WO2021202463A1 - Anti-rsv antibodies - Google Patents

Anti-rsv antibodies Download PDF

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Publication number
WO2021202463A1
WO2021202463A1 PCT/US2021/024793 US2021024793W WO2021202463A1 WO 2021202463 A1 WO2021202463 A1 WO 2021202463A1 US 2021024793 W US2021024793 W US 2021024793W WO 2021202463 A1 WO2021202463 A1 WO 2021202463A1
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WIPO (PCT)
Prior art keywords
antibody
rsv
antigen
antibodies
seq
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PCT/US2021/024793
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French (fr)
Inventor
David A. Estell
Amy Deming Liu
Michael C. Miller
Jeffrey Wayne MUNOS
Jonathan David PETERSON
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Danisco Us Inc
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Publication of WO2021202463A1 publication Critical patent/WO2021202463A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • RSV Respiratory Syncytial Virus
  • Respiratory syncytial virus is the leading cause of severe respiratory illness in infants and young children and is the major cause of infantile bronchiolitis (Welliver (2003) J Pediatr 143 : S 112). An estimated 64 million cases of respiratory illness and 160,000 deaths worldwide are attributable to RSV induced disease. In the United States alone, tens of thousands of infant hospitalizations are due to infections by paramyxoviruses, such as RSV and parainfluenza virus (PIV) (Shay et al. (1999) JAMA 282:1440-1446).
  • PIV parainfluenza virus
  • RSV infection occurs most often in children and infants, especially in premature infants. Underlying health problems such as chronic lung disease or congenital heart disease can significantly increase the risk of serious illness. RSV infections also can cause serious illness in the elderly, individuals with chronic pulmonary disease and immunocompromised adults, such as bone marrow transplant recipients.
  • isolated polypeptides, antibodies or antigen-binding fragments thereof for the prophylaxis and treatment of Respiratory syncytial virus (RSV) infection and RSV-mediated diseases or conditions as well as methods for making and using the same.
  • RSV Respiratory syncytial virus
  • an isolated anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising: (a) a heavy chain variable region which has at least 90% sequence identity to
  • AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein optionally the antibody differs by at least one amino acid from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO:6.
  • an isolated anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising: (a) a light chain variable region which has at least 90% sequence identity to
  • AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G
  • the antibody differs by at least one amino acid from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO: 6; and (ii) wherein said antibody exhibits one or more improved properties comprising increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the light chain encoded by the amino acid sequence of SEQ ID NO: 6.
  • the antibody or functional fragment thereof further comprises: (a) a heavy chain variable region which has at least 90% sequence identity to QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RETI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, T, I, Y, W, C, or S; X 2 is T, N, or V; X3 is S, V, L, or W; X41S A, G, S, or T; X5 is P or Y; Xe is A or S; and X71S A or G; and/or (b) a heavy chain constant region comprising
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, wherein Xi is R, T, I,
  • X 2 is T, N, or V
  • X 3 is S, V, L, or W
  • X 4 is A, G, S, or T
  • X 5 is P or Y
  • Xe is A or S
  • X7 is A or G.
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:4, wherein Xi2 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X 2 ois A, S, V, orW; X 2i is L, S, or G; X 22 is G or A; X 23 i s T or Y ; and X 24 is F or C.
  • said functional fragment is selected from the group consisting of Fab, Fab , F(ab') 2 and Fv fragments.
  • said antibody is chimeric, humanized, or fully human. In some embodiments of any of the embodiments disclosed herein, said antibody competitively inhibits the binding of palivizumab to the surface of RSV.
  • nucleic acid encoding the heavy chain variable region of SEQ ID NO: 1 and/or the heavy chain constant region of SEQ ID NO:2.
  • nucleic acid encoding the light chain variable region of SEQ ID NO:4 and/or the light chain constant region of SEQ ID NO:5.
  • a vector comprising any of the nucleic acids disclosed herein.
  • a recombinant cell comprising any of the vectors disclosed herein.
  • the cell is a mammalian cell, a bacterial cell, or a fungal cell.
  • the fungal cell is T. reesei.
  • an anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising providing an isolated cell with a nucleic acid encoding said antibody or functional part thereof, wherein said antibody or functional part thereof comprises one or more of (a) a heavy chain variable region comprising
  • AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3 and/or a light chain encoded by the amino acid sequence of SEQ ID NO: 6.
  • the cell is a mammalian cell, a bacterial cell, or a fungal cell.
  • the fungal cell is T. reesei.
  • the antibody or functional fragment thereof exhibits one or more improved properties selected from the group consisting of increased manufacturability, thermostability, and protease resistance compared to an antibody that does not differ by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3 and/or a light chain encoded by the amino acid sequence of SEQ ID NO: 6.
  • a method for treating or preventing a respiratory syncytial virus (RSV) infection in an individual in need thereof comprising administering a therapeutically effective amount of the antibody or functional fragment thereof of any of the isolated anti-respiratory syncytial virus (RSV) antibodies or functional fragments thereof disclosed herein to the individual.
  • the antibody or functional fragment thereof is administered parenterally or intravenously.
  • the individual is a human.
  • the human is a preterm infant (under 35 weeks gestation) infant, infant with congenital heart defects (CHD), infant with bronchopulmonary dysplasia (BPD), and/or infant with congenital malformations of the airway.
  • the individual is less than four years old. In some embodiments of any of the embodiments disclosed herein, the individual is further administered oxygen therapy.
  • a pharmaceutical composition comprising any of the isolated anti-respiratory syncytial virus (RSV) antibodies or functional fragments thereof disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • RSV anti-respiratory syncytial virus
  • anti-RSV antibodies or antigen-binding fragments thereof that bind to and neutralize respiratory syncytial virus.
  • the anti-RSV antibodies provided herein are neutralizing antibodies that recognize one or more epitopes on the surface of RSV.
  • the antibodies provided herein can be used in prophylaxis therapies.
  • the antibodies provided herein also can be used as therapeutics.
  • the antibodies provided can be employed for the prevention and/or spread of pathogenic disease, including, but not limited to the inhibition of viral transmission between subjects, inhibition of establishment of viral infection in a host, and reduction of viral load in a subject.
  • the antibodies also can be employed for preventing, treating, and/or alleviating one or more symptoms of a RSV infection or for reducing the duration of a RSV infection. Accordingly, treatment of patients with antibodies provided herein can decrease the mortality and/or morbidity rate associated with RSV infection.
  • RSV persistence is associated with the generation of escape mutants that cannot be neutralized by an antibody.
  • the main challenges to development of therapeutic anti viral antibodies are the generation or identification of antibodies that have a neutralization epitope that is 1) conserved across various strains or serotypes and 2) is difficult for the evolving virus to generate escape mutants against.
  • Antibodies provided herein also exhibit one or more improved properties including one or more of increased manufacturability, thermostability, and/or protease resistance compared to existing antibodies in the prior art.
  • the provided anti-RSV antibodies in addition to prophylaxis therapy, also are useful for the treatment of RSV infection.
  • the anti-RSV antibodies provided herein have the ability to inhibit or reduce one or more activities of the virus, such as, for example, association of the virus with a target cell membrane, fusion of the virus with the target cell membrane and/or cell entry, production of new viral particles, including inhibition of viral replication, or cell to cell fusion of an infected cell with another cell (i.e. syncytia formation).
  • the provided anti- RSV antibodies also can be employed to increase the immune the response against a RSV infection.
  • antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin.
  • an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site).
  • Antibodies include antibody fragments, such as anti-RSV antibody fragments.
  • the term antibody thus, includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti- id) antibodies, or antigen-binding fragments of any of the above.
  • Fab fragments Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-
  • Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • immunoglobulin type e.g., IgG, IgM, IgD, IgE, IgA and IgY
  • any class e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass e.g., IgG2a and IgG2b.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody that binds antigen (e.g. one or more CDRs and/or one or more antibody combining sites) and thus retains the binding specificity, and at least a portion of the specific binding ability of the full-length antibody.
  • an antigen binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full- length antibodies, as well as synthetically, e.g.
  • An antibody fragment is included among antibodies.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers.
  • An antibody fragment generally contains at least or about 50 amino acids and typically at least or about 200 amino acids.
  • An antigen-binding fragment includes any antibody fragment that when inserted into an antibody framework (such as by replacing a corresponding region) results in an antibody that immunospecifically binds (i.e. exhibits Ka of at least or at least about 10 7 - 10 8 M 1 ) to the antigen.
  • a "therapeutic antibody” refers to any antibody or antigen-binding fragment thereof that is administered for treatment of an animal, including a human.
  • Such antibodies can be prepared by any known methods for the production of polypeptides, and hence, include, but are not limited to, recombinantly produced antibodies, synthetically produced antibodies, and therapeutic antibodies extracted from cells or tissues and other sources.
  • therapeutic antibodies can be heterogeneous in length or differ in post-translational modification, such as glycosylation (i.e. carbohydrate content). Heterogeneity of therapeutic antibodies also can differ depending on the source of the therapeutic antibodies.
  • reference to therapeutic antibodies refers to the heterogeneous population as produced or isolated. When a homogeneous preparation is intended, it will be so-stated. References to therapeutic antibodies herein are to their monomeric, dimeric or other multimeric forms, as appropriate.
  • neutralizing antibody is any antibody or antigen-binding fragment thereof that binds to a pathogen and interferes with the ability of the pathogen to infect a cell and/or cause disease in a subject.
  • neutralizing antibodies are neutralizing antibodies that bind to viruses, bacteria, and fungal pathogens.
  • the neutralizing antibodies provide herein bind to the surface of the pathogen.
  • a neutralizing antibody that binds to the virus typically binds to a protein on the surface of the virus.
  • the surface protein can be a capsid protein (e.g.
  • a capsid protein of a non-enveloped virus or a viral envelope protein (e.g., a viral envelope protein of an enveloped virus).
  • the protein is a glycoprotein.
  • the ability of the virus to inhibit virus infectivity can be measure for example, by an in vitro neutralization assay, such as, for example, a plaque reduction assay using Vero host cells.
  • a "conventional antibody” refers to an antibody that contains two heavy chains (which can be denoted H and H') and two light chains (which can be denoted L and L') and two antibody combining sites, where each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding capability (e.g . heavy chains include, but are not limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or any functional region of (e.g. light chains include, but are not limited to, VL chains and VL-CL chains).
  • a full-length antibody is an antibody having two full-length heavy chains (e.g. VH-CH 1 -CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and hinge regions, such as human antibodies produced naturally by antibody secreting B cells and antibodies with the same domains that are synthetically produced.
  • VH-CH 1 -CH2-CH3 or VH-CH1-CH2-CH3-CH4 two full-length light chains
  • VL-CL full-length light chains
  • the term "derivative” refers to a polypeptide that contains an amino acid sequence of an anti-RSV antibody or a fragment thereof which has been modified, for example, by the introduction of amino acid residue substitutions, deletions or additions, by the covalent attachment of any type of molecule to the polypeptide (e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein).
  • a derivative of an anti-RSV antibody or antigen-binding fragment thereof can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation. Further, a derivative of an anti-RSV antibody or antigen-binding fragment thereof can contain one or more non-classical amino acids. Typically, a polypeptide derivative possesses a similar or identical function as an anti-RSV antibody or antigen-binding fragment thereof provided herein (e.g., neutralization of RSV).
  • antibody fragments e.g., Fab, F(ab'), F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments
  • Such fragments can be derived by a variety of methods known in the art, including, but not limited to, enzymatic cleavage, chemical crosslinking, recombinant means or combinations thereof.
  • the derived antibody fragment shares the identical or substantially identical heavy chain variable region (VH) and light chain variable region (VL) of the parent antibody, such that the antibody fragment and the parent antibody bind the same epitope
  • a "parent antibody” or “source antibody” refers the to an antibody from which an antibody fragment (e.g., Fab, F(ab'), F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments) is derived.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants typically contain chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • a chimeric polypeptide refers to a polypeptide that contains portions from at least two different polypeptides or from two non-contiguous portions of a single polypeptide.
  • a chimeric polypeptide generally includes a sequence of amino acid residues from all or part of one polypeptide and a sequence of amino acids from all or part of another different polypeptide.
  • the two portions can be linked directly or indirectly and can be linked via peptide bonds, other covalent bonds or other non-covalent interactions of sufficient strength to maintain the integrity of a substantial portion of the chimeric polypeptide under equilibrium conditions and physiologic conditions, such as in isotonic pH 7 buffered saline.
  • chimeric polypeptides include those containing all or part of an anti- RSV antibody linked to another polypeptide, such as, for example, a multimerization domain, a heterologous immunoglobulin constant domain or framework region, or a diagnostic or therapeutic polypeptide.
  • a fusion protein is a polypeptide engineered to contain sequences of amino acids corresponding to two distinct polypeptides, which are joined together, such as by expressing the fusion protein from a vector containing two nucleic acids, encoding the two polypeptides, in close proximity, e.g., adjacent, to one another along the length of the vector.
  • a fusion protein provided herein refers to a polypeptide that contains a polypeptide having the amino acid sequence of an antibody or antigen-binding fragment thereof and a polypeptide or peptide having the amino acid sequence of a heterologous polypeptide or peptide, such as, for example, a diagnostic or therapeutic polypeptide.
  • a fusion protein refers to a chimeric protein containing two, or portions from two, or more proteins or peptides that are linked directly or indirectly via peptide bonds.
  • the two molecules can be adjacent in the construct or separated by a linker, or spacer polypeptide.
  • the spacer can encode a polypeptide that alters the properties of the polypeptide, such as solubility or intracellular trafficking.
  • linker or “spacer” peptide refers to short sequences of amino acids that join two polypeptide sequences (or nucleic acid encoding such an amino acid sequence).
  • “Peptide linker” refers to the short sequence of amino acids joining the two polypeptide sequences.
  • Exemplary of polypeptide linkers are linkers joining a peptide transduction domain to an antibody or linkers joining two antibody chains in a synthetic antibody fragment such as an scFv fragment. Linkers are well-known and any known linkers can be used in the provided methods.
  • Exemplary of polypeptide linkers are (Gly-Ser) amino acid sequences, with some Glu or Lys residues dispersed throughout to increase solubility. Other exemplary linkers are described herein; any of these and other known linkers can be used with the provided compositions and methods.
