from any suitable acceptor human immunoglobulin.
  • a suitable human antibody may be one selected from a conventional database, e.g., the Kabat database, Los
  • 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 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 human antibody.
  • the heterologous framework and constant regions are selected from the human immunoglobulin classes and isotypes, such as IgG
  • the acceptor antibody need not comprise only human immunoglobulin protein sequences.
  • a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding the amino acid sequence of a polypeptide effector or reporter molecule.
  • an engineered antibody may be encoded by a synthetic nucleic acid sequence encoding CDRs of the variable light chain region of NFS2 or a functional fragment thereof in place of at least a part of the nucleic acid sequence encoding the light chain variable region of an acceptor mAb, and a nucleic acid sequence encoding CDRs of the variable heavy chain region of NFS2 or a functional fragment thereof in place of at least a part of the nucleic acid sequence encoding the heavy chain variable region of an acceptor mAb, such as a human antibody.
  • the resulting humanized antibody is characterized by the antigen binding specificity of mAb NFS2.
  • the engineered antibody (or the other monoclonal antibodies) of the invention may have attached to it an effector or reporter molecule.
  • the procedure of recombinant DNA technology may be used to produce an engineered antibody of the invention in which the F c fragment or CH3 domain of a complete antibody molecule has been replaced by an enzyme or toxin molecule.
  • engineered 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 may be substituted by other amino acids either in the variable domain frameworks or CDRs or both.
  • Such engineered antibodies can be effective in prevention of productive malaria (e.g., by P. falciparum) infection in humans.
  • the invention provides fusion proteins which are chimeric antibodies, as defined above.
  • Such antibodies differ from the humanized antibodies described above by providing the entire donor antibody heavy chain and light chain variable regions, including framework regions, e.g., Figs. 1 [SEQ ID NOS: 3 and 4] and 4 [SEQ ID NOS: 9 and 10], fused to acceptor constant regions for both chains.
  • a fusion molecule, recombinant antibody or fusion protein of this invention is desirably constructed by recombinant DNA technology using genetic engineering techniques.
  • the same or similar techniques may also be employed to generate other embodiments of this invention, e.g., 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.
  • compositions of this invention is set out in Example 3 below using the CDRs of murine NFS2 and one or more selected human antibody light and heavy chain framework regions.
  • a hybridoma producing the murine antibody NFS2 is conventionally cloned, and the cDNA of its heavy and light chain variable regions is obtained by techniques known to one of skill in the art, e.g., the techniques described in Sambrook et al. , Molecular Cloning (A Laboratory Manual) . 2nd edition, Cold Spring Harbor Laboratory (1989) .
  • the variable regions of the NFS2 are obtained using PCR primers, and the CDRs are 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 were identified using the same databases, e.g., Kabat, and a human antibody having homology to NFS2 was selected as the acceptor antibody.
  • the sequences of synthetic heavy chain variable regions containing the NFS2 CDRs within the human antibody frameworks were designed with optional nucleotide replacements in the framework regions for restriction sites. This designed sequence was synthesized by overlapping oligonucleotides, amplified by polymerase chain reaction (PCR) , and corrected for errors.
  • a suitable light chain variable framework region was designed in a similar manner, resulting in two synthetic light chain variable sequences containing the NFS2 CDRs. See, Figs. 2 [SEQ ID NOS: 5 and 6] and 3 [SEQ ID NOS: 7 and 8]. As stated above, the source of the light chain is not a limiting factor of this invention.
  • variable light and/or heavy chain sequences and the CDRs of mAb NFS2, and their encoding nucleic acid sequences are employed in the construction of fusion proteins and engineered antibodies, preferably humanized antibodies, of this invention, by the following process.
  • a DNA sequence is obtained which encodes the donor antibody variable heavy or light chain regions. wherein at least the CDRs and those minimal portions of the acceptor mAb light and/or heavy variable domain framework region required in order to retain donor mAb binding specificity as well as the remaining immunoglobulin-derived parts of the antibody chain derived from a human immunoglobulin.
  • a conventional expression vector or recombinant plasmid is produced by placing these sequences encoding the fusion protein in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell.
  • regulatory sequences may be readily selected by one of skill in the art and are not intended as a limitation of the present invention.
  • Regulatory sequence include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived by one of skill in the art from antibodies.
  • a first vector can contain a sequence encoding a light chain-derived polypeptide.
  • a second expression vector is produced having a similar DNA sequence which encodes a complementary antibody light or heavy chain.
  • the CDRs (and those minimal portions of the acceptor mAb light and/or heavy variable domain framework region required in order to retain donor mAb binding specificity) of the variable domain are derived from a donor antibody and the remaining immunoglobulin-derived parts of the antibody chain are derived from a human immunoglobulin in these vectors.
  • this second expression vector is identical to the first, with the exception of the coding sequences and selectable markers, to ensure that each polypeptide chain is functionally expressed.
  • a single vector of the invention may be used, the vector including the sequence encoding both light chain and heavy chain-derived 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 (or the single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention.
  • the 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 followed by the Inhibition of Sporozoite Invasion (ISI) assay described in the examples below. Similar conventional techniques may be employed to construct other fusion proteins of this invention.
  • the invention also includes a recombinant plasmid containing the coding sequence of the fusion molecule or engineered antibody of the invention.
  • a vector is prepared by conventional techniques and suitably comprises the above-described DNA sequences and a suitable promoter operatively linked to the DNA sequences which encode the engineered antibody.
  • Such a vector is transfected into a mammalian cell via conventional techniques.
  • Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art.
  • the conventional pUC series of cloning vectors may be used.
  • One vector used is pUC19, which is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden) .
  • any vector which is capable of replicating readily, has an abundance of cloning sites and marker genes, and is easily manipulated may be used for cloning.
  • the selection of the cloning vector is not a limiting factor in this invention.
  • the vectors employed for expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vector.
  • the 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 vectors contain the above described DNA sequences which code for the engineered antibody or other fusion protein.
  • 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, e.g., the mammalian dihydrofolate reductase gene (DHFR) or neomycin resistance gene (neo R ) .
  • marker genes suitable for amplifying expression of the heterologous DNA sequences e.g., the mammalian dihydrofolate reductase gene (DHFR) or neomycin resistance gene (neo R ) .
  • Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) , and the betaglobin promoter sequence (betaglupro) .
  • BGH bovine growth hormone
  • betaglupro betaglobin promoter sequence
  • replicons e.g. replicons, selection genes, enhancers, promoters, and the like
  • selection genes e.g. replicons, selection genes, enhancers, promoters, and the like
  • Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
  • Two exemplary expression vectors employed in the following examples for expression of the synthetic heavy and light chain sequences are the mammalian vectors Pfhzhc2-3-Pcd and Pfhzlcl-1-Pcn (see Figs. 7 and 8) .
  • this invention is not limited to the use of these illustrative pUC19-based vectors.
  • the present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or other fusion protein described by this invention.
  • Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. coli are used for replication of the cloning vectors and other steps in the construction of the mAbs of this invention.
  • Suitable host cells or cell lines for the expression of the engineered antibody or other protein of the invention of this invention are preferably a eukaryotic cell, and most preferably a mammalian cell, such as a CHO cell or a myeloid cell.
  • Other primate cells may be used as host cells and, most desirably, human cells are used, thus enabling the protein to be modified with human glycosylation patterns.
  • other eukaryotic cell lines may be employed.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al. , cited above.
  • Bacterial cells may prove useful as host cells suitable for the expression of the recombinant mAbs of the present invention.
  • any recombinant mAb produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the protein expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host.
  • various strains of E. coli used for expression are well-known as host cells in the field of biotechnology.
  • Various strains of B. subtilis are well-known as host cells in the field of biotechnology.
  • Streptomyces, other bacilli and the like may also be employed in this method.
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells and viral expression systems. See, e.g. Miller et al. , Genetic Engineering. 8.:277-298, Plenum Press (1986) and references cited therein.
  • transfection methods required to produce the host cells of the invention and culture methods necessary to produce the fusion protein, and preferably an engineered antibody of the invention from such host cell are all conventional techniques.
  • the fusion proteins, preferably the engineered antibodies of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention.
  • the engineered antibody is then examined for in vitro activity by use of an assay appropriate for the selected pathogen.
  • an assay appropriate for the selected pathogen Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the engineered antibody to the R32tet32 epitope [SEQ ID NO: 2].
  • the ISI assay described in Example 6 may also be employed.
  • a similar assay, the inhibition of hepatocyte invasion assay (ILSDA) may be performed [S. Mellouk et al. , Bull. WHO, Suppl. 68:52-58 (1990)].
  • assays currently being developed in the SCID mouse model may also be used to verify efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the engineered antibody in the body despite the usual clearance mechanisms.
  • the fusion proteins may be employed as prophylactic agents, capable of conferring short-term passive immunity to infection by the pathogen from which the original antigenic substance derives, e.g., P. falciparum , to a subject.
  • the protective effect conferred by the use of the engineered antibodies of this invention is produced by binding of the immunoglobulin to the pathogen and the subsequent removal of this bound complex by the normal function of macrophages.
  • the engineered antibodies of the present invention when in preparations and formulations appropriate for prophylactic use, are highly desirable for persons anticipating short-term exposure to the pathogen, e.g., travelers, tourists, or military personnel anticipating travel in endemic areas.
  • this invention also relates to a method of prophylactic treatment of human P. falciparum infection in a human in need thereof which comprises administering an effective, protective dose of antibodies including one or more of the engineered antibodies or other fusion proteins described herein, or fragments thereof, to a human anticipating exposure to a species of Plasmodium.