  • antibody hinge region refers to a polypeptide region that exists naturally in the heavy chain of the gamma, delta and alpha antibody isotypes, between the CHI and CH2 domains that has no homology with the other antibody domains. This region is rich in proline residues and gives the IgG, IgD and IgA antibodies flexibility, allowing the two "arms" (each containing one antibody combining site) of the Fab portion to be mobile, assuming various angles with respect to one another as they bind antigen. This flexibility allows the Fab arms to move in order to align the antibody combining sites to interact with epitopes on cell surfaces or other antigens.
  • the synthetically produced antibody fragments contain one or more hinge regions, for example, to promote stability via interactions between two antibody chains. Hinge regions are exemplary of dimerization domains.
  • humanized antibodies refer to antibodies that are modified to include "human” sequences of amino acids so that administration to a human does not provoke an immune response.
  • a humanized antibody typically contains complementarily determining regions (CDRs) derived from a non-human species immunoglobulin and the remainder of the antibody molecule derived mainly from a human immunoglobulin.
  • CDRs complementarily determining regions
  • Methods for preparation of such antibodies are known. For example, DNA encoding a monoclonal antibody can be altered by recombinant DNA techniques to encode an antibody in which the amino acid composition of the non-variable regions is based on human antibodies. Methods for identifying such regions are known, including computer programs, which are designed for identifying the variable and non-variable regions of immunoglobulins.
  • an “Ig domain” is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
  • VL variable region domain
  • CL constant region domain
  • CH constant region domain
  • variable domain or “variable region” is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity, and thus are responsible for antigen recognition. Each variable region contains CDRs that are part of the antigen-binding site domain and framework regions (FRs).
  • FRs framework regions
  • antigen-binding domain As used herein, "antigen-binding domain,” “antigen-binding site,” “antigen combining site” and “antibody combining site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with cognate antigen.
  • a native conventional full-length antibody molecule has two conventional antigen-binding sites, each containing portions of a heavy chain variable region and portions of a light chain variable region.
  • a conventional antigen-binding site contains the loops that connect the anti-parallel beta strands within the variable region domains.
  • the antigen combining sites can contain other portions of the variable region domains.
  • Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions also are called complementarity-determining regions (CDRs).
  • variable region domain contains three CDRs, named CDR1, CDR2 and CDR3.
  • the three CDRs are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide.
  • the CDRs are located within the loops that join the parallel strands of the beta sheets of the variable domain.
  • FRs framework regions
  • a "constant region” or “constant domain” is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved than that of the variable region domain.
  • each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, which include, CHI, CH2, CH3 and CH4.
  • CH heavy chain constant region
  • Full-length IgA, IgD and IgG isotypes contain CHI, CH2, CH3 and a hinge region, while IgE and IgM contain CHI, CH2, CH3 and CH4.
  • CHI and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms.
  • Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g., through interactions with various cells, biomolecules and tissues.
  • a functional region of an antibody is a portion of the antibody that contains at least a VH, VL, CH (e.g. CHI, CH2 or CH3), CL or hinge region domain of the antibody, or at least a functional region thereof.
  • telomere binding fragment As used herein, “specifically bind” or “immunospecifically bind” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of the antibody and the antigen.
  • the antigen can be an isolated antigen or presented in a virus.
  • an antibody that immunospecifically binds (or that specifically binds) to a virus antigen or virus is one that binds to the virus antigen (or to the antigen in the virus or to the virus) with an affinity constant Ka of about or 1 xlO 7 M-l or lx 108 M-l or greater (or a dissociation constant (Kd) of lx 10 7 M or 1 xlO 8 M or less).
  • Ka affinity constant
  • Kd dissociation constant
  • Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst.
  • ITC isothermal titration calorimetry
  • other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of anti-RSV antibodies).
  • Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).
  • An antibody that immunospecifically binds to a virus antigen can bind to other peptides, polypeptides, or proteins or viruses with equal or lower binding affinity.
  • an antibody or antigen-binding fragment thereof provided herein that binds immunospecifically to a RSV protein (or RSV virus) does not cross-react with other antigens or cross reacts with substantially (at least 10-100 fold) lower affinity for such antigens.
  • Antibodies or antigen-binding fragments that immunospecifically bind to a particular virus antigen e.g.
  • a RSV protein can be identified, for example, by immunoassays, such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELIS As), surface plasmon resonance, or other techniques known to those of skill in the art.
  • An antibody or antigen-binding fragment thereof that immunospecifically binds to an epitope on a RSV protein typically is one that binds to the epitope (presented in the protein or virus) with a higher binding affinity than to any cross-reactive epitope as determined using experimental techniques, such as, but not limited to, immunoassays, surface plasmon resonance, or other techniques known to those of skill in the art.
  • Immunospecific binding to an isolated RSV protein does not necessarily mean that the antibody will exhibit the same immunospecific binding and/or neutralization of the virus. Such measurements and properties are distinct.
  • the affinity for the antibody or antigen-binding fragments for virus or the antigen as presented in the virus can be determined.
  • the target such as the isolated protein or the virus, will be identified.
  • Fc or “Fc region” or “Fc domain” refers to a polypeptide containing the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgE, or the last three constant region immunoglobulin domains of IgE and IgM.
  • an Fc domain can include all or part of the flexible hinge N- terminal to these domains.
  • Fc can include the J chain.
  • Fc contains immunoglobulin domains Cy2 and Cy3, and optionally, all or part of the hinge between Cyl and Cy2.
  • Fc region can vary, but typically, include at least part of the hinge region.
  • Fc also includes any allelic or species variant or any variant or modified form, such as any variant or modified form that alters the binding to an FcR or alters an Fc-mediated effector function.
  • a "tag” or an “epitope tag” refers to a sequence of amino acids, typically added to the N- or C- terminus of a polypeptide, such as an antibody provided herein.
  • the inclusion of tags fused to a polypeptide can facilitate polypeptide purification and/or detection.
  • a tag or tag polypeptide refers to polypeptide that has enough residues to provide an epitope recognized by an antibody or can serve for detection or purification, yet is short enough such that it does not interfere with activity of chimeric polypeptide to which it is linked.
  • the tag polypeptide typically is sufficiently unique so an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked.
  • Suitable tag polypeptides generally have at least 5 or 6 amino acid residues and usually between about 8-50 amino acid residues, typically between 9-30 residues.
  • the tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture.
  • Such tags are well known and can be readily synthesized and designed.
  • Exemplary tag polypeptides include those used for affinity purification and include, His tags, the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5, (Field et al. (1988) Mol. Cell. Biol.
  • polypeptide refers to two or more amino acids covalently joined.
  • polypeptide and “protein” are used interchangeably herein.
  • peptide refers to a polypeptide that is from 2 to about or 40 amino acids in length.
  • amino acid is an organic compound containing an amino group and a carboxylic acid group.
  • a polypeptide contains two or more amino acids.
  • amino acids contained in the antibodies provided include the twenty naturally- occurring amino acids, non-natural amino acids, and amino acid analogs (e.g., amino acids wherein the a-carbon has a side chain).
  • amino acids which occur in the various amino acid sequences of polypeptides appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations.
  • the nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard single-letter designations used routinely in the art.
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are generally in the "L” isomeric form. Residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NFh refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residues are used throughout. All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus.
  • amino acid residue is defined to include natural, modified, non-natural and unusual amino acids.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NFh or to a carboxyl- terminal group such as COOH.
  • Naturally occurring amino acids refer to the 20 L-amino acids that occur in polypeptides.
  • an "activity" or a "functional activity" of a polypeptide, such as an antibody refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, ability to interact with a biomolecule, for example, through antigen-binding, DNA binding, ligand binding, or dimerization, enzymatic activity, for example, kinase activity or proteolytic activity.
  • activities include, but are not limited to, the ability to specifically bind a particular antigen, affinity of antigen-binding (e.g.
  • Activity can be assessed in vitro or in vivo using recognized assays, such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure on- or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry and binding assays (e.g., panning assays).
  • activities can be assessed by measuring binding affinities, avidities, and/or binding coefficients (e.g., for on-/off-rates), and other activities in vitro or by measuring various effects in vivo, such as immune effects, e.g. antigen clearance, penetration or localization of the antibody into tissues, protection from disease, e.g.
  • results of such assays that indicate that a polypeptide exhibits an activity can be correlated to activity of the polypeptide in vivo, in which in vivo activity can be referred to as therapeutic activity, or biological activity.
  • Activity of a modified polypeptide can be any level of percentage of activity of the unmodified polypeptide, including but not limited to, 1 % of the activity, 2 %, 3 %, 4 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 100 %, 200 %, 300 %, 400 %, 500 %, or more of activity compared to the unmodified polypeptide.
  • Assays to determine functionality or activity of modified (e.g. variant) antibodies are well known in the art.
  • “exhibits at least one or more improved properties” refers to the activity exhibited by a modified polypeptide, such as a variant polypeptide produced according to the provided methods, such as a modified, e.g. variant antibody or other therapeutic polypeptide (e.g. a modified anti-RSV antibody or antigen-binding fragment thereof), compared to the target or unmodified polypeptide, that does not contain the modification.
  • a modified, or variant, polypeptide that retains an activity of a target polypeptide can exhibit improved activity (for example one or more of improved manufacturability, thermostability, or protease resistance) or maintain the activity of the unmodified polypeptide.
  • a modified, or variant, polypeptide can retain an activity that is increased compared to an target or unmodified polypeptide. In some cases, a modified, or variant, polypeptide can retain an activity that is decreased compared to an unmodified or target polypeptide.
  • Activity of a modified, or variant, polypeptide can be any level of percentage of activity of the unmodified or target polypeptide, including but not limited to, 1 % of the activity, 2 %, 3 %, 4 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 100 %, 200 %, 300 %, 400 %, 500 %, or more activity compared to the unmodified or target polypeptide.
  • the change in activity is at least about 2 times, 3 times, 4 times, 5 times,
  • Assays for retention of an activity depend on the activity to be retained. Such assays can be performed in vitro or in vivo. Activity can be measured, for example, using assays known in the art and described in the Examples below for activities such as but not limited to ELISA and panning assays. Activities of a modified, or variant, polypeptide compared to an unmodified or target polypeptide also can be assessed in terms of an in vivo therapeutic or biological activity or result following administration of the polypeptide.
  • nucleic acid refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof.
  • PNA peptide nucleic acid
  • Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid).
  • a nucleotide analog or a "backbone" bond other than a phosphodiester bond for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid).
  • RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded nucleic acids.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • uracil base is uridine.
  • regulatory region of a nucleic acid molecule means a cis-acting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.
  • Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.
  • Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.
  • IRIS internal ribosome binding site
  • operably linked with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • nucleic acid encoding a leader peptide can be operably linked to nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide.
  • the nucleic acid encoding a first polypeptide is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed.
  • an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide.
  • a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.
  • synthetic with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.
  • production by recombinant means by using recombinant DNA methods” means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.
  • expression refers to the process by which polypeptides are produced by transcription and translation of polynucleotides.
  • the level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.
  • a "host cell” is a cell that is used in to receive, maintain, reproduce and amplify a vector.
  • a host cell also can be used to express the polypeptide encoded by the vector.
  • the nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.
  • the host cell is a genetic package, which can be induced to express the variant polypeptide on its surface.
  • the host cell is infected with the genetic package.
  • the host cells can be phage- display compatible host cells, which can be transformed with phage or phagemid vectors and accommodate the packaging of phage expressing fusion proteins containing the variant polypeptides.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation.
  • Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression/display of the polypeptide encoded by the nucleic acid.
  • the vectors typically remain episomal but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome.
  • vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
  • an "expression vector” includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • similarity between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. "Identity” refers to the extent to which the amino acid or nucleotide sequences are invariant.
  • Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).
  • polypeptide or nucleic acid molecule or region thereof contains or has "identity" or “homology” to another polypeptide or nucleic acid molecule or region
  • the two molecules and/or regions share greater than or equal to at or about 40 % sequence identity, and typically greater than or equal to at or about 50 % sequence identity, such as at least or about 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % sequence identity; the precise percentage of identity can be specified if necessary.
  • a nucleic acid molecule, or region thereof, that is identical or homologous to a second nucleic acid molecule or region can specifically hybridize to a nucleic acid molecule or region that is 100 % complementary to the second nucleic acid molecule or region. Identity alternatively can be compared between two theoretical nucleotide or amino acid sequences or between a nucleic acid or polypeptide molecule and a theoretical sequence.
  • Sequence "identity,” per se, has an art-recognized meaning and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, e.g.
  • identity is well known to skilled artisans (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).
  • Sequence identity compared along the full length of two polynucleotides or polypeptides refers to the percentage of identical nucleotide or amino acid residues along the full-length of the molecule. For example, if a polypeptide A has 100 amino acids and polypeptide B has 95 amino acids, which are identical to amino acids 1-95 of polypeptide A, then polypeptide B has 95 % identity when sequence identity is compared along the full length of a polypeptide A compared to full length of polypeptide B. Alternatively, sequence identity between polypeptide A and polypeptide B can be compared along a region, such as a 20 amino acid analogous region, of each polypeptide.
  • sequence identity for the regions is 100 %.
  • sequence identity can be compared along the length of a molecule, compared to a region of another molecule.
  • sequence identity between polypeptide A and polypeptide B can be compared along the same length polypeptide but with amino acid replacements, such as conservative amino acid replacements or non conservative amino acid replacements.
  • amino acid replacements such as conservative amino acid replacements or non conservative amino acid replacements.
  • nucleic acid or polypeptide molecules have nucleotide sequences that are at least or about 60 %, 70 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 % or 99 % "identical” can be determined using known computer algorithms such as the "FASTA" program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J. et al. (1984) Nucleic Acids Research 12(I):387), BLASTP, BLASTN, FASTA (Altschul, S.F.
  • Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al.
  • a "modification” is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively.
  • Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.
  • substitution refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule.
  • substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule.
  • Substitution mutations compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence.
  • the phase "having the same binding specificity" when used to describe an antibody in reference to another antibody means that the antibody specifically binds (immunospecifically binds or specifically binds to the virus) to all or a part of the same antigenic epitope as the reference antibody.
  • the epitope can be in the isolated protein, or in the protein in the virus.
  • the ability of two antibodies to bind to the same epitope can be determined by known assays in the art such as, for example, surface plasmon resonance assays and antibody competition assays.
  • antibodies that immunospecifically bind to the same epitope can compete for binding to the epitope, which can be measured, for example, by an in vitro binding competition assay (e.g.
  • a first antibody that immunospecifically binds to the same epitope as a second antibody can compete for binding to the epitope by about or 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 100 %, where the percentage competition is measured ability of the second antibody to displace binding of the first antibody to the epitope.