  • the fusion proteins including the engineered antibqdies or fragments thereof of this invention, may also be used in conjunction with other antibodies, particularly human mAbs reactive with other epitopes responsible for the disease against which the engineered antibody of the invention is directed.
  • mAbs reactive with other 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. Any antibody that is capable of operating without interfering with the Plasmodium antibody of this invention, e.g., antibodies to other malaria stages or to different epitopes, are useful in these compositions.
  • the prophylactic agents of this invention are believed to be desirable to confer protection to exposure to the pathogen for from about 4 days to about 8 weeks, without requiring booster dosages of the agent.
  • This definition of 'short-term 1 relates to the relative duration of the recombinant antibodies of the present invention in the human circulation.
  • the mode of administration of the prophylactic agent of the invention may be any suitable route which delivers the agent to the host.
  • the fusion proteins, including the engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously.
  • the agent is preferably administered by intramuscular injection.
  • Prophylactic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the engineered antibody of the invention as an active ingredient in a nontoxic and sterile pharmaceutically acceptable carrier.
  • an aqueous suspension or solution containing the engineered antibody preferably buffered at physiological pH, in a form ready for injection is preferred.
  • the compositions for parenteral administration will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers may be employed, e.g., saline, glycine, and the like. These solutions are sterile and generally free of particulate matter.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for parenteral e.g., intramuscular injection
  • a pharmaceutical composition of the invention for intravenous infusion could be prepared to contain 1 mL sterile buffered water, and between about 50 to about 100 mg of an engineered antibody of the invention.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of an engineered antibody of the invention.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science. 15th ed., Mack Publishing Company, Easton, Pennsylvania.
  • the prophylactic agent of the invention when in a pharmaceutical preparation, be present in unit dose forms.
  • the appropriate therapeutically effective dose can be determined readily by those of skill in the art.
  • one dose of approximately 1 mg/kg to approximately 20 mg/kg of a protein or an antibody of this invention should be administered parenterally, preferably intramuscularly (i.m.) and possibly intravenously (i.v.). Such dose may be repeated at appropriate intervals during exposure.
  • the antibodies, engineered antibodies or fragments thereof described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.
  • the pharmaceutical composition of the invention should provide a quantity of the engineered antibodies of the invention sufficient to effectively prevent infection.
  • the following examples illustrate the construction of exemplary engineered antibodies and expression thereof in suitable vectors and host cells, and are not to be construed as limiting the scope of this invention. All amino acids are identified by conventional codes or by full name, unless otherwise indicated. All 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 performed in Sambrook et al. , cited previously, or the first edition thereof.
  • Murine IgG mAb NFS2 was made by repeated injection of P. falciparum sporozoites into mice followed by B cell fusion with a myeloma cell line.
  • the murine mAb NFS2 is characterized by an antigen binding specificity to the repeat region of the P. falciparum CS protein. Specifically, the NFS2 mAb binds to the epitope, Pro Asn Ala Asn Pro Asn SEQ ID NO: 27. It is possible that the antibody also binds to a larger, or overlapping epitope on the repeat region.
  • This murine mAb in in vitro assays, prevented invasion of sporozoites into human hepatocytes and hepatoma cells.
  • Analogous antibodies in the mouse model have conferred passive protection against malaria and have been observed to be highly potent [see, e.g., R. A. Wirtz et al.. Bull WHO. 65:39-45 (1987), incorporated herein by reference] . This antibody is available from the U. S. Naval Medical Research Institute.
  • V H lg heavy chain variable region cDNA
  • V L light chain variable region cDNA
  • V H primers #2621 and #2853, extended from Kpnl through PstI and were made to conserved regions of murine RNA.
  • CCCCAG3' SEQ ID NO: 31 PCR as described by Saiki et al. , Science, 239:487-491 (1988), was performed on the RNA template.
  • DNA/primer mixtures consisted of 5 ⁇ l RNA and 0.5 ⁇ M of the primers.
  • DNA/primer mixtures consisted of 5 ⁇ l RNA and 0.5 ⁇ M of the primers.
  • V H DNA was purified on a low melting point agarose gel and by Elutip-d column chromatography [Schleicher and Schuell-Dussel, Germany] and cloned into pUCl ⁇ [New England Biolabs] .
  • Elutip-d column chromatography [Schleicher and Schuell-Dussel, Germany] and cloned into pUCl ⁇ [New England Biolabs] .
  • the general cloning and ligation methodology was as described in Maniatis et al., cited above.
  • V H DNA was cloned as KpnI-PstI fragments into similarly-digested pUC18.
  • V L DNA was cloned as Hindlll- Xbal fragments into pUC18 digested with the same enzymes. Representative clones were sequenced by the dideoxy method [Sanger et al. , Proc. Natl. Acad. Sci. USA. 74:5463-5467 (1977)] using T7 DNA polymerase [US Biologicals] . From the sequences of NFS2 V H and V L domains, the CDR sequences were elucidated in accordance with the methodology of Kabat et al.
  • the source of the donor CDRs utilized to prepare these engineered antibodies was the murine mAb, NFS2, described in Examples 1 and 2 above.
  • the sequenced NFS2 variable framework regions were employed to again search through the Kabat database to identify homologous framework regions of a human antibody.
  • NFS2 CDRs (Example 2) and the sequence of the human antibody 18/17
  • a synthetic heavy chain variable region was made, and PCR performed to fill in and amplify DNA.
  • the NFS2 CDR sequences and the 18/17 V H framework regions were synthesized by the following overlapping oligonucleotides: SEQ ID NO:44:TAGTGAAGAATTCGAGGACGCCAGCAACATGGTGTTGCAGAC CCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGAGGTGCAG (Base 1-97) ;
  • SEQ ID NO:46 GAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAG GACACGTCTCTGTTAAGGTTCTTGTGCGACATAGACGTTTACTGCAGTATATTAC TGTGCGAAACTCATCTACTATGGTTACGACGGGTATGCTATGGACTAGCTGCCCA TACGATACCTGATC (Base 316-421) ;
  • SEQ ID NO:47 TTCTTGAAAGCTTGGGCCCTTGGTACTAGCTGAGCTCACGG TGACCAGGGTACCCTGGCCCCAGTAGTCCATAGCATACCCGTCG (Base 484- 400) ;
  • SEQ ID NO:48 CATTTGCAGATACAGCGTGTTCTTGGAATTGTCTCTGGATA TCGTGAACCGGCCCGTCACAGTGTCTGGATAGTAGGTGTAACTACCACCATCACT AATTTC (Base 337-236) ;
  • SEQ ID NO:49 CTAAAGGTGAATCCGCTAGCTGCACAGGAGAGTCTCAGGGA CCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCGAGCAGCTGCACCTCCCCGTAG GCACC (Base 177-77).
  • primers are annealed together and DNA is filled in using Taq polymerase, followed by PCR amplification using the following 5' primer: SEQ ID NO:50: CCGCGAATTCGAGGACGCCAGCAAC and 3' primer: SEQ ID NO:51: CCGCAAGCTTGGGCCCTTGGTACTAGCT. Any errors in the mapped sequence which were inserted by PCR were corrected. In addition, conservative nucleotide replacements were placed in the framework regions to introduce selected restriction sites suitable for enzymatic cleavage. These alterations in the framework regions are indicated by boxes in the sequences of Figs.
  • Two synthetic heavy chain variable regions were obtained, namely, Pfhzhc2-3 SEQ ID NOS: 13 and 14, and Pfhzhc2-4 SEQ ID NOS: 11 and 12. These sequences are described in detail in Figs. 5 and 6.
  • Each of these synthetic heavy chain variable regions is characterized by one or two nucleotide, or amino acid, differences.
  • Pfhzhc2-3 [SEQ ID NOS: 13 and 14] has a Ser at position 98
  • Pfhzhc2-4 [SEQ ID NOS: 11 and 12] has a Lys at position 98. Otherwise, these heavy chain variable regions are identical.
  • the NFS2 light chain CDRs and the light chain variable framework sequence of the human antibody identified in H. G. Klobeck et al. , Nucl. Acids Res.. 11:6515-6529 (1985) were used to make a suitable synthetic light chain sequence by the same methods.
  • the oligonucleotides used were as follows.
  • SEQ ID NO:52 TAAGCGGAATTCGTAGTCGGATATCGTGATGACCCAGTC TCCAGACTCGCTAGCTGTGTCTCTGGGCGAGAGGGC (Base 1-75)
  • SEQ ID NO:53 TTACTTGGCCTGGTATCAGCAGAAACCCGGGCAGTCTCC TAAGTTGCTCATAGTTTTCTTAATGAACCGGACTTACTGGGCGTCAACTAG (Base 130-198) ;
  • SEQ ID NO:54 GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAA
  • SEQ ID NO:55 CAGTTGGAAAGCTTGGCGCCGCCACAGTACGTTTGATCTCCA CCTTGGTCCCTCCGCCGAACGTCCGCGGATAGCTATAATATTGC (Base 389- 304);
  • SEQ ID NO:56 GTGAAATCTGTCCCAGACCCGCTGCCACTGAATCGG TCAGGTACCCCAGATTCCCTAGTTGACGCC (Base 252-187) ;
  • SEQ ID NO:57 CAGGCCAAGTAATTCTTTTGATTGCTCGAGTATAAA AGGCTCTGAGAGCTCTTGCAGTTGATGGTGGCCCTCTCGCCC (Base 141-64) .
  • the primers were annealed together and DNA filled in using Taq polymerase, followed by PCR amplifcation with the following 5' SEQ ID NO:58: GCGGAATTCGTAGTCGGATATCGTGATGAC and 3' SEQ ID NO:59: TGGAAAGCTTGGCGCCGCCACAGTACGTTTGATC primers.
  • Pfhzlcl-1 SEQ ID NOS: 5 and 6 Two synthetic light chain variable sequences containing the NFS2 CDRs were designed and synthesized as described above for the synthetic heavy chains and referred to as Pfhzlcl-1 SEQ ID NOS: 5 and 6, and Pfhzlcl-2 SEQ ID NOS: 7 and 8. These two sequences differed in amino acid sequence at only one amino acid position, 49.
  • Pfhzlcl-1 [SEQ ID NOS: 5 and 6] has a Ser at position 49;
  • Pfhzlcl-2 [SEQ ID NOS: 7 and 8] has a Pro at the same position.
  • the construct is digested with EcoRI and EcoRV, and the same signal sequence was ligated onto the variable sequence.
  • Other signal sequences are well known to those of skill in the art and may be substituted for this exemplary sequence.
  • Selected constant regions of the human IgG 1 antibodies selected for the heavy and light chain were synthesized and confirmed by PCR. These constant region sequences were then inserted into pUC19-based expression vectors.
  • the above-described synthetic variable constructs, containing the signal and variable regions of the light and heavy chains, were thereafter inserted into these pUC19-based expression vectors containing CMV promoters and the constant regions and fused in frame to the previously inserted human heavy and light chain constant regions by conventional methods [Maniatis et al.. cited above].
  • plasmids shown in Figs. 7 and 8 resulted. These plasmids were then co-transfected into a selected host cell and, following incubation, the media was assayed for antibody activity via ELISA as described in Example 4 below.
  • Another exemplary humanized antibody is constructed using the synthesized heavy chain sequence Pfhzhc2-3 [SEQ ID NO:13 and 14] (Fig. 6) and the synthetic light chain sequence Pfhzlcl-2 [SEQ ID NO:7 and 8] .
  • Example 4 A High Affinity Humanized Antibody
  • the amino acid differences in the variable regions of the frameworks of the original murine antibody NSF2 described in Examples 1 and 2 and the Pfhzhc2.3 were determined, and several changes were made to increase the level of conservation of the original antibody conformation.
  • the Ser of the humanized heavy chain Pfhzhc2.3 was changed to Ala, which is the amino acid found at this position in the native murine NSF2.
  • the replacement employed conventional genetic engineering technology, e.g., by making a synthetic DNA fragment containing the appropriate nucleotide changes to alter the amino acid.
  • a fragment of Pfhzhc2.3 was digested with Xbal and EcoRV and the synthetic fragment bearing the nucleotide change encoding Ala in place of a Ser codon, is inserted to make the amino acid replacement.
  • the resulting synthetic heavy chain was termed Pfhzhc2.6.
  • This synthetic heavy chain was expressed as previously described for the Pfhzhc2.3 synthetic heavy chain.
  • the expression plasmid for this humanized heavy chain sequence is essentially identical to the expression plasmid illustrated in Fig. 7, with the exception of the single amino acid difference described previously.
  • humanized antibodies consisting of the Pfhzhc2.6 heavy chain and Pfhzlcl.l light chain and the Pfhzhc2.6 heavy chain and the Pfhzlcl.2 light chain were assembled via co-transfection of mammalian cells and assayed for antibody activity by ELISA, as described in Example 5 below.