  • the antigen is incubated in the presence a predetermined limiting dilution of a labeled antibody (e.g., 50- 70% saturation concentration), and serial dilutions of an unlabeled competing antibody. Competition is determined by measuring the binding of the labeled antibody to the antigen for any decreases in binding in the presence of the competing antibody. Variations of such assays, including various labeling techniques and detection methods including, for example, radiometric, fluorescent, enzymatic and colorimetric detection, are known in the art.
  • the ability of a first antibody to bind to the same epitope as a second antibody also can be determined, for example, by virus neutralization assays using Monoclonal Antibody- Resistant Mutants (MARMs).
  • MAMs Monoclonal Antibody- Resistant Mutants
  • a second antibody that neutralizes the wild- type RSV but not the particular mutant RSV generally binds the same epitope on RSV as the first antibody.
  • a first anti-RSV antibody neutralizes wild-type RSV but not a particular mutant RSV a second antibody that neutralizes the wild-type RSV and the particular mutant RSV generally does not bind the same epitope on RSV as the first antibody.
  • disease or disorder refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms.
  • Diseases and disorders of interest herein are those involving RSV infection or those that increase the risk of a RSV infection.
  • infection and RSV infection refer to all stages of a RSV life cycle in a host (including, but not limited to the invasion by and replication of RSV in a cell or body tissue), as well as the pathological state resulting from the invasion by and replication of a RSV.
  • the invasion by and multiplication of a RSV includes, but is not limited to, the following steps: the docking of the RSV particle to a cell, fusion of a virus with a cell membrane, the introduction of viral genetic information into a cell, the expression of RSV proteins, the production of new RSV particles and the release of RSV particles from a cell.
  • a RSV infection can be an upper respiratory tract RSV infection (URI), a lower respiratory tract RSV infection (LRI), or a combination thereof.
  • the pathological state resulting from the invasion by and replication of a RSV is an acute RSV disease.
  • acute RSV disease refers to clinically significant disease in the lungs or lower respiratory tract as a result of a RSV infection, which can manifest as pneumonia and/or bronchiolitis, where such symptoms can include, for example, hypoxia, apnea, respiratory distress, rapid breathing, wheezing, and cyanosis.
  • Acute RSV disease requires an affected individual to obtain medical intervention, such as hospitalization, administration of oxygen, intubation and/or ventilation.
  • treating means that the subject's symptoms are partially or totally alleviated or remain static following treatment.
  • treatment encompasses prophylaxis, therapy and/or cure.
  • Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease.
  • Treatment also encompasses any pharmaceutical use of any antibody or antigen-binding fragment thereof provided or compositions provided herein.
  • prevention or prophylaxis, and grammatically equivalent forms thereof, refers to methods in which the risk of developing disease or condition is reduced.
  • a “pharmaceutically effective agent” includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.
  • a “therapeutic effect” means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition.
  • a “therapeutically effective amount” or a “therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
  • “therapeutic efficacy” refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the an agent, compound, material, or composition containing a compound has been administered.
  • a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection.
  • the full prophylactic effect does not necessarily occur by administration of one dose and can occur only after administration of a series of doses.
  • a prophylactically effective amount can be administered in one or more administrations.
  • the terms “immunotherapeutically” or “immunotherapy” in conjunction with antibodies provided denotes prophylactic as well as therapeutic administration.
  • the therapeutic antibodies provided can be administered to a subject at risk of contracting a virus infection (e.g. a RSV infection) in order to lessen the likelihood and/or severity of the disease or administered to subjects already evidencing active virus infection (e.g. a RSV infection).
  • amelioration of the symptoms of a particular disease or disorder by a treatment refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.
  • a “label” or “detectable moiety” is a detectable marker (e.g., a fluorescent molecule, chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be attached or linked directly or indirectly to a molecule (e.g., an anti -RSV antibody or antigen-binding fragment thereof provided herein) or associated therewith and can be detected in vivo and/or in vitro.
  • a detectable marker e.g., a fluorescent molecule, chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of
  • the detection method can be any method known in the art, including known in vivo and/or in vitro methods of detection (e.g., imaging by visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signal, X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET) scanning, single-photon emission computed tomography (SPECT)), fluorescence spectroscopy or absorption).
  • MR magnetic resonance
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • Indirect detection refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of an atom, molecule or composition that binds directly or indirectly to the detectable moiety (e.g., detection of a labeled secondary antibody or antigen-binding fragment thereof that binds to a primary antibody (e.g., an anti-RSV antibody or antigen-binding fragment thereof provided herein).
  • a physical phenomenon such as energy or particle emission or absorption
  • the term “individual” refers to an animal, including a mammal, such as a human being.
  • animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; pigs and other animals.
  • rodents such as mice and rats
  • fowl such as chickens
  • ruminants such as goats, cows, deer, sheep
  • pigs and other animals Non-human animals exclude humans as the contemplated animal.
  • a “human infant” refers to a human less than or about 24 months (e.g., less than or about 16 months, less than or about 12 months, less than or about 6 months, less than or about 3 months, less than or about 2 months, or less than or about 1 month of age).
  • the human infant is bom at more than 38 weeks of gestational age.
  • a “human infant bom prematurely” refers to a human bom at less than or about 40 weeks gestational age, typically, less than or about 38 weeks gestational age.
  • an “isolated” or “purified” polypeptide or protein e.g. an isolated antibody or antigen-binding fragment thereof
  • biologically-active portion thereof e.g. an isolated antigen-binding fragment
  • Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art.
  • a substantially chemically pure compound can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
  • a “cellular extract” or “lysate” refers to a preparation or fraction which is made from a lysed or disrupted cell.
  • “combination therapy” refers to administration of two or more different therapeutics, such as two or more different anti-RSV antibodies and/or anti- RSV antibodies and antigen-binding fragments thereof.
  • the different therapeutic agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.
  • composition that optionally contains additional exogenous enzymes means that the enzymes can be present or not present in the composition.
  • composition comprising the component(s) can further include other non-mandatory or optional component(s).
  • anti -RSV antibodies or antigen-binding fragments thereof that can be employed for therapeutic, prophylactic and diagnostic use.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be used, for example, for passive immunization of a subject against RSV or for treatment of a subject with a viral infection.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are used for prophylaxis, i. e.. the prevention of RSV infection.
  • the anti- RSV antibodies or antigen-binding fragments thereof provided herein are used as therapeutic antibodies, i.e., for treatment of a RSV viral infection.
  • the anti- RSV antibodies or antigen-binding fragments thereof provided herein are used for passive immunization of a subject against RSV.
  • the provided anti-RSV antibodies or antigen-binding fragments thereof also can be used for detection of a RSV infection or for monitoring RSV infection in vitro and in vivo.
  • Full-length antibodies contain multiple chains, domains and regions.
  • a full length conventional antibody contains two heavy chains and two light chains, each of which contains a plurality of immunoglobulin (Ig) domains.
  • An Ig domain is characterized by a structure called the Ig fold, which contains two beta-pleated sheets, each containing anti parallel beta strands connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond.
  • the Ig domains in the antibody chains are variable (V) and constant (C) region domains.
  • Each heavy chain is linked to a light chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. Linkage of the heavy chains is mediated by a flexible region of the heavy chain, known as the hinge region.
  • Each full-length conventional antibody light chain contains one variable region domain (VL) and one constant region domain (CL).
  • Each full-length conventional heavy chain contains one variable region domain (VH) and three or four constant region domains (CH) and, in some cases, hinge region.
  • nucleic acid sequences encoding the variable region domains differ among antibodies and confer antigen-specificity to a particular antibody.
  • the constant regions are encoded by sequences that are more conserved among antibodies. These domains confer functional properties to antibodies, for example, the ability to interact with cells of the immune system and serum proteins in order to cause clearance of infectious agents.
  • Different classes of antibodies for example IgM, IgD, IgG, IgE and IgA, have different constant regions, allowing them to serve distinct effector functions.
  • Each variable region domain contains three portions called complementarity determining regions (CDRs) or hypervariable (HV) regions, which are encoded by highly variable nucleic acid sequences.
  • the CDRs are located within the loops connecting the beta sheets of the variable region Ig domain.
  • CDR1 complementarity determining regions
  • HV hypervariable regions
  • CDR2 and CDR3 and three light chain CDRs make up a conventional antigen-binding site (antibody combining site) of the antibody, which physically interacts with cognate antigen and provides the specificity of the antibody.
  • a whole antibody contains two identical antibody combining sites, each made up of CDRs from one heavy and one light chain. Because they are contained within the loops connecting the beta strands, the three CDRs are non-contiguous along the linear amino acid sequence of the variable region. Upon folding of the antibody polypeptide, the CDR loops are in close proximity, making up the antigen combining site.
  • the beta sheets of the variable region domains form the framework regions (FRs), which contain more conserved sequences that are important for other properties of the antibody, for example, stability.
  • anti-RSV antibodies provided herein exhibit one or more properties that are advantageous or different from anti-RSV antibodies in the art (such as, for example, improved manufacturability, thermostability, and/or protease resistance).
  • isolated anti-respiratory syncytial virus (RSV) antibodies or functional fragments thereof having a heavy chain variable region which has at least about 90% (such as any of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to RSV respiratory syncytial virus
  • the antibody can differ by at least one amino acid (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids) from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO: 3. Additionally, the antibody can exhibit one or more improved properties including, but not limited to, increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3.
  • amino acid such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids
  • the antibody or functional fragment thereof can comprise or further comprise a light chain variable region which has at least 90% (such as any of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to DIQMX12 X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21LIYDTSK LASGVPSRFSGSGSX22X23E
  • X24TLTIS SLQPDDF ATYY CFQGS GYPFTF GGGTKLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X201S A, S, V, orW; X211S L, S, or G; X221S G or A; X231S T or Y ; and X24 is F or C; and/or a light chain constant region comprising AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:
  • the antibody exhibits increased or improved manufacturability compared to a parent antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and or the light chain encoded by the amino acid sequence of SEQ ID NO:6.
  • the antibody may display reduced aggregation-propensity, and/or increased productivity upon expression, relative to the parent antibody.
  • the ant-RSV antibody may display an increase in productivity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% relative to the parent antibody and/or a decrease in aggregation ( i.e .
  • the anti-RSV antibody can display a decrease in aggregation of up to 100% relative to the parent immunoglobulin (i.e. complete abolition of aggregation).
  • Improvements in manufacturability may result fully or partially from reduced aggregation-propensity relative to the parent immunoglobulin.
  • “Aggregation-propensity” relates to the tendency of the anti-RSV antibody to form insoluble aggregates after expression in a recombinant system.
  • Reductions in aggregation propensity reduce the proportion of molecules in the native state ensemble of the immunoglobulin which exist in an aggregated form (Carpenter et al, 2009 J Pharm Sci. April; 98(4): 1201-5). In other words, the proportion of molecules within the native state ensemble of the anti-RSV antibody which exist in an aggregated or insoluble form is lower than the proportion within the native state ensemble of the parent antibody.
  • An anti-RSV antibody disclosed herein may show less self-association or aggregation compared to a parent antibody either under native conditions or at increased temperature (e.g. 60° C.) (i.e. conditions under which the antigen binding site of an immunoglobulin does not unfold).
  • the anti-RSV antibody shows less self-association or aggregation than parent antibody under native conditions (e.g. conditions which do not lead to unfolding of the antibody).
  • Aggregation propensity as described herein is distinct from thermal refolding efficiency (TRE), which relates to the ability of a protein to correctly refold after thermal denaturation and is typically measured using circular dichroism (CD) (Tanha et al Protein Eng Des Sel. 2006 November; 19(11):503-9). Reductions in the aggregation propensity of an antibody as described herein may have little or no effect on the thermal refolding efficiency of the immunoglobulin. Thermal refolding efficiency is therefore independent of aggregation propensity and does not have a significant impact on the manufacturability of an antibody.
  • TRE thermal refolding efficiency
  • Aggregation may be measured by conventional methods. Suitable techniques include GP-HPLC, HPLC and AUC (Gabrielson J P et al J Pharm Sci 2007 96(2): 268-79), protein loss after filtration; turbidity; fluorescent dye binding (e.g. Nile Red, thioflavin T or 8- anilino-l-napthalenesulfonic acid; see for example Hawe, A. et al Pharmaceutical Research 200825 (7) 1487-99 or Demeule, B et al 2007 Int J Pharm 329: 37-45), field-flow fractionation (FFF; Demeule, B et al.
  • FFF field-flow fractionation
  • the variant immunoglobulin may show increased yields or titers compared to a parent immunoglobulin when expressed in a recombinant system, e.g. bacterial or mammalian cells.
  • Productivity may be measured using standard techniques, such as the Bradford assay, spectrophotometry and ELISA.
  • An anti-RSV antibody disclosed herein may also display one or more of improved purification yields; reduced formulation problems; reduced immunogenicity and increased bioavailability relative to the parent antibody. In some embodiments, improvements in manufacturability may result from both reduced aggregation-propensity and increased productivity relative to the parent antibody. In some embodiments, an anti-RSV antibody disclosed herein displays the same or substantially the same activity as the parent antibody (i.e. antigen binding activity).
  • the antibody exhibits increased or improved or enhanced thermostability compared to a parent antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and or the light chain encoded by the amino acid sequence of SEQ ID NO:6.
  • thermostabilized or “thermostability” refers to the quality of a protein or antibody to resist chemical or physical change as a result of increasing temperature.
  • alterations to the amino acid sequence of an antibody may be made to increase the thermostability of said antibody compared to the parent antibody. Thermostability may be determined by any known method in the field, including the measurement of the antibody melting temperature (TM).
  • thermostability include increases in the TM by greater than or equal to 0.1 °C to greater than or equal to 10.0 °C.
  • the antibody may display increased thermostability, relative to the parent antibody.
  • the anti-RSV antibody may display an increase in thermostability of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% relative to the parent antibody.
  • the antibody exhibits increased or improved or enhanced protease resistance compared to a parent antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and or the light chain encoded by the amino acid sequence of SEQ ID NO: 6.
  • protease resistance refers to the ability of a molecule comprised of peptide bonds, to resist hydrolytic cleavage of one or more of its peptide bonds in the presence of a proteolytic enzyme.
  • the resistance to proteolytic enzymes is a relative property and is compared to a molecule (such as a parent antibody) which is less able to withstand hydrolytic cleavage of one or more of its peptide bonds over a specified time period and under specified conditions, including the pH and or temperature at which the cleavage resistance is tested.
  • a molecule such as a parent antibody
  • proteolytic cleavage indicative that cleavage has occurred is the generation of smaller fragments (lower molecular weight) as compared to the molecular weight of the intact, non-cleaved parent molecule.
  • An anti-RSV antibody or a fragment thereof disclosed herein comprising a hinge, a CH2 domain and a CH3 domain is “protease resistant” or “resistant to proteolysis” or has “increased resistance to proteolysis” when more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a full length antibody (such as any of the anti-RSV antibodies disclosed herein) remains intact for a given period of time (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) when digested by a protease (such as, but not limited to, pepsin, matrix metalloprotease-3 (MMP-3), matrix metalloprotease-12 (MMP- 12), glutamyl endopeptidase V8 of Staphylococcus aureus (GluV8), or immunoglobulin degrading enzyme of Streptococcus pyogenes
  • anti-RSV antibodies or antigen-binding fragments thereof provided herein can be further modified. Modifications of an anti-RSV antibody or antigen-binding fragment can improve one or more properties of the antibody, including, but not limited to, decreasing the immunogenicity of the antibody or antigen-binding fragment, improving the half-life of the antibody or antigen-binding fragment, such as reducing the susceptibility to proteolysis and/or reducing susceptibility to oxidation, and altering or improving of the binding properties of the antibody or antigen-binding fragment thereof.
  • Exemplary modifications include, but are not limited to, modifications of the primary amino acid sequence of the anti- RSV antibody or antigen-binding fragment thereof and alteration of the post-translational modification of the anti-RSV antibody or antigen-binding fragment thereof.
  • Exemplary post- translational modifications include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with protecting/blocking group, proteolytic cleavage, linkage to a cellular ligand or other protein.
  • Other exemplary modifications include attachment of one or more heterologous peptides to the anti-RSV antibody or antigen-binding fragment to alter or improve one or more properties of the antibody or antigen-binding fragment thereof.
  • modified antibodies or antigen-binding fragments thereof can be assayed for binding to RSV by methods such as, but not limited to, ELISA, surface plasmon resonance (SPR), or through in vitro microneutralization assays.
  • Increasing the half-life of the anti- RSV antibodies or antigen-binding fragments thereof provided herein can increase the therapeutic effectiveness of the anti-RSV antibodies or antigen-binding fragments thereof and allow for less frequent administration of the antibodies or antigen-binding fragments thereof for prophylaxis and/or treatment, such as preventing or treating a RSV infection, preventing, treating, and/or alleviating of one or more symptoms of a RSV infection, or reducing the duration of a RSV infection.
  • Modification of the anti-RSV antibodies or antigen-binding fragments thereof produced herein can include one or more amino acid substitutions, deletions or additions, either from natural mutation or human manipulation from the parent antibody.
  • Methods for modification of polypeptides, such as antibodies, are known in the art and can be employed for the modification of any antibody or antigen-binding fragment thereof provided herein.
  • the pharmacokinetic properties of the anti-RSV antibodies or antigen binding fragments thereof provided herein can be enhanced through Fc modifications by techniques known to those skilled in the art.
  • Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide molecule encoding an antibody or an antigen-binding fragment provided herein in order to produce an polypeptide with one or more amino acid substitutions.
  • Exemplary techniques for introducing mutations include, but are not limited to, site-directed mutagenesis and PCR-mediated mutagenesis.
  • the anti-RSV antibodies and antigen-binding fragments thereof provided herein can be modified by the attachment of a heterologous peptide to facilitate purification.
  • a heterologous peptide Generally, such peptides are expressed as a fusion protein containing the antibody fused to the peptide at the C- or N-terminus of the antibody or antigen-binding fragment thereof.
  • Exemplary peptides commonly used for purification include, but are not limited to, hexa-histidine peptides, hemagglutinin (HA) peptides, and flag tag peptides (see e.g., Wilson et al.
  • the linker peptide contains a protease cleavage site which allows for removal of the purification peptide following purification by cleavage with a protease that specifically recognizes the protease cleavage site.
  • the anti-RSV antibodies and antigen-binding fragments thereof provided herein also can be modified by the attachment of a heterologous polypeptide that targets the antibody or antigen-binding fragment to a particular cell type (e.g., respiratory epithelial cells), either in vitro or in vivo.
  • a heterologous polypeptide that targets the antibody or antigen-binding fragment e.g., respiratory epithelial cells
  • an anti-RSV antibody or antigen-binding fragment thereof provided herein can be targeted to a particular cell type by fusing or conjugating the antibody or antigen-binding fragment thereof to an antibody specific for a particular cell surface receptor or other polypeptide that interacts with a specific cell receptor.
  • an anti-RSV antibody or antigen-binding fragment thereof provided herein can be targeted to a target cell surface and/or taken up by the target cell by fusing or conjugating the antibody or antigen-binding fragment thereof to a peptide that binds to cell surface glycoproteins, such as a protein transduction domain (e.g., a TAT peptide).
  • a protein transduction domain e.g., a TAT peptide.
  • Exemplary protein transduction domains include, but are not limited to, PTDs derived from proteins such as human immunodeficiency virus 1 (HIV-1) TAT (Ruben et al. (1989) J.
  • Virol. 63:1-8 e.g., SEQ ID NOS:326-337, such as for example, GRKKRRQRRR (TAT 48- 57) SEQ ID NO:330)
  • the herpes virus tegument protein VP22 Elliott and O'Hare (1997) Cell 88:223-233; e.g., SEQ ID NO:342
  • the homeotic protein of Drosophila melanogaster Antennapedia (Antp) protein Penetratin PTD; Derossi et al. (1996) J. Biol. Chem.
  • PTDs also include synthetic PTDs, such as, but not limited to, polyarginine peptides (Futaki et al. (2003) J. Mol.
  • the anti-RSV antibodies and antigen-binding fragments thereof provided herein can be modified by the attachment of diagnostic and/or therapeutic moiety to the antibody or antigen-binding fragment thereof.
  • the anti-RSV antibodies and antigen-binding fragments thereof provided herein can be modified by the covalent attachment of any type of molecule, such as a diagnostic or therapeutic molecule, to the antibody or antigen-binding fragment thereof such that covalent attachment does not prevent the antibody or antigen-binding fragment thereof from binding to its corresponding epitope.
  • an anti- RSV antibody or antigen-binding fragment thereof provided herein can be further modified by covalent attachment of a molecule such that the covalent attachment does not prevent the antibody or antigen-binding fragment thereof from binding to RSV.
  • the antibodies or antigen-binding fragments thereof can be recombinantly fused to a heterologous polypeptide at the N terminus or C terminus or chemically conjugated, including covalent and non-covalent conjugation, to a heterologous polypeptide or other composition.
  • the heterologous polypeptide or composition can be a diagnostic polypeptide or other diagnostic moiety or a therapeutic polypeptide or other therapeutic moiety.
  • diagnostic and therapeutic moieties include, but are not limited to, drugs, radionucleotides, toxins, fluorescent molecules (see, e.g. International PCT Publication Nos. WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.
  • Diagnostic polypeptides or diagnostic moieties can be used, for example, as labels for in vivo or in vitro detection.
  • Therapeutic polypeptides or therapeutic moieties can be used, for example, for therapy of a viral infection, such as RSV infection, or for treatment of one or more symptoms of a viral infection.
  • Additional fusion proteins of the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be generated through the techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”).
  • DNA shuffling can be employed to alter the activities of anti-RSV antibodies or antigen-binding fragments thereof provided herein, for example, to produce antibodies or antigen-binding fragments thereof with higher affinities and lower dissociation rates (see, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al. (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama (1998) Trends Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo and Blasco (1998) Biotechniques 24(2):308-13).
  • the provided anti-RSV antibodies or antigen-binding fragments thereof can also be attached to solid supports, which are useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the antibodies or antigen-binding fragments thereof provided herein can be further modified to reduce the immunogenicity in a subject, such as a human subject.
  • a subject such as a human subject.
  • one or more amino acids in the antibody or antigen-binding fragment thereof can be modified to alter potential epitopes for human T-cells in order to eliminate or reduce the immunogenicity of the antibody or antigen-binding fragment thereof when exposed to the immune system of the subject.
  • Exemplary modifications include substitutions, deletions and insertion of one or more amino acids, which eliminate or reduce the immunogenicity of the antibody or antigen-binding fragment thereof.
  • such modifications do not alter the binding specificity of the antibody or antigen-binding fragment thereof for its respective antigen.
  • Reducing the immunogenicity of the antibody or antigen binding fragment thereof can improve one or more properties of the antibody or antigen binding fragment thereof, such as, for example, improving the therapeutic efficacy of the antibody or antigen-binding fragment thereof and/or increasing the half-life of the antibody or antigen-binding fragment thereof in vivo.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be conjugated to polymer molecules such as high molecular weight polyethylene glycol (PEG) to increase half-life and/or improve their pharmacokinetic profiles. Conjugation can be carried out by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function (see, e.g., Deckert et al., Int. J. Cancer 87: 382-390, 2000; Knight et al., Platelets 15: 409-418, 2004; Leong et al., Cytokine 16: 106-119, 2001; and Yang et al., Protein Eng.
  • PEG polyethylene glycol
  • PEG can be attached to the antibodies or antigen binding fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or antigen-binding fragments or via epsilon-amino groups present on lysine residues.
  • Linear or branched polymer derivatization that results in minimal loss of biological activity can be used.
  • the degree of conjugation can be monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies.
  • Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.
  • PEG-derivatized antibodies or antigen-binding fragments thereof can be tested for binding activity to RSV antigens as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.
  • the anti-RSV antibodies and antibody fragments provided herein can be further modified by conjugation to a detectable moiety.
  • the detectable moieties can be detected directly or indirectly. Depending on the detectable moiety selected, the detectable moiety can be detected in vivo and/or in vitro.
  • the detectable moieties can be employed, for example, in diagnostic methods for detecting exposure to RSV or localization of RSV or binding assays for determining the binding affinity of the anti-RSV antibody or antigen binding fragment thereof for RSV.
  • the detectable moieties also can be employed in methods of preparation of the anti-RSV antibodies, such as, for example, purification of the antibody or antigen-binding fragment thereof.
  • detectable moieties are selected such that conjugation of the detectable moiety does not interfere with the binding of the antibody or antigen-binding fragment thereof to the target epitope.
  • the choice of the detectable moiety depends on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • One of skill in the art is familiar with labels and can identify a detectable label suitable for and compatible with the assay employed. Methods of labeling antibodies with detectable moieties are known in the art and include, for example, recombinant and chemical methods.
  • the detectable moiety can be any material having a detectable physical or chemical property.
  • detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied in the methods provided.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), in particular, gamma and positron emitting radioisotopes (e.g., 157 Gd, 55 Mn, 162 Dy, 52 Cr, and 56 Fe), metallic ions, enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), electron transfer agents (e.g., including metal binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), magnetic beads (e.g., DYNABEADSTM), and colorimetric labels such as colloidal gold or colored glass or plastic
  • the anti-RSV antibodies and antigen-binding fragments provided herein can be further modified by conjugation to a therapeutic moiety.
  • exemplary therapeutic moieties include, but are not limited to, a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive metal ion (e.g., alpha-emitters).
  • Exemplary cytotoxin or cytotoxic agents include, but are not limited to, any agent that is detrimental to cells, such as, but not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • any agent that is detrimental to cells such as, but not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, ten
  • Exemplary therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracy dines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), anti-mitotic agents (e.
  • the anti-RSV antibodies and antigen-binding fragments provided herein can be further modified by conjugation to a therapeutic moiety that is a therapeutic polypeptide.
  • a therapeutic polypeptide include, but are not limited to, a toxin, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; or an immunostimulatory agent, such as a cytokine, such as, but not limited to, an interferon (e.g., IFN-a, b, g, w), a lymphokine, a hematopoietic growth factor, such as, for example, GM-CSF (granulocyte macrophage colony stimulating factor), Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin- 4 (IL-4), Interleukin-7 (IL-7), Interleukin- 10 (IL-10), Interleukin- 12 (IL-12), Interleukin- 14 (IL-14),
  • compositions containing an anti-RSV antibody or antigen-binding fragment thereof provided herein can be used for therapeutic, prophylactic, and/or diagnostic applications.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be formulated with a pharmaceutical acceptable carrier or diluent.
  • a pharmaceutical acceptable carrier or diluent e.g., a pharmaceutical acceptable carrier or diluent.
  • such pharmaceutical compositions utilize components which will not significantly impair the biological properties of the antibody or antigen-binding fragment thereof, such as the binding of to its specific epitope (e.g. binding to an epitope on a RSV protein).
  • Each component is pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • the formulations can conveniently be presented in unit dosage form and can be prepared by methods well known in the art of pharmacy, including but not limited to, tablets, pills, powders, liquid solutions or suspensions (e.g., including injectable, ingestible and topical formulations (e.g., eye drops, gels or ointments), aerosols (e.g., nasal sprays), liposomes, suppositories, injectable and infusible solution and sustained release forms.
  • injectable, ingestible and topical formulations e.g., eye drops, gels or ointments
  • aerosols e.g., nasal sprays
  • liposomes e.g., suppositories
  • injectable and infusible solution and sustained release forms e.g., e., et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17
  • the therapeutic composition When administered systematically, the therapeutic composition is sterile, pyrogen-free, generally free of particulate matter, and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art.
  • parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, e.g., “Remington: The Science and Practice of Pharmacy (Formerly Remington's Pharmaceutical Sciences),” 19th ed., Mack Publishing Company, Easton, Pa. (1995).
  • compositions provided herein can be in various forms, e.g., in solid, semi-solid, liquid, powder, aqueous, or lyophilized form.
  • suitable pharmaceutical carriers include but are not limited to water, buffering agents, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, powders, among others.
  • compositions provided herein can contain other additives including, for example, antioxidants, preservatives, antimicrobial agents, analgesic agents, binders, disintegrants, coloring, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents, flavoring agents, emulsions, such as oil/water emulsions, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth
  • compositions suitable for use include compositions wherein one or more anti-RSV antibodies are contained in an amount effective to achieve their intended purpose. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Therapeutically effective dosages can be determined by using in vitro and in vivo methods as described herein. Accordingly, an anti-RSV antibody or antigen binding fragment thereof provided herein, when in a pharmaceutical preparation, can be present in unit dose forms for administration.
  • An anti-RSV antibody or antigen-binding fragment thereof provided 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 protein preparations and art-known lyophilization and reconstitution techniques can be employed.
  • An anti-RSV antibody or antigen-binding fragment thereof provided herein can be provided as a controlled release or sustained release composition.
  • Polymeric materials are known in the art for the formulation of pills and capsules which can achieve controlled or sustained release of the antibodies or antigen-binding fragments thereof provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983) I, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.
  • polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and poly orthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • sustained release formulation containing one or more anti-RSV antibodies or antigen-binding fragments provided herein.
  • the pharmaceutical composition contains an anti-RSV antibody or antigen-binding fragment thereof provided herein and one or more additional antibodies.
  • the one or more additional antibodies includes, but is not limited to, palivizumab (SYNAGIS®), and derivatives thereof, such as, but not limited to, motavizumab (NUMAX®), AFFF, P12f2, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, IX- 493L1, FRH3-3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, and A4B4-F52S (see U.S. Pat. Nos.
  • palivizumab SYNAGIS®
  • derivatives thereof such as, but not limited to, motavizumab (NUMAX®), AFFF, P12f2, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B
  • rsv6, rsvll, rsvl3, rsvl9, rsv21, rsv22, rsv23 see, e.g. U.S. Pat. Nos. 5,824,307, 6,685,942 and 6,818,216
  • ahuman anti-RSV antibody such as, but not limited to, rsv6, rsvll, rsvl3, rsvl9 (i.e. Fab 19), rsv21, rsv22, rsv23, RF-1, RF-2 (see, e.g. U.S. Pat. Nos.
  • a humanized antibody derived from an anti-RSV mouse monoclonal antibody such as, but not limited to, MAbs 1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112, 1269, 1269, 1243 (Beeler et al. (1989) J. Virology 63(7):2841-2950), MAM51 (Mufson et al. (1987) J. Clin. Microbiol. 25:1635-1539), MAbs 43-1 and 13-1 (Femie et al. (1982) Proc. Soc. Exp. Biol. Med.
  • compositions of anti-RSV antibodies or nucleic acids encoding anti- RSV antibodies, or a derivative or a biologically active portion thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for prophylaxis (i.e. vaccination, passive immunization) and/or treating the RSV- mediated disease or disorder, and a label that indicates that the antibody or nucleic acid molecule is to be used for vaccination and/or treating the disease or disorder.
  • the pharmaceutical compositions can be packaged in unit dosage forms contain an amount of the pharmaceutical composition for a single dose or multiple doses.
  • the packaged compositions can contain a lyophilized powder of the pharmaceutical compositions containing the anti- RSV antibodies or antigen-binding fragments thereof provided, which can be reconstituted (e.g. with water or saline) prior to administration.
  • the articles of manufacture provided herein contain packaging materials.
  • Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art.
  • Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g, jet or ultrasonic nebulizers) and other single breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • MDI pressurized metered dose inhalers
  • DPI dry powder inhalers
  • nebulizers e.g, jet or ultrasonic nebulizers
  • the pharmaceutical composition also can be incorporated in, applied to or coated on a barrier or other protective device that is used for contraception from infection.
  • kits can optionally include one or more components such as instructions for use, devices and additional reagents (e.g, sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods.
  • additional reagents e.g, sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein
  • components such as tubes, containers and syringes for practice of the methods.
  • kits can include the anti-RSV antibodies or antigen-binding fragments thereof provided herein, and can optionally include instructions for use, a device for administering the anti- RSV antibodies or antigen-binding fragments thereof to a subject, a device for detecting the anti-RSV antibodies or antigen-binding fragments thereof in a subject, a device for detecting the anti-RSV antibodies or antigen-binding fragments thereof in samples obtained from a subject, and a device for administering an additional therapeutic agent to a subject.
  • the kit can, optionally, include instructions. Instructions typically include a tangible expression describing the anti-RSV antibodies or antigen-binding fragments thereof and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, dosing regimens, and the proper administration method for administering the anti-RSV antibodies or antigen-binding fragments thereof. Instructions also can include guidance for monitoring the subject over the duration of the treatment time [0143] Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of the selected anti-RSV antibody or antigen-binding fragment thereof in a subject.
  • the anti-RSV antibody or antigen-binding fragment thereof is provided in a diagnostic kit for the detection of RSV in an isolated biological sample (e.g., a fluid sample, such as blood, sputum, lavage, lung intubation sample, saliva, urine or lymph obtained from a subject).
  • the diagnostic kit contains a panel of one or more anti-RSV antibodies or antigen-binding fragments thereof and/or one or more control antibodies (i.e. non-RSV binding antibodies), where one or more antibodies in the panel is an anti-RSV antibody or antigen-binding fragment provided herein.
  • Kits provided herein also can include a device for administering the anti-RSV antibodies or antigen-binding fragments thereof to a subject.
  • a device for administering the anti-RSV antibodies or antigen-binding fragments thereof to a subject Any of a variety of devices known in the art for administering medications to a subject can be included in the kits provided herein.
  • Exemplary devices include, but are not limited to, an inhaler (e.g., pressurized metered dose inhaler (MDI), dry powder inhaler (DPI), nebulizer (e.g., jet or ultrasonic nebulizers) and other single breath liquid system), a hypodermic needle, an intravenous needle, a catheter, and a liquid dispenser such as an eyedropper.
  • MDI pressurized metered dose inhaler
  • DPI dry powder inhaler
  • nebulizer e.g., jet or ultrasonic nebulizers
  • other single breath liquid system e.g.,
  • the device for administering the anti-RSV antibodies or antigen-binding fragments thereof of the kit will be compatible with the desired method of administration of the anti-RSV antibodies or antigen-binding fragments thereof.
  • an anti-RSV antibody or antigen-binding fragment thereof to be delivered by pulmonary administration can be included in a kit with or contained in an inhaler or a nebulizer.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be generated by any suitable method known in the art for the preparation of antibodies, including chemical synthesis and recombinant expression techniques.
  • Various combinations of host cells and vectors can be used to receive, maintain, reproduce and amplify nucleic acids (e.g. nucleic acids encoding antibodies such as the anti-RSV antibodies or antigen- binding fragments thereof provided), and to express polypeptides encoded by the nucleic acids.
  • nucleic acids e.g. nucleic acids encoding antibodies such as the anti-RSV antibodies or antigen- binding fragments thereof provided
  • polypeptides encoded by the nucleic acids e.g. nucleic acids encoding antibodies such as the anti-RSV antibodies or antigen- binding fragments thereof provided
  • the choice of host cell and vector depends on whether amplification, polypeptide expression, and/or display on a genetic package, such as a phage, is desired. Methods for transforming host cells are
  • Any known transformation method e.g., transformation, transfection, infection, electroporation and sonoporation
  • transformation, transfection, infection, electroporation and sonoporation can be used to transform the host cell with nucleic acids.
  • Procedures for the production of antibodies such as monoclonal antibodies and antibody fragments, such as, but not limited to, Fab fragments and single chain antibodies are well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, but not limited to, the use of hybridoma, recombinant expression, phage display technologies or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et ak, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, Monoclonal Antibodies and T-Cell Hybridomas 5630681 (Elsevier N.Y. 1981).
  • Polypeptides such as any set forth herein, including the anti-RSV antibodies or antigen-binding fragments thereof provided herein, can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired polypeptides can be expressed in any organism suitable to produce the required amounts and forms of the proteins, such as for example, needed for analysis, administration and treatment.
  • Expression hosts include prokaryotic and eukaryotic organisms such as E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals (e.g., rabbits, mice, rats, and livestock, such as, but not limited to, goats, sheep, and cattle), including production in serum, milk and eggs.
  • Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.
  • isolated nucleic acid molecules encoding an anti-RSV antibody or antigen-binding fragment thereof provided herein.
  • the isolated nucleic acid molecule provided encodes an antibody or antigen-binding fragment having an amino acid sequence set forth in SEQ ID NOs:l-2 and/or 4-5.
  • Nucleic acid molecules encoding the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be prepared using well-known recombinant techniques for manipulation of nucleic acid molecules (see, e.g., techniques described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology, John Wiley & Sons, NY).
  • methods such as, but not limited to, recombinant DNA techniques, site directed mutagenesis, and polymerase chain reaction (PCR) can be used to generate modified antibodies or antigen-binding fragments thereof having a different amino acid sequence, for example, to create amino acid substitutions, deletions, and/or insertions.
  • PCR polymerase chain reaction
  • one or more of the CDRs of an anti-RSV antibody or antigen binding fragment thereof provided herein is inserted within framework regions using routine recombinant DNA techniques.
  • the framework regions can be selected from naturally occurring or consensus framework regions, including human framework regions (see, e.g., Chothia et al. (1998) J. Mol. Biol. 278: 457-479 for exemplary framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody or antigen-binding fragment thereof that maintains the antigen binding specificity of the parent anti-RSV antibody or antigen-binding fragment thereof.
  • Alterations to the polynucleotide can be made to improve one or more properties of the encoded antibody or antigen-binding fragment thereof and within the skill of the art.
  • one or more modifications of the polynucleotide can be made to produce amino acid substitutions within the framework regions, which, for example, improve binding of the antibody or antigen-binding fragment thereof to its antigen.
  • such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • vectors that contain nucleic acid or nucleic acids (encoding the heavy and light chain) encoding the anti-RSV antibodies or antigen-binding fragments thereof.
  • nucleic acid encoding the heavy chain of an antibody is cloned into a vector and the nucleic acid encoding the light chain of an antibody is cloned into the vector.
  • the genes can be cloned into a single vector for dual expression thereof, or into separate vectors. If desired, the vectors also can contain further sequences encoding additional constant region(s) or hinge regions to generate other antibody forms.
  • expression vectors are available and known to those of skill in the art and can be used for expression of polypeptides.
  • the choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan.
  • expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals.
  • Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells.
  • an origin of replication can be used to amplify the copy number of the vector in the cells.
  • Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g. a hexa-his tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association.
  • an epitope tag such as for localization, e.g. a hexa-his tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association.
  • Expression of the antibodies or antigen-binding fragments thereof can be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan.
  • Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bemoist and Chambon, (1981) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., (1981) Proc. Natl. Acad. Sci.
  • promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., (1984) Cell 38:639-646; Omitz et al., (1986) Cold Spring Harbor Symp. Quant.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the antibody, or portion thereof, in host cells.
  • a typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the antibody chain and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers.
  • the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.
  • Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter.
  • vectors are provided that contain a sequence of nucleotides that encodes the heavy chain and/or light chain variable region of an anti-RSV antibody.
  • vectors provided herein contain a sequence of nucleotides that encodes the constant region of an antibody operably linked to the nucleic acid sequence encoding the variable region of the antibody.
  • the vector can include the sequence for one or all of a CHI, CH2, hinge, CH3 or CH4 and/or CL.
  • the vector contains the sequence for a CHI or CL (kappa or lambda light chains).
  • the sequences of constant regions or hinge regions are known to one of skill in the art (see e.g. U.S. Published Application No. 20080248028) and described herein.
  • any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding an antibody or antigen-binding fragment thereof provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.
  • Exemplary plasmid vectors useful to produce the antibodies or antigen-binding fragments provided herein contain a strong promoter, such as the HCMV immediate early enhancer/promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 polyA signal.
  • the plasmid can be multi cistronic to enable expression of the full-length heavy and light chains of the antibody, a single chain Fv fragment or other immunoglobulin fragments.
  • Nucleic acids encoding the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be expressed in a suitable host.
  • Cells containing the vectors and nucleic acids encoding the anti-RSV antibodies or antigen-binding fragments thereof provided herein are provided.
  • any cell type that can be engineered to express heterologous DNA and has a secretory pathway is suitable.
  • Expression hosts include prokaryotic and eukaryotic organisms, such as bacterial cells ( e.g . E. coli ), yeast cells, fungal cells, Archae, plant cells, insect cells and animal cells including human cells. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins.
  • the choice of expression host is often related to the choice of vector and transcription and translation elements used.
  • the choice of expression host is often, but not always, dependent on the choice of precursor sequence utilized.
  • many heterologous signal sequences can only be expressed in a host cell of the same species (i.e., an insect cell signal sequence is optimally expressed in an insect cell).
  • other signal sequences can be used in heterologous hosts such as, for example, the human serum albumin (hHSA) signal sequence which works well in yeast, insect, or mammalian host cells and the tissue plasminogen activator pre/pro sequence which has been demonstrated to be functional in insect and mammalian cells (Tan et ak,
  • Expression in eukaryotic hosts can include expression in yeasts such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as Drosophila cells and lepidopteran cells, plants and plant cells such as tobacco, com, rice, algae, and lemna, or fungal cells such as T. reesei.
  • Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells.
  • Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs.
  • Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated.
  • transformation, transfection, infection, electroporation and sonoporation so that many copies of the gene sequence are generated.
  • standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large quantity of antibody chains, which is then purified using standard techniques (see e.g., Colley et al.
  • any of the well-known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors (e.g., baculovirus, vaccinia virus, adenovirus and other viruses), and any other the other well known methods for introducing cloned genomic DNA, cDNA, plasmid DNA, cosmid DNA, synthetic DNA or other foreign genetic material into a host cell.
  • viral vectors e.g., baculovirus, vaccinia virus, adenovirus and other viruses
  • host cells are transfected with a first vector encoding at least a VH chain or heavy chain of an anti-RSV antibody and a second vector encoding at least a VL chain or light chain of the anti-RSV antibody.
  • host cells also can be transfected with a single vector encoding both the heavy and light chain or portion thereof.
  • nucleic acid encoding the heavy chain of an antibody is ligated into a first expression vector and nucleic acid encoding the light chain of an antibody is ligated into a second expression vector.
  • the expression vectors can be the same or different, although generally they are sufficiently compatible to allow comparable expression of proteins (heavy and light chain) therefrom.
  • the first and second expression vectors are generally co transfected into host cells, typically at a 1:1 ratio. Exemplary of vectors include, but are not limited to, p01HC and pKLC (Tiller et al. (2008) Journal of Immunological Methods, 329:112-24).
  • Other expression vectors include the L chain expression vector pAG4622 and the heavy chain expression vector pAH4604 (Coloma et al. (1992) J. Immunol. Methods, 152:89-104).
  • the pAG4622 vector contains the genomic sequence encoding the C- region domain of the human K L chain and the gpt selectable marker.
  • the pAH4604 vectors contain the hisD selectable marker and sequences encoding the human H chain g ⁇ C-region domain.
  • the heavy and light chain can be cloned into a single vector that has expression cassettes for both the heavy and light chain.
  • genes encoding the heavy and light chains can be cloned into the mammalian expression vector pTT5 (NRC Biotechnology Research).
  • genes encoding the heavy and light chains, or portions thereof, can be cloned into pCALM (SEQ ID NO: 102).