  • an amino acid is in the framework of the donor antibody but not in the framework of the engineered antibody, then an alternative heavy chain gene comprising the engineered antibody is produced.
  • an alternative heavy chain gene comprising the original amino acid at that position is reproduced.
  • alternative plasmids produced on this basis are tested for production of high affinity engineered antibodies.
  • the presence of humanized antibody in the medium of wells containing transfected clones is measured by conventional ELISA techniques.
  • Micro-titer plates are coated overnight at 4°C with goat anti-human IgG (F c specific) antibodies [Sigma, St. Louis, MO] at 0.1 ⁇ g per well.
  • PBS pH 7.5
  • 50 ⁇ l of culture medium from the wells containing transfectants is added to each microtitre well for 2 hours at room temperature.
  • the wells are then emptied, washed with PBS and peroxidase-conjugated goat anti-human IgG antibodies [BioRad, Richmond, CA] are added at 50 ⁇ L of a 1/1000 dilution per well.
  • the wells are then emptied, washed with PBS and peroxidase-conjugated goat anti-human IgG antibodies [BioRad, Richmond, CA] are added at 50 ⁇ L of a 1/1000 dilution per well. Plates are then incubated at room temperature for l hour. The wells are then emptied and washed with PBS. 100 ⁇ l 2.2'-azino-di[3-ethyl- benzthiazoline sulfonate(6) ] are added per well. Reactions were allowed to continue for 1 hour at room temperature. The absorbance at 405 n is then measured spectrophotometrically.
  • Example 4 In preliminary studies, an increase in affinity was observed for the humanized antibodies of Example 4, which contained the Pfhzhc2-6 heavy chain construct, as compared to the Pfhzhc2-3 heavy chain construct.
  • a chimeric antibody of the invention was constructed essentially as described above.
  • a chimeric antibody contains the native murine NSF2 variable framework and CDR regions on the human constant regions selected for the heavy chain [H. Dersimonian et al. , J. Immunol.. 139:2496-2501 (1987) and light chain [Klobeck et al.. Nucl. Acids. Res... 11:6515-6529 (1985)], with the exception that the variable regions were obtained by PCR of the RNA of the murine antibody obtained from the NFS2 hybridoma and the entire constant regions of the human IgG 2 antibodies were synthesized by overlapping oligonucleotides and amplified by PCR. Any errors which were inserted by PCR were corrected.
  • the resulting chimeric heavy chain and chimeric light chain were expressed as described above for the humanized antibody.
  • This chimeric antibody is advantageous in that it is characterized by activity substantially identical to that of the native murine antibody, but contains enough human sequences that it is anticipated to be useful in human therapy.
  • the Inhibition of Sporozoite Invasion assay is performed as described in M. R. Hollingdale et al. , J. Immunol.. 132:909-913 (1984) to be used to assess neutralizing effect against live P. falciparum sporozoites.
  • the human hepatoma cloned cell line HepG2-Al6 2 was grown to near confluency on 1% C0 2 glass cover slips in MEM and 10% bovine fetal serum. Antisera or purified antibodies were diluted in culture medium (see table below) and added to the cell cultures.
  • 30,000 P. falciparum sporozoites isolated from dissected mosquito salivary glands are counted, diluted and added to each cell culture.
  • the cultures are incubated at 37°C for 2.5 hours, rinsed with PES, and fixed with methanol. Fixed cultures are reacted in an immunoperoxidase antibody assay using a labelled mAb which recognizes the P. falciparum CS protein to visualize invaded sporozoites. Then, the number of invading sporozoites are counted by phase microscopy at 40Ox.
  • the ISI is the percent reduction in invasion in the presence of the test antibody, the humanized antibody, as compared to a control (i.e. non-related) antibody.
  • the assay ranks antibodies in order according to their relative potency.
  • recombinant antibodies capable of neutralizing pathogens other than P. falciparum may be provided according to the teachings of this invention for the development of prophylactic agents capable of conferring passive immunity to other human diseases.
  • engineered antibodies capable of recognizing repeat regions on other malaria pathogens or engineered antibodies to any region on the surface of any stages of the life-cycle of the plasmodium species or capable of neutralizing any stage in the life cycle of the parasite are desirable starting materials to develop passive immunity agents according to this invention.
  • Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.
  • MOLECULE TYPE DNA (genomic)
  • CAG CAG AAA CCA GGG CAG TCT CCT AAA CTG CTG ATT TAC TGG 168 Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr Trp 45 50 55
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAG CAG AAA CCC GGG CAG CCT CCT AAG TTG CTC ATT TAC TGG 168 Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Tyr Trp 45 50 55
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:23: TGGGCGTCAA CTAGGGAATC T 21
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:25: CAGCAATATT ATAGCTATCC GCGGACG 27
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:30: GGGGTACCAG GTCCARCTKC TCGAGTCWGG 30
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:40: TGGGCGTCGA CTAGGGAATC T 21
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GAAACTCATC TACTATGGTT ACGACGGGTA TGCTATGGAC TAGCTGCCCA 150
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/US1993/008435 1992-09-09 1993-09-08 Novel antibodies for conferring passive immunity against infection by a pathogen in humans WO1994005690A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP6507519A JPH08500979A (ja) 1992-09-09 1993-09-08 ヒトにおいて病原体による感染に対する受動免疫を付与するための新規の抗体
AU48511/93A AU4851193A (en) 1992-09-09 1993-09-08 Novel antibodies for conferring passive immunity against infection by a pathogen in humans
EP93921412A EP0659192A4 (en) 1992-09-09 1993-09-08 PASSIVE IMMUNITY DELIVERING ANTIBODIES AGAINST INFECTION IN PEOPLE Caused by PATHOGENS.
KR1019950700936A KR950703573A (ko) 1992-09-09 1995-03-09 병원체에 의한 인간의 감염에 대한 수동 면역을 부여하는 신규의 항체(novel antibodies for conferring passive immunity against infection by a pathogen in humans)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94165492A 1992-09-09 1992-09-09
US07/941,654 1992-09-09