  • sequences encoding the VH-CHl-hinge-CH2-Cn3 can be cloned into a first expression vector and sequences encoding the VL-CL domains can be cloned into a second expression vector.
  • sequences encoding the VH- CH1 can be cloned into a first expression vector and sequences encoding the VL-CL domains can be cloned into a second expression vector.
  • a heavy chain pairs with a light chain and a Fab monomer is generated.
  • Expression can be in any cell expression system known to one of skill in the art.
  • Exemplary cells for expression include, but are not limited to, 293FS cells, HEK293-6E cells or CHO cells. Other expression vectors and host cells are described below.
  • antibody heavy and light chains pair by disulfide bond to form a full-length antibody or fragments thereof.
  • polypeptides including the anti-RSV antibodies or antigen-binding fragments thereof provided herein, from host cells will depend on the chosen host cells and expression systems.
  • proteins generally are purified from the culture media after removing the cells.
  • cells can be lysed and the proteins purified from the extract.
  • polypeptides are isolated from the host cells by centrifugation and cell lysis (e.g. by repeated freeze-thaw in a dry ice/ethanol bath), followed by centrifugation and retention of the supernatant containing the polypeptides.
  • transgenic organisms such as transgenic plants and animals
  • tissues or organs can be used as starting material to make a lysed cell extract.
  • transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary further the proteins can be extracted and further purified using standard methods in the art.
  • Proteins such as the anti-RSV antibodies or antigen-binding fragments thereof provided herein, can be purified, for example, from lysed cell extracts, using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation and ionic exchange chromatography, such as anion exchange. Affinity purification techniques also can be utilized to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind proteases can be used in affinity purification.
  • Expression constructs also can be engineered to add an affinity tag to a protein such as a myc epitope, GST fusion or His6 and affinity purified with myc antibody, glutathione resin and Ni- resin, respectively. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques.
  • antibodies and portions thereof are purified by any procedure known to one of skill in the art.
  • the antibodies can be purified to substantial purity using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography or column chromatography.
  • antibodies can be purified by column chromatography.
  • Exemplary of a method to purify antibodies is by using column chromatography, wherein a solid support column material is linked to Protein G, a cell surface-associated protein from Streptococcus, that binds immunoglobulins with high affinity.
  • the antibodies can be purified to 60%, 70%, 80% purity and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity.
  • Purity can be assessed by standard methods such as by SDS-PAGE and coomassie staining.
  • the isolated polypeptides then can be analyzed, for example, by separation on a gel (e.g . SDS-Page gel), size fractionation (e.g. separation on a SephacrylTM S-200 HiPrepTM 16x60 size exclusion column (Amersham from GE Healthcare Life Sciences, Piscataway, N.J.).
  • Isolated polypeptides also can be analyzed in binding assays, typically binding assays using a binding partner bound to a solid support, for example, to a plate (e.g. ELISA-based binding assays) or a bead, to determine their ability to bind desired binding partners.
  • binding assays described in the sections below which are used to assess binding of precipitated phage displaying the polypeptides, also can be used to assess polypeptides isolated directly from host cell lysates.
  • binding assays can be carried out to determine whether antibody polypeptides bind to one or more antigens, for example, by coating the antigen on a solid support, such as a well of an assay plate and incubating the isolated polypeptides on the solid support, followed by washing and detection with secondary reagents, e.g. enzyme-labeled antibodies and substrates.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be characterized in a variety of ways well-known to one of skill in the art.
  • the anti- RSV antibodies or antigen-binding fragments thereof provided herein can be assayed for the ability to immunospecifically bind to a protein of human Respiratory Syncytial Virus (RSV).
  • RSV Respiratory Syncytial Virus
  • Such assays can be performed, for example, in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), on beads (Lam (1991) Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), on bacteria (U.S. Pat. No.
  • Antibodies or antigen-binding fragments thereof that have been identified to immunospecifically bind to a RSV antigen or a fragment thereof also can be assayed for their specificity and affinity for a RSV antigen.
  • the binding specificity, or epitope can be determined, for example, by competition assays with other anti- RSV antibodies and/or virus neutralization assays using Monoclonal Antibody-Resistant Mutants (MARMs).
  • MAMs Monoclonal Antibody-Resistant Mutants
  • in vitro assays and in vivo animal models using the anti- RSV antibodies or antigen-binding fragments thereof provided herein can be employed for measuring the level of RSV neutralization effected by contact or administration of the anti- RSV antibodies or antigen-binding fragments thereof.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be assessed for their ability to bind a selected target (e.g., RSV virus) and the specificity for such targets by any method known to one of skill in the art. Exemplary assays are provided in the Examples and described herein below. Binding assays can be performed in solution, suspension or on a solid support. For example, target antigens can be immobilized to a solid support (e.g. a carbon or plastic surface, a tissue culture dish or chip) and contacted with antibody or antigen-binding fragment thereof. Unbound antibody or target protein can be washed away and bound complexes can then be detected.
  • a solid support e.g. a carbon or plastic surface, a tissue culture dish or chip
  • Binding assays can be performed under conditions to reduce nonspecific binding, such as by using a high ionic strength buffer (e.g., 0.3-0.4 M NaCl) with nonionic detergent (e.g. 0.1% TRITON X®-100 or TWEEN®
  • a high ionic strength buffer e.g., 0.3-0.4 M NaCl
  • nonionic detergent e.g. 0.1% TRITON X®-100 or TWEEN®
  • Binding affinities can be determined using Scatchard analysis (Munson et ak, (1980) Anal. Biochem., 107:220), surface plasmon resonance, isothermal calorimetry, or other methods known to one of skill in the art.
  • immunoassays which can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as, but not limited to, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), Meso Scale Discovery (MSD, Gaithersburg, Md.), “sandwich” immunoassays, immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays.
  • competitive and non-competitive assay systems using techniques such as, but not limited to, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), Meso Scale Discovery (MSD, Gaithersburg, Md.), “sandwich”
  • Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety).
  • Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., (1986) Amer. Clin. Prod. Rev. 5:34-41). Exemplary immunoassays not intended by way of limitation are described briefly below.
  • Immunoprecipitation protocols generally involve lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or TRITON® X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody or antigen-binding fragment thereof of interest to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or
  • the ability of the antibody or antigen-binding fragment thereof of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art is knowledgeable as to the parameters that can be modified to increase the binding of the antibody or antigen-binding fragment thereof to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally involves preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody or antigen-binding fragment thereof (i.e., the antibody or antigen-binding fragment thereof of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti -human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I
  • ELISAs involve preparing antigen, coating the well of a 96-well microtiter plate with the antigen, adding the antibody or antigen-binding fragment thereof of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • the antibody can be coated to the well.
  • a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
  • the binding affinity of an antibody or antigen-binding fragment thereof to an antigen and the off-rate of an antibody-antigen interaction can be determined, for example, by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) with the antibody or antigen-binding fragment thereof of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody or antigen-binding fragment thereof bound to the labeled antigen.
  • labeled antigen e.g., 3H or 1251
  • the affinity of an anti-RSV antibody or antigen-binding fragment thereof provided herein for a RSV antigen and the binding off-rates can be determined from the data by Scatchard plot analysis.
  • Radioimmunoassays competition with a second antibody can also be determined using radioimmunoassays.
  • a RSV antigen is incubated with an anti-RSV antibody or antigen-binding fragment thereof provided herein conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
  • a labeled compound e.g., 3H or 1251
  • surface plasmon resonance e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335) kinetic analysis can be used to determine the binding on and off rates of antibodies or antigen-binding fragments thereof to a RSV antigen.
  • Surface plasmon resonance kinetic analysis involves analyzing the binding and dissociation of a RSV antigen from chips with immobilized antibodies or fragments thereof on their surface.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be assayed for their ability to inhibit the binding of RSV to its host cell receptor using techniques known to those of skill in the art. For example, cells expressing the receptor for RSV can be contacted with RSV in the presence or absence of an antibody or antigen binding fragment thereof and the ability of the antibody or fragment thereof to inhibit RSV's binding can measured by, for example, flow cytometry or a scintillation assay.
  • RSV e.g., a RSV antigen such as F glycoprotein or G glycoprotein
  • the antibody or antibody fragment can be labeled with a detectable compound such as a radioactive label (e.g., 32P, 35S, and 1251) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between RSV and its host cell receptor.
  • a detectable compound such as a radioactive label (e.g., 32P, 35S, and 1251) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between RSV and its host cell receptor.
  • RSV or a RSV antigen such as F glycoprotein can be contacted with an antibody or fragment thereof and the ability of the antibody or antibody fragment to inhibit RSV or the RSV antigen from binding to its host cell receptor can be determined.
  • the antibody or the antigen-binding fragment is immobilized on a solid support and RSV or a RSV antigen is labeled with a detectable compound.
  • RSV or a RSV antigen is immobilized on a solid support and the antibody or fragment thereof is labeled with a detectable compound.
  • the RSV or RSV antigen can be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate.
  • a RSV antigen can be a fusion protein comprising the RSV antigen and a domain such as glutathionine-5 -transferase.
  • a RSV antigen can be biotinylated using techniques well known to those of skill in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).
  • the binding specificity, or epitope, of the anti-RSV antibodies or antigen binding fragments thereof provided herein can be determined by any assay known to one of skill in the art, including, but not limited to surface plasmon resonance assays, competition assays and virus neutralization assays using Monoclonal Antibody-Resistant Mutants (MARMs).
  • the epitope can be in the isolated protein, or in the protein in the virus.
  • the ability of two antibodies to bind to the same epitope can be determined by known assays in the art such as, for example, surface plasmon resonance assays and antibody competition assays.
  • antibodies that immunospecifically bind to the same epitope can compete for binding to the epitope, which can be measured, for example, by an in vitro binding competition assay (e.g. competition ELISA), using techniques known the art.
  • a first antibody that immunospecifically binds to the same epitope as a second antibody can compete for binding to the epitope by about or 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, where the percentage competition is measured ability of the second antibody to displace binding of the first antibody to the epitope.
  • the antigen is incubated in the presence a predetermined limiting dilution of a labeled antibody (e.g., 50-70% saturation concentration), and serial dilutions of an unlabeled competing antibody. Competition is determined by measuring the binding of the labeled antibody to the antigen for any decreases in binding in the presence of the competing antibody. Variations of such assays, including various labeling techniques and detection methods including, for example, radiometric, fluorescent, enzymatic and colorimetric detection, are known in the art.
  • the ability of a first antibody to bind to the same epitope as a second antibody also can be determined, for example, by virus neutralization assays using Monoclonal Antibody -Resistant Mutants.
  • a MARM is a mutant respiratory syncytial virus (RSV) that not neutralized by a monoclonal antibody that neutralizes the wildtype RSV virus, i. e.. a MARM is an RSV escape mutant.
  • MARMs are generated by culturing wildtype RSV in the presence of a monoclonal antibody for successive rounds of viral replication in the presence of the antibody such that after each successive round of virus replication, cytopathic effects (CPE) are observed in the presence of increasing concentrations of antibodies until a mutant virus results that is not neutralized by the antibody. If a first antibody can neutralize a MARM generated against a second antibody, one can conclude that the antibodies specifically bind to or interact with different epitopes.
  • CPE cytopathic effects
  • a second antibody that neutralizes the wild-type RSV but not the particular mutant RSV generally binds the same epitope on RSV as the first antibody.
  • a first anti-RSV antibody neutralizes wild-type RSV but not a particular mutant RSV
  • a second antibody that neutralizes the wild-type RSV and the particular mutant RSV generally does not bind the same epitope on RSV as the first antibody.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be analyzed by any suitable method known in the art for the detection of viral neutralization.
  • Methods for detection of viral neutralization include, but are not limited to, plaque assays and assays for inhibition of syncytium formation.
  • Such assays can be employed to assess, for example, inhibition of viral attachment, viral entry and cell-to-cell spread of the virus (see, e.g. Burioni et al., (1994 ) Proc. Natl. Acad. Sci. U.S.A. 91:355-359; Sanna et al.
  • Standard plaque assays include, for example, plaque reduction assays, plaque size reduction assays, neutralization assays and neutralization kinetic assays. These assays measure the formation of viral plaques (i.e. areas of lysed cells) following infection of target cell monolayers by a virus.
  • Exemplary target cell lines that can be used in plaque reduction assays include, but are not limited to, Vero cells, MRC-5 cells, RC-37 cells, BHK-21/C13 cells and HEp-2 cells.
  • Vero cells MRC-5 cells
  • RC-37 cells i.e. areas of lysed cells
  • BHK-21/C13 cells HEp-2 cells.
  • One of skill in the art can identify appropriate target cell lines for use in a plaque assay.
  • Selection of an appropriate cell line for a plaque assay can depend on known factors, such as, for example, cell infectivity and the ability of the virus to propagate in and lyse the target cell. Examples 6 and 9 exemplify in vitro neutralization assays.
  • Plaque reduction assays can be used to measure the ability of the anti-RSV antibody or antigen-binding fragment thereof to effect viral neutralization in solution.
  • the antibody or antigen-binding fragment thereof and the virus are pre-incubated prior to the addition of target cells.
  • Target cells are then infected with the antibody/virus mixture and a plaque assay is performed following a predetermined infection period.
  • One of skill in the art can determine the incubation times required based on known examples in the art.
  • a reduction in the number of virus plaques produced following infection of the target cells indicates the ability of the antibody or antigen-binding fragment thereof to prevent binding of the virus to the target cells independent of antibody or antigen-binding fragment thereof attachment to the target cell and/or antibody, or antigen-binding fragment thereof, internalization.
  • Plaque size reduction assays can be used to measure the ability of the anti- RSV antibody or antigen-binding fragment thereof to inhibit of viral cell-to-cell spread.
  • the target cells are first infected with the virus for a predetermined infection period and then the antibody or antigen-binding fragment thereof is added to the infected cell.
  • One of skill in the art can determine the incubation times required based on known examples in the art.
  • a reduction in the size (i.e. diameter) of the virus plaques indicates that the antibody or antigen-binding fragment thereof is capable of preventing viral cell-to-cell spread.
  • Virus neutralization assays can be used to measure the ability of the anti- RSV antibody or antigen-binding fragment thereof to effect viral neutralization at the target cell surface by association of the antibody or antigen-binding fragment thereof with the target cell prior to virus exposure.
  • the antibody or antigen binding fragment thereof and target cells are pre-incubated for a predetermined period of time to allow for binding of the antibody or antigen-binding fragment thereof to the targeted cell. Following the pre-incubation period, the unbound antibody is removed and the target cells are infected with the virus.
  • a reduction in the number of plaques in this assay indicates the ability of the antibody or antigen-binding fragment thereof to prevent viral infection dependent upon attachment to the target cell and/or internalization of the antibody or antigen binding fragment thereof.
  • This assay also can be used to measure neutralization kinetics by varying antibody or antigen-binding fragment concentrations and pre-incubation times.
  • Exemplary assays for inhibition of syncytium formation can be employed to measure antibody-mediated inhibition of viral cytopathic effects by blocking the formation of syncytia when using a fusogenic viral strain.
  • One of skill in the art can identify an appropriate fusogenic viral strain for use in the assay.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be assayed for their ability to inhibit or downregulate RSV replication using techniques known to those of skill in the art.
  • RSV replication can be assayed by a plaque assay such as described, e.g., by Johnson et al. (1997) Journal of Infectious Diseases 176:1215-1224.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be assayed for their ability to inhibit or downregulate the expression of RSV polypeptides. Techniques known to those of skill in the art, including, but not limited to, Western blot analysis, Northern blot analysis, and RT-PCR can be used to measure the expression of RSV polypeptides.
  • In vivo studies using animal models can be performed to assess the efficacy of the anti-RSV antibodies or antigen-binding fragments thereof provided herein. In vivo studies using animal models can be performed to assess any toxicity of administration of such antibodies or antigen-binding fragments thereof.
  • a variety of assays, such as those employing in vivo animal models, are available to those of skill in the art for evaluating the ability of the anti-RSV antibodies to inhibit or treat RSV virus infection and for assaying any toxicity.
  • the therapeutic effect of the anti-RSV antibodies can be assessed using animal models of the pathogenic infection, including animal models of viral infection.
  • Such animal models are known in the art, and include, but are not limited to, animal models for RSV infection, such as but not limited to cotton rat, inbred mouse, calf, ferret, hamster, guinea pig, chimpanzee, owl monkey, rhesus monkey, African green monkey, cebus monkey, squirrel monkey, bonnet monkey, baboon, (see, e.g., Prince et al. (1978 )Am. J. Pathol. 93:771-791; Prince et al. (1979) Infect. Immunol. 26:764-766; Byrd and Prince (1997) Clinical Infectious Diseases 25:1363-1368, including references cited therein, for exemplary models of RSV infection).
  • any animal model system known in the art can be used, including, but not limited to, rats, mice, cows, monkeys, and rabbits.
  • Efficacy in treating or preventing viral infection can be demonstrated by detecting the ability of a anti-RSV antibody or antigen-binding fragment thereof provided herein to inhibit the replication of the virus, to inhibit transmission or prevent the virus from establishing itself in its host, to reduce the incidence of RSV infection, or to prevent, ameliorate or alleviate one or more symptoms associated with RSV infection.
  • the treatment is considered therapeutic if there is, for example, a reduction is viral load, amelioration of one or more symptoms, a reduction in the duration of a RSV infection, or a decrease in mortality and/or morbidity following administration of an antibody or composition provided herein. Further, the treatment is considered therapeutic if there is an increase in the immune response following the administration of one or more antibodies or antigen-binding fragments thereof which immunospecifically bind to one or more RSV antigens.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be tested in vitro and in vivo for the ability to induce the expression of cytokines such as IFN-a, IFN-b, IFN-g, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-15. Techniques known to those of skill in the art can be used to measure the level of expression of cytokines.
  • the level of expression of cytokines can be measured by analyzing the level of RNA of cytokines by, for example, RT-PCR and Northern blot analysis, and by analyzing the level of cytokines by, for example, immunoprecipitation followed by Western blot analysis or ELISA.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be tested in vitro and in vivo for their ability to modulate the biological activity of immune cells, including human immune cells (e.g., T-cells, B-cells, and Natural Killer cells).
  • human immune cells e.g., T-cells, B-cells, and Natural Killer cells.
  • the ability of an anti-RSV antibody or antigen-binding fragment to modulate the biological activity of immune cells can be assessed by detecting the expression of antigens, detecting the proliferation of immune cells, detecting the activation of signaling molecules, detecting the effector function of immune cells, or detecting the differentiation of immune cells. Techniques known to those of skill in the art can be used for measuring these activities.
  • cellular proliferation can be assayed by 3H-thymidine incorporation assays and trypan blue cell counts.
  • Antigen expression can be assayed, for example, by immunoassays including, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, immunohistochemistry radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and FACS analysis.
  • the activation of signaling molecules can be assayed, for example, by kinase assays and electrophoretic shift assays (EMSAs).
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be tested for their ability to inhibit viral replication or reduce viral load in in vitro, ex vivo and in vivo assays.
  • the anti-RSV antibodies or antigen-binding fragments thereof also can be assayed for their ability to decrease the time course of RSV infection.
  • the anti- RSV antibodies or antigen-binding fragments thereof also can be assayed for their ability to increase the survival period of humans suffering from RSV infection by at least or about 25%, at least or about 50%, at least or about 60%, at least or about 75%, at least or about 85%, at least or about 95%, or at least or about 99%.
  • anti-RSV antibodies or antigen binding fragments thereof can be assayed for their ability reduce the hospitalization period of humans suffering from RSV infection by at least or about 60%, at least or about 75%, at least or about 85%, at least or about 95%, or at least or about 99%.
  • Techniques known to those of skill in the art can be used to analyze the function of the anti-RSV antibodies or antigen binding fragments thereof provided herein in vivo.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be used in diagnostic assays for the detection, purification, and/or neutralization of RSV.
  • Exemplary diagnostic assays include in vitro and in vivo detection of RSV.
  • assays using the anti-RSV antibodies or antigen-binding fragments thereof provided herein for qualitatively and quantitatively measuring levels of RSV in an isolated biological sample (e.g., sputum) or in vivo are provided.
  • the anti-RSV antibodies or antigen-binding fragments thereof can be conjugated to a detectable moiety for in vitro or in vivo detection.
  • Such antibodies can be employed, for example, to evaluate the localization and/or persistence of the anti- RSV antibody or antigen-binding fragment thereof at an in vivo site, such as, for example, a mucosal site.
  • the anti-RSV antibodies or antigen-binding fragments thereof which are coupled to a detectable moiety can be detected in vivo by any suitable method known in the art.
  • the anti-RSV antibodies or antigen-binding fragments thereof which are coupled to a detectable moiety also can be detected in isolated biological samples, such as tissue or fluid samples obtained from the subject following administration of the antibody or antigen binding fragment thereof.
  • RSV can be detected in a subject or patient based on the presence of one or more RSV proteins and/or polynucleotides encoding such proteins in a biological sample (e.g., blood, sera, sputum urine and/or other appropriate cells or tissues) obtained from a subject or patient.
  • a biological sample e.g., blood, sera, sputum urine and/or other appropriate cells or tissues
  • Such proteins can be used as markers to indicate the presence or absence of RSV in a subject or patient.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be employed for detection of the level of antigen and/or epitope that binds to the agent in the biological sample.
  • a variety of assay formats are known to those of ordinary skill in the art for using a anti-RSV antibody or antigen-binding fragment thereof to detect polypeptide markers in a sample (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • the presence or absence of RSV in a subject or patient can be determined by contacting a biological sample obtained from a subject or patient with an anti- RSV antibody or antigen-binding fragment thereof provided herein and detecting in the sample a level of polypeptide that binds to the anti-RSV antibody or antigen-binding fragment thereof.
  • the assay involves the use of an anti-RSV antibody or antigen binding fragment thereof provided herein immobilized on a solid support to bind to and remove the target polypeptide from the remainder of the sample.
  • the bound polypeptide can then be detected using a detection reagent that contains a reporter group and specifically binds to the antibody/polypeptide complex.
  • detection reagents can contain, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent.
  • a competitive assay can be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized anti-RSV antibody or antigen-binding fragment thereof after incubation of the anti-RSV antibody or antigen binding fragment thereof with the sample.
  • the extent to which components of the sample inhibit the binding of the labeled polypeptide to the anti-RSV antibody or antigen-binding fragment thereof is indicative of the reactivity of the sample with the immobilized anti- RSV antibody or antigen-binding fragment thereof.
  • Suitable polypeptides for use within such assays include full length RSV proteins and portions thereof, to which an anti-RSV antibody or antigen-binding fragment thereof binds, as described above.
  • the solid support can be any material known to those of ordinary skill in the art to which the protein can be attached.
  • the solid support can be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support also can be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support also can be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681.
  • the anti-RSV antibody or antigen binding fragment thereof can be immobilized on the solid support using a variety of techniques known to those of skill in the art.
  • the anti-RSV antibody or antigen-binding fragment thereof can be immobilized by adsorption to a well in a microtiter plate or to a membrane. In such cases, adsorption can be achieved by contacting the anti-RSV antibody or antigen-binding fragment thereof, in a suitable buffer, with the solid support for a suitable amount of time.
  • the contact time varies with temperature, but is typically between about 1 hour and about 1 day.
  • contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of anti-RSV antibody or antigen-binding fragment thereof ranging from about 10 ng to about 10 jag, and typically about 100 ng to about 1 jag, is sufficient to immobilize an adequate amount of anti-RSV antibody or antigen binding fragment thereof.
  • a plastic microtiter plate such as polystyrene or polyvinylchloride
  • Covalent attachment of anti-RSV antibody or antigen-binding fragment thereof to a solid support can generally be achieved by first reacting the support with a bifunctional reagent that will react with the support and a functional group, such as a hydroxyl or amino group, on the anti-RSV antibody or antigen-binding fragment thereof.
  • a bifunctional reagent that will react with the support and a functional group, such as a hydroxyl or amino group, on the anti-RSV antibody or antigen-binding fragment thereof.
  • the anti- RSV antibody or antigen-binding fragment thereof can be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
  • the assay is performed in a flow-through or strip test format, wherein the anti-RSV antibody or antigen-binding fragment thereof is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • polypeptides within the sample bind to the immobilized anti-RSV antibody or antigen-binding fragment thereof as the sample passes through the membrane.
  • a second, labeled binding agent then binds to the anti- RSV antibody or antigen-binding fragment thereof-polypeptide complex as a solution containing the second binding agent flows through the membrane.
  • RSV protein markers can be assayed within a given sample. It will be apparent that anti-RSV antibodies or antigen-binding fragments thereof specific for different RSV polypeptides can be combined within a single assay. Further, multiple primers or probes can be used concurrently. The selection of RSV protein markers can be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for RSV proteins provided herein can be combined with assays for other known RSV antigens.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be employed as an in vivo diagnostic agent.
  • the anti-RSV antibodies or antigen binding fragments thereof can provide an image of infected tissues (e.g., RSV infection in the lungs) using detection methods such as, for example, magnetic resonance imaging, X-ray imaging, computerized emission tomography and other imaging technologies.
  • the antibody portion of the anti-RSV antibody generally will bind to RSV (e.g., binding a RSV protein epitope), and the imaging agent will be an agent detectable upon imaging, such as a paramagnetic, radioactive or fluorescent agent that is coupled to the anti-RSV antibody or antigen-binding fragment thereof.
  • the imaging agent will be an agent detectable upon imaging, such as a paramagnetic, radioactive or fluorescent agent that is coupled to the anti-RSV antibody or antigen-binding fragment thereof.
  • the anti-RSV antibody or antigen-binding fragment thereof is coupled directly or indirectly to the imaging agent.
  • Imaging agents are known in the art, as are methods for their attachment to the anti-RSV antibodies or antigen-binding fragments (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509).
  • Exemplary attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the antibody or antigen-binding fragment thereof (U.S. Pat. No. 4,472,509).
  • the antibodies also can be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of such coupling agents or by reaction with an isothiocyanate.
  • the type of detection instrument available is considered when selecting a given radioisotope.
  • the radioisotope selected has a type of decay which is detectable for a given type of instrument.
  • Another factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized
  • a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which can be readily detected by conventional gamma cameras.
  • radioisotopes can be bound to the antibodies or antigen-binding fragments thereof provided herein either directly or indirectly by using an intermediate functional group.
  • exemplary intermediate functional groups which can be used to bind radioisotopes, which exist as metallic ions, to antibodies include bifunctional chelating agents, such as diethylene-triamine pentaacetic acid (DTP A) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
  • DTP A diethylene-triamine pentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • Examples of metallic ions which can be bound to the anti-RSV antibodies or antigen-binding fragments thereof provided include, but are not limited to, 72 Arsenic, 211 Astatine, 14 Carbon, 51 Chromium, 36 Chlorine, 57 Cobalt, 58Cobalt, 67 Copper, 15 2 Europium, 67 Gallium, 68 Gallium, 3 Hydrogen, 123 Iodine, 125 Iodine, 131 Iodine, m Indium, 59 Iron
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic imaging can be utilized.
  • gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
  • Elements which are particularly useful in such techniques include, but are not limited to, 157Gd, 55Mn, 162Dy, 52Cr, and 56Fe.
  • Exemplary paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).
  • Ions useful, for example, in X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and bismuth (III).
  • the concentration of detectably labeled anti-RSV antibody or antigen-binding fragment thereof which is administered is sufficient such that the binding to RSV is detectable compared to the background. Further, it is desirable that the detectably labeled anti-RSV antibody or antigen-binding fragment thereof be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of detectably labeled anti-RSV antibody or antigen-binding fragment thereof for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual.
  • the dosage of a human monoclonal antibody can vary, for example, from about 0.01 mg/m 2 to about 500 mg/m 2 , 0.1 mg/m 2 to about 200 mg/m 2 , or about 0.1 mg/m 2 to about 10 mg/m 2 . Such dosages can vary, for example, depending on whether multiple injections are given, tissue, and other factors known to those of skill in the art.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein and pharmaceutical compositions containing anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject for prophylaxis and therapy.
  • the antibodies or antigen-binding fragments thereof provided can be administered for treatment of a disease or condition, such as a RSV infection.
  • the antibodies or antigen-binding fragments thereof provided can be administered to a subject for prophylactic uses, such as the prevention and/or spread of RSV infection, including, but not limited to the inhibition of establishment of RSV infection in a host or inhibition of RSV transmission between subjects.
  • the antibodies or antigen-binding fragments thereof provided can be administered to a subject for the reduction of RSV viral load in the subject.
  • the antibodies or antigen-binding fragments thereof also can be administered to a subject for preventing, treating, and/or alleviating of one or more symptoms of a RSV infection or reduce the duration of a RSV infection.
  • administering inhibits the incidence of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about
  • administering decreases the severity of one or more symptoms of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the severity of the one or more symptoms of RSV infection in the absence of the anti-RSV antibody or antigen binding fragment.