Publications (1)

Publication Number Publication Date
WO1994005690A1 true WO1994005690A1 (en) 1994-03-17

Family

ID=25476840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/008435 WO1994005690A1 (en) 1992-09-09 1993-09-08 Novel antibodies for conferring passive immunity against infection by a pathogen in humans

Country Status (9)

Country Link
EP (1) EP0659192A4 (ja)
JP (1) JPH08500979A (ja)
KR (1) KR950703573A (ja)
CN (1) CN1087681A (ja)
AU (1) AU4851193A (ja)
CA (1) CA2143417A1 (ja)
NZ (1) NZ256232A (ja)
WO (1) WO1994005690A1 (ja)
ZA (1) ZA936260B (ja)

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009398A1 (en) * 1994-09-19 1996-03-28 Rijkslandbouwuniversiteit Wageningen Gene constructs encoding crop-protecting agents, as well as transformed plants containing and expressing such constructs, and methods of controlling plague organisms and pathogens in crops
WO1996022310A1 (en) * 1995-01-18 1996-07-25 Bioinvent International Ab Antibodies for use in cancer therapy and diagnosis
EP0953639A1 (en) * 1998-04-30 1999-11-03 Boehringer Ingelheim International GmbH FAPalpha-specific antibody with improved producibility
US6455677B1 (en) 1998-04-30 2002-09-24 Boehringer Ingelheim International Gmbh FAPα-specific antibody with improved producibility
WO2002072600A3 (en) * 2001-01-26 2003-11-06 Inhibitex Inc Monoclonal antibodies to the clfa protein and method of use in treating or preventing infections
EP1361892A1 (en) * 2001-01-17 2003-11-19 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US7166697B1 (en) 1998-03-06 2007-01-23 Diatech Pty. Ltd. V-like domain binding molecules
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US7754209B2 (en) 2003-07-26 2010-07-13 Trubion Pharmaceuticals Binding constructs and methods for use thereof
US7829084B2 (en) 2001-01-17 2010-11-09 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US8163283B2 (en) * 2009-09-03 2012-04-24 Vancouver Biotech Ltd. Monoclonal antibodies against gonadotropin-releasing hormone receptor
AU2008200400B2 (en) * 2001-01-17 2012-06-07 Aptevo Research And Development Llc Binding domain-immunoglobulin fusion proteins
US8333966B2 (en) 2008-04-11 2012-12-18 Emergent Product Development Seattle, Llc CD37 immunotherapeutics and uses thereof
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
US8597911B2 (en) 2003-06-11 2013-12-03 Chugai Seiyaku Kabushiki Kaisha Process for producing antibodies
US8945543B2 (en) 2005-06-10 2015-02-03 Chugai Seiyaku Kabushiki Kaisha Stabilizer for protein preparation comprising meglumine and use thereof
US9005612B2 (en) 2001-01-17 2015-04-14 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
US9493569B2 (en) 2005-03-31 2016-11-15 Chugai Seiyaku Kabushiki Kaisha Structural isomers of sc(Fv)2
US9670269B2 (en) 2006-03-31 2017-06-06 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US20170190774A1 (en) * 2014-09-10 2017-07-06 Repropharm Ligands that potentiate the bioactivity of gonadotropins
US9975966B2 (en) 2014-09-26 2018-05-22 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing theraputic agent
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US10066018B2 (en) 2009-03-19 2018-09-04 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
US10150808B2 (en) 2009-09-24 2018-12-11 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant regions
US10253091B2 (en) 2009-03-19 2019-04-09 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US10435458B2 (en) 2010-03-04 2019-10-08 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variants with reduced Fcgammar binding
US10450381B2 (en) 2010-11-17 2019-10-22 Chugai Seiyaku Kabushiki Kaisha Methods of treatment that include the administration of bispecific antibodies
US11046784B2 (en) 2006-03-31 2021-06-29 Chugai Seiyaku Kabushiki Kaisha Methods for controlling blood pharmacokinetics of antibodies
US11072666B2 (en) 2016-03-14 2021-07-27 Chugai Seiyaku Kabushiki Kaisha Cell injury inducing therapeutic drug for use in cancer therapy
US11124576B2 (en) 2013-09-27 2021-09-21 Chungai Seiyaku Kabushiki Kaisha Method for producing polypeptide heteromultimer
US11142587B2 (en) 2015-04-01 2021-10-12 Chugai Seiyaku Kabushiki Kaisha Method for producing polypeptide hetero-oligomer
US11248053B2 (en) 2007-09-26 2022-02-15 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substitution in CDR
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies
US11332533B2 (en) 2007-09-26 2022-05-17 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant region
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
US11649262B2 (en) 2015-12-28 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for promoting efficiency of purification of Fc region-containing polypeptide
US11851476B2 (en) 2011-10-31 2023-12-26 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule having regulated conjugation between heavy-chain and light-chain

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200530269A (en) 2003-12-12 2005-09-16 Chugai Pharmaceutical Co Ltd Anti-Mpl antibodies
WO2014164553A1 (en) 2013-03-13 2014-10-09 Imaginab, Inc. Antigen binding constructs to cd8
CA2994951A1 (en) 2015-08-07 2017-02-16 Imaginab, Inc. Antigen binding constructs to target molecules
CN117854601B (zh) * 2024-03-04 2024-05-14 鲁东大学 一种基于基因类型和氨基酸序列的决定性互补区分类方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0270077A2 (en) * 1986-12-03 1988-06-08 Sumitomo Chemical Company, Limited Antibody to exotoxin of pseudomonas aeruginosa and gene therefor

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4632909A (en) * 1983-10-31 1986-12-30 The United States Of America As Represented By The Department Of Health And Human Services Monoclonal antibodies which block infectivity of malarial parasites to mosquitoes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0270077A2 (en) * 1986-12-03 1988-06-08 Sumitomo Chemical Company, Limited Antibody to exotoxin of pseudomonas aeruginosa and gene therefor

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Bull World Health Organ, 68 Suppl., issued 1990, MELLOUK et al., "Evaluation of an in Vitro Assay Aimed at Measuring Protective Antibodies against Sporozoites", pages 52-59, see entire Abstract. *
Proceedings of the National Academy of Sciences, Volume 86, issued December 1989, QUEEN et al., "A Humanized Antibody that Binds to the Interleukin 2 Receptor", pages 10029-10033, see entire document. *
See also references of EP0659192A4 *
The EMBO Journal, Volume 5, No. 7, issued 1986, ANDREW J. CATON et al., "Structural and Functional Implications of a Restricted Antibody Response to a Defined Antigenic Region on the Influenza Virus Hemagglutinin", pages 1577-1587, see figures 4 and 5. *
The Journal of Immunology, Volume 139, No. 7, issued 01 October 1987, HAROUT DERSIMONIAN et al., "Relationship of Human Variable Region Heavy Chain Germ-Line Genes to Genes Encoding Anti-DNA Autoantibodies", pages 2496-2501, see page 2498. *

Cited By (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009398A1 (en) * 1994-09-19 1996-03-28 Rijkslandbouwuniversiteit Wageningen Gene constructs encoding crop-protecting agents, as well as transformed plants containing and expressing such constructs, and methods of controlling plague organisms and pathogens in crops
WO1996022310A1 (en) * 1995-01-18 1996-07-25 Bioinvent International Ab Antibodies for use in cancer therapy and diagnosis
US7405288B2 (en) 1998-03-06 2008-07-29 Diatech Pty. Ltd. V-like domain binding molecules and polynucleotides encoding therefor
US7166697B1 (en) 1998-03-06 2007-01-23 Diatech Pty. Ltd. V-like domain binding molecules
EA005401B1 (ru) * 1998-04-30 2005-02-24 Бёрингер Ингельхайм Интернациональ Гмбх Антитела повышенной продуктивности к белку активации фибробластов и способы их применения
EP0953639A1 (en) * 1998-04-30 1999-11-03 Boehringer Ingelheim International GmbH FAPalpha-specific antibody with improved producibility
WO1999057151A2 (en) * 1998-04-30 1999-11-11 Boehringer Ingelheim International Gmbh FAP α-SPECIFIC ANTIBODY WITH IMPROVED PRODUCIBILITY
WO1999057151A3 (en) * 1998-04-30 2000-03-16 Boehringer Ingelheim Int FAP α-SPECIFIC ANTIBODY WITH IMPROVED PRODUCIBILITY
US6455677B1 (en) 1998-04-30 2002-09-24 Boehringer Ingelheim International Gmbh FAPα-specific antibody with improved producibility
EP1361892A4 (en) * 2001-01-17 2004-10-13 Trubion Pharmaceuticals Inc BINDING DOMAIN FUSION PROTEIN Immunoglobulin
US8853366B2 (en) 2001-01-17 2014-10-07 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US7829084B2 (en) 2001-01-17 2010-11-09 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US9005612B2 (en) 2001-01-17 2015-04-14 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
AU2008200400B2 (en) * 2001-01-17 2012-06-07 Aptevo Research And Development Llc Binding domain-immunoglobulin fusion proteins
EP1361892A1 (en) * 2001-01-17 2003-11-19 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
AU2002256985B2 (en) * 2001-01-26 2007-10-11 Inhibitex, Inc Monoclonal antibodies to the ClfA protein and method of use in treating or preventing infections
AU2002256985B8 (en) * 2001-01-26 2008-03-20 Inhibitex, Inc Monoclonal antibodies to the ClfA protein and method of use in treating or preventing infections
US6979446B2 (en) 2001-01-26 2005-12-27 Inhibitex, Inc. Monoclonal antibodies to the ClfA protein and method of use in treating or preventing infections
WO2002072600A3 (en) * 2001-01-26 2003-11-06 Inhibitex Inc Monoclonal antibodies to the clfa protein and method of use in treating or preventing infections
US8597911B2 (en) 2003-06-11 2013-12-03 Chugai Seiyaku Kabushiki Kaisha Process for producing antibodies
US7754209B2 (en) 2003-07-26 2010-07-13 Trubion Pharmaceuticals Binding constructs and methods for use thereof
US11168344B2 (en) 2005-03-31 2021-11-09 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US9493569B2 (en) 2005-03-31 2016-11-15 Chugai Seiyaku Kabushiki Kaisha Structural isomers of sc(Fv)2
US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
US8945543B2 (en) 2005-06-10 2015-02-03 Chugai Seiyaku Kabushiki Kaisha Stabilizer for protein preparation comprising meglumine and use thereof
US9777066B2 (en) 2005-06-10 2017-10-03 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
US10307481B2 (en) 2005-07-25 2019-06-04 Aptevo Research And Development Llc CD37 immunotherapeutics and uses thereof
US9670269B2 (en) 2006-03-31 2017-06-06 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US10934344B2 (en) 2006-03-31 2021-03-02 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US11046784B2 (en) 2006-03-31 2021-06-29 Chugai Seiyaku Kabushiki Kaisha Methods for controlling blood pharmacokinetics of antibodies
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
US11332533B2 (en) 2007-09-26 2022-05-17 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant region
US11248053B2 (en) 2007-09-26 2022-02-15 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substitution in CDR
US8333966B2 (en) 2008-04-11 2012-12-18 Emergent Product Development Seattle, Llc CD37 immunotherapeutics and uses thereof
US9101609B2 (en) 2008-04-11 2015-08-11 Emergent Product Development Seattle, Llc CD37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
US10066018B2 (en) 2009-03-19 2018-09-04 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US10253091B2 (en) 2009-03-19 2019-04-09 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US8163283B2 (en) * 2009-09-03 2012-04-24 Vancouver Biotech Ltd. Monoclonal antibodies against gonadotropin-releasing hormone receptor
US10150808B2 (en) 2009-09-24 2018-12-11 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant regions
US10435458B2 (en) 2010-03-04 2019-10-08 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variants with reduced Fcgammar binding
US10450381B2 (en) 2010-11-17 2019-10-22 Chugai Seiyaku Kabushiki Kaisha Methods of treatment that include the administration of bispecific antibodies
US11851476B2 (en) 2011-10-31 2023-12-26 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule having regulated conjugation between heavy-chain and light-chain
US11124576B2 (en) 2013-09-27 2021-09-21 Chungai Seiyaku Kabushiki Kaisha Method for producing polypeptide heteromultimer
US10703815B2 (en) 2014-09-10 2020-07-07 Igyxos Ligands that potentiate the bioactivity of gonadotropins
US10584166B2 (en) * 2014-09-10 2020-03-10 Repropharm Vet Ligands that potentiate the bioactivity of gonadotropins
US20170190774A1 (en) * 2014-09-10 2017-07-06 Repropharm Ligands that potentiate the bioactivity of gonadotropins
US11001643B2 (en) 2014-09-26 2021-05-11 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing therapeutic agent
US9975966B2 (en) 2014-09-26 2018-05-22 Chugai Seiyaku Kabushiki Kaisha Cytotoxicity-inducing theraputic agent
US11142587B2 (en) 2015-04-01 2021-10-12 Chugai Seiyaku Kabushiki Kaisha Method for producing polypeptide hetero-oligomer
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
US11649262B2 (en) 2015-12-28 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for promoting efficiency of purification of Fc region-containing polypeptide
US11072666B2 (en) 2016-03-14 2021-07-27 Chugai Seiyaku Kabushiki Kaisha Cell injury inducing therapeutic drug for use in cancer therapy
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies

Also Published As

Publication number Publication date
NZ256232A (en) 1997-02-24
CN1087681A (zh) 1994-06-08
KR950703573A (ko) 1995-09-20
EP0659192A1 (en) 1995-06-28
EP0659192A4 (en) 1996-09-11
AU4851193A (en) 1994-03-29
JPH08500979A (ja) 1996-02-06
ZA936260B (en) 1994-03-18
CA2143417A1 (en) 1994-03-17

Similar Documents

Publication Publication Date Title
EP0659192A1 (en) Novel antibodies for conferring passive immunity against infection by a pathogen in humans
CA2153135C (en) Method for obtaining modified immunoglobulins with reduced immunogenicity of murine antibody variable domains and compositions containing them
RU2112037C1 (ru) Гибридное моноклональное антитело, взаимодействующее с cd4-антигеном т-хелперных клеток человека, и способ его получения
US5840526A (en) Immunoglobulins devoid of light chains
RU2126046C1 (ru) Рекомбинантное антитело, полинуклеотид (варианты), способ получения тяжелых и/или легких цепей рекомбинантного антитела (варианты)
WO1995001997A1 (en) RECOMBINANT AND HUMANIZED IL-1β ANTIBODIES FOR TREATMENT OF IL-1 MEDIATED INFLAMMATORY DISORDERS IN MAN
US20070238133A1 (en) Immunoglobulins devoid of light chains
AU679440B2 (en) Antibodies for treatment and prevention of respiratory syncytial virus infection
KR20050049470A (ko) 항 수초 관련 당단백질 항체
EP1169352A1 (en) Erythropoietin receptor antibodies
RU2223785C2 (ru) Антитромботическое средство и гуманизированное моноклональное антитело против фактора фон виллебранда
WO1993012220A1 (en) RECOMBINANT AND CHIMERIC ANTIBODIES TO c-erbB-2
US7635476B2 (en) Anti-hepatitis a virus antibodies
WO1993020210A9 (en) Antibodies for treatment and prevention of respiratory syncytial virus infection
US6998124B1 (en) Erythropoietin receptor antibodies
JPH07501806A (ja) ヒトｐｄｇｆベータ・レセプタに対する阻害性免疫グロブリン・ポリペプチド
EP0937153B1 (en) Human monoclonal antibodies specific for hepatitis c virus (hcv) e2 antigen
AU673347B2 (en) Genetically engineered antibodies
WO1994028933A1 (en) Bispecific human monoclonal antibodies specific for human immunodeficiency virus
EP1272205B1 (en) Sialoadhesin factor-2 antibodies
JP2001510329A (ja) ヒトモノクローナル抗体
Chin et al. Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell
US6399062B1 (en) Murine monoclonal antibody protective against Plasmodium vivax malaria
WO1994003604A1 (en) PfEMP3 MALARIA ANTIGEN, ANALOGS, ANTIBODIES AND USES THEREOF
MXPA01001814A (en) Antithrombotic agent and humanized anti-von willebrand factor monoclonal antibody

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR NZ US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2143417

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 256232

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 1993921412

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 1995 397128

Country of ref document: US

Date of ref document: 19950420

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 1993921412

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1993921412

Country of ref document: EP