  • a subject or candidate for therapy with an anti-RSV antibody or antigen-binding fragment thereof provided herein includes, but is not limited to, a subject, such as a human patient, that has been exposed to a RSV virus, a subject, such as a human patient, who exhibits one or more symptoms of a RSV infection and a subject, such as a human patient, who is at risk of a RSV infection.
  • RSV virus infections include those caused by RSV viruses, such as, but not limited to, acute RSV disease, RSV upper respiratory tract infection (URI) and/or RSV lower respiratory tract infection (LRI), including, for example, bronchiolitis and pneumonia.
  • the subject for therapy with an anti-RSV antibody or antigen binding fragment thereof provided herein is a mammal.
  • the subject for therapy with an anti-RSV antibody or antigen-binding fragment thereof provided herein is a primate.
  • the subject for therapy with an anti-RSV antibody or antigen binding fragment thereof provided herein is a human.
  • the provided anti-RSV antibodies or antigen-binding fragments thereof can be administered to a subject, such as a human patient, for the treatment of any RSV-mediated disease.
  • a subject such as a human patient
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject to alleviate one or more symptoms or conditions associated with a RSV virus infection, including, but not limited to, asthma, wheezing, reactive airway disease (RAD), and chronic obstructive pulmonary disease (COPD).
  • RAD reactive airway disease
  • COPD chronic obstructive pulmonary disease
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject, such a human patient, having a RSV virus infection for the maintenance or suppression therapy of recurring RSV virus-mediated disease.
  • the provided anti-RSV antibodies or antigen-binding fragments thereof can be administered to a subject, such as a human patient, at risk of a RSV virus infection, including, but not limited to, a prematurely bom (pre-term) infant (e.g.
  • a human infant bom less than 38 weeks of gestational age such as, for example, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, or 37 weeks gestational age
  • an infant e.g., a human infant bom more than 37 weeks gestational age
  • a subject having cystic fibrosis, bronchopulmonary dysplasia, congenital heart disease, congenital immunodeficiency, or acquired immunodeficiency e.g, an AIDS patient
  • leukemia non-Hodgkin lymphoma
  • an immunosuppressed patient such as, for example, a recipient of a transplant (e.g.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject, such as a pre-term infant or infant exposed to one or more environmental risk factors, such as, but not limited to attending daycare, having school aged siblings, exposure to environmental air pollutants, congenital airway abnormalities, and/or severe neuromuscular disease.
  • the provided anti-RSV antibodies or antigen-binding fragments thereof can be administered to a subject, such an infant or child who is younger than two years, having chronic lung disease or congenital heart disease, including congestive heart failure, pulmonary hypertension, and cyanotic heart disease.
  • Tests for various pathogens and pathogenic infection are known in the art and can be employed for the assessing whether a subject is a candidate for therapy with an anti- RSV antibody or antigen-binding fragment thereof provided herein.
  • tests for RSV virus infection are known and include for example, viral culture plaque assays, antigen detection test, polymerase chain reaction (PCR) tests, and various antibody serological tests.
  • Tests for viral infection can be performed on samples obtained from tissue or fluid samples, such as spinal fluid, blood, or urine.
  • Additional tests include, but are not limited to chest X-rays, which can show signs of pneumonia, other blood tests, such as a chemistry screening, a complete blood count, or arterial blood gases (ABGs) analysis, and oximetry, to measure the amount of oxygen in the blood.
  • chest X-rays which can show signs of pneumonia
  • other blood tests such as a chemistry screening, a complete blood count, or arterial blood gases (ABGs) analysis, and oximetry, to measure the amount of oxygen in the blood.
  • ABSGs arterial blood gases
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject, who is at an increased risk of RSV infection during particular times of the year. RSV season typically extends from October through May. Subjects, who exhibit increased susceptibility to virus infection during this time, such as infants the elderly or immunocompromised patients, can be administered an anti-RSV antibody or antigen-binding fragment thereof provided herein for the prophylaxis and/or treatment of RSV infection just prior to and/or during RSV season.
  • the anti-RSV antibody or antigen binding fragment thereof provided herein is administered one time, two times, three times, four times or five times during RSV season.
  • the anti-RSV antibody or antigen-binding fragment thereof provided herein is administered one time, two times, three times, four times or five times within one month, two months or three months, prior to a RSV season.
  • the anti-RSV antibody or antigen-binding fragment thereof provided herein is administered in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration of an anti-RSV antibody or antigen-binding fragment thereof can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by using the assays provided herein or known in the art.
  • an effective amount of antibody or antigen-binding fragment thereof to be administered therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient.
  • the attending physician takes into consideration various factors known to modify the action of drugs, including severity and type of disease, patient's health, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Accordingly, it will be necessary for the therapist to titer the dosage of the antibody or antigen-binding fragment thereof and modify the route of administration as required to obtain the optimal therapeutic effect.
  • the clinician will administer the antibody or antigen-binding fragment thereof until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays. Exemplary assays for monitoring treatment of a viral infection are know in the art and include for example, viral titer assays.
  • the dosage ranges for the administration of the anti-RSV antibodies or antigen-binding fragments thereof provided herein are those large enough to produce the desired effect in which the symptom(s) of the pathogen-mediated disease (e.g . viral disease) are ameliorated or the likelihood of virus infection is decreased.
  • the anti- RSV antibodies or antigen-binding fragments thereof provided herein are administered in an amount effective for inducing an immune response in the subject.
  • the dosage is not so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema or congestive heart failure.
  • the dosage will vary with the age, condition, sex and the extent of the disease in the patient and can be determined by one of skill in the art.
  • Exemplary dosages for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection include, but are not limited to, about or 0.01 mg/kg to about or 300 mg/kg, such as for example, about or 0.01 mg/kg, about or 0.1 mg/kg, about or 0.5 mg/kg, about or 1 mg/kg, about or 5 mg/kg, about or 10 mg/kg, about or 15 mg/kg, about or 20 mg/kg, about or 25 mg/kg, about or 30 mg/kg, about or 35 mg/kg, about or 40 mg/kg, about or 45 mg/kg, about or 50 mg/kg, about or 100 mg/kg, about or 150 mg/kg, about or 200 mg/kg, about or 250 mg/kg, or about or 300 mg/kg.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered to a subject at a dosage effective to achieve a desired serum titer.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a serum titer of at least or about 1 pg/ml, at least or about 2 pg/ml, at least or about 3 pg/ml, at least or about 4 pg/ml, at least or about 5 pg/ml, at least or about 6 pg/ml, at least or about 7 pg/ml, at least or about 8 pg/ml, at least or about 9 pg/ml, at least or about 10 pg/ml, at least or about 15 pg/ml, at least or about 20 pg/m
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered by pulmonary delivery to a subject at a dosage effective to achieve a desired titer in an intubation sample, sputum or lavage from the lungs.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a titer of 10 pg/mg (ng anti-RSV antibody or antigen-binding fragment thereof per mg lung protein) or about 10 pg/mg, 15 pg/mg or about 15 pg/mg, 20 pg/mg or about 20 pg/mg, 25 pg/mg or about 25 pg/mg, 30 pg/mg or about 30 pg/mg, 40 pg/mg or about 40 pg/mg, 50 pg/mg or about 50 pg/mg, 60 pg/mg or about 60 pg/mg, 70 pg/mg or about 70 pg/mg, 80 pg/mg or about 80 pg/mg, 90
  • the dosage of the anti-RSV antibodies or antigen binding fragments thereof can vary depending on the type and severity of the disease.
  • the anti-RSV antibodies or antigen-binding fragments thereof can be administered single dose, in multiple separate administrations, or by continuous infusion.
  • the treatment can be repeated until a desired suppression of disease symptoms occurs or the desired improvement in the patient's condition is achieved.
  • Repeated administrations can include increased or decreased amounts of the anti-RSV antibody or antigen-binding fragment thereof depending on the progress of the treatment.
  • Other dosage regimens also are contemplated.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered one time, two times, three times, four times, five times, six time, seven times, eight times, nine times, ten times or more per day or over several days.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered one time, two times, three times, four times, five times, six time, seven times, eight times, nine times, ten times or more for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a serum titer of at least or about 1 pg/ml, at least or about 2 pg/ml, at least or about 3 pg/ml, at least or about 4 pg/ml, at least or about 5 pg/ml, at least or about 6 pg/ml, at least or about 7 pg/ml, at least or about 8 pg/ml, at least or about 9 pg/ml, at least or about 10 mg/ml, at least or about 15 pg/ml, at least or about 20 pg/ml, at least or about 25 pg/ml.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered four times for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a serum titer of at least or about 72 pg/ml at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days or 40 days following administration of the fourth dose of the antibody or antigen-binding fragment thereof and prior to a subsequent dose of the antibody or antigen-binding fragment thereof.
  • the anti-RSV antibodies or antigen-binding fragments thereof are administered in a sequence of two or more administrations, where the administrations are separated by a selected time period.
  • the selected time period is at least or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months.
  • a prophylactically effective amount of an anti-RSV antibody or antigen-binding fragment thereof provided herein is administered one or more times just prior to RSV season. In some examples, a prophylactically effective amount of an anti- RSV antibody or antigen-binding fragment thereof provided herein is administered one or more times just prior to RSV season and/or one or more times during RSV season.
  • Therapeutic efficacy of a particular dosage or dosage regimen also can be assessed, for example, by measurement of viral titer in the subject prior to and following administration of one or more doses of the anti-RSV antibody or antigen-binding fragment thereof. Dosage amounts and/or frequency of administration can be modified depending on the desired rate of clearance of the virus in the subject.
  • the optimal treatment regimen will vary and it is within the scope of the treatment methods to evaluate the status of the disease under treatment and the general health of the patient prior to, and following one or more cycles of therapy in order to determine the optimal therapeutic dosage and frequency of administration.
  • an anti-RSV antibody or antigen-binding fragment thereof to be administered for the treatment of a disease or condition can be determined by standard clinical techniques (e.g. viral titer or antigen detection assays).
  • in vitro assays and animal models can be employed to help identify optimal dosage ranges. Such assays can provide dosages ranges that can be extrapolated to administration to subjects, such as humans. Methods of identifying optimal dosage ranges based on animal models are well known by those of skill in the art.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject by any method known in the art for the administration of polypeptides, including for example systemic or local administration.
  • the anti- RSV antibodies or antigen-binding fragments thereof can be administered by routes, such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavity), topical, epidural, or mucosal (e.g. intranasal or oral).
  • routes such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavity), topical, epidural, or mucosal (e.g. intranasal or oral).
  • the anti-RSV antibodies or antigen-binding fragments thereof can be administered externally to a subject, at the site of the disease for exertion of local or transdermal action.
  • compositions containing anti- RSV antibodies or antigen-binding fragments thereof can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa).
  • Compositions containing anti-RSV antibodies or antigen-binding fragments can be administered together with other biologically active agents.
  • the mode of administration can include topical or other administration of a composition on, in or around areas of the body that may come on contact with fluid, cells, or tissues that are infected, contaminated or have associated therewith a virus, such as a RSV virus.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered by topical or aerosol routes for delivery directly to target organs, such as the lungs (e.g. by pulmonary aerosol).
  • the provided anti- RSV antibodies or antigen-binding fragments thereof can be administered as a controlled release formulation as such as by apump (see, e.g., Langer (1990) Science 249:1527-1533; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al. (1980) Surgery 88:507; and Saudek et al. (1989) N. Engl. J. Med.
  • a controlled or sustained release system can be placed in proximity of the therapeutic target, for examples, the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • the provided anti-RSV antibodies or antigen-binding fragments thereof are administered by pulmonary delivery (see, e.g., U.S. Pat. Nos.
  • exemplary methods of pulmonary delivery include, but are not limited to, aerosol methods, such as inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems), intratracheal instillation and insufflation.
  • inhalers e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems
  • nebulizers e.g., jet or ultrasonic nebulizers
  • pulmonary delivery can be enhanced by co-administration of or administration of a co-formulation containing the anti-RSV antibodies or antigen-binding fragments thereof provided herein and a permeation enhancer, such as, for example, surfactants, fatty acids, saccharides, chelating agents and enzyme inhibitors, such as protease inhibitors.
  • a permeation enhancer such as, for example, surfactants, fatty acids, saccharides, chelating agents and enzyme inhibitors, such as protease inhibitors.
  • Appropriate methods for delivery can be selected by one of skill in the art based on the properties of the dosage amount of the anti-RSV antibody or antigen-binding fragment thereof or the pharmaceutical composition containing the antibody or antigen-binding fragment thereof.
  • properties include, but are not limited to, solubility, hygroscopicity, crystallization properties, melting point, density, viscosity, flow, stability and degradation profile.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein increase the efficacy mucosal immunization against a virus.
  • the anti-RSV antibodies or antigen-binding fragments thereof are administered to a mucosal surface.
  • the anti-RSV antibodies or antigen-binding fragments thereof can be delivered via routes such as oral (e.g., buccal, sublingual), ocular (e.g., comeal, conjunctival, intravitreally, intra-aqueous injection), intranasal, genital (e.g., vaginal), rectal, pulmonary, stomachic, or intestinal.
  • the anti-RSV antibodies or antigen binding fragments thereof provided herein can be administered systemically, such as parenterally, for example, by injection or by gradual infusion over time or enterally (i.e., digestive tract).
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered topically, such as for example, by topical installation or application (e.g., intratracheal instillation and insufflation using a bronchoscope or other artificial airway) of liquid solutions, gels, ointments, powders or by inhalation (e.g., nasal sprays, inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems)).
  • Administration can be effected prior to exposure to the virus or subsequent to exposure to the virus.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered alone or in combination with one or more therapeutic agents or therapies for the prophylaxis and/or treatment of a disease or condition.
  • the provided anti- RSV antibodies or antigen-binding fragments thereof can be administered in combination with one or more antiviral agents for prophylaxis.
  • the provided anti- RSV antibodies or antigen-binding fragments thereof can be administered in combination with one or more antiviral agents treatment of a viral infection, such as a respiratory viral infection.
  • the respiratory viral infection is a RSV infection.
  • the antiviral agents can include agents to decrease and/or eliminate the pathogenic infection or agents to alleviate one or more symptoms of a pathogenic infection.
  • a plurality of antibodies or antigen-binding fragments thereof also can be administered in combination, where at least one of the antibodies is an anti- RSV antibody or antigen-binding fragment thereof provided herein.
  • a plurality of antibodies can be administered in combination for the prophylaxis, where at least one of the antibodies is an anti-RSV antibody or antigen-binding fragment thereof provided herein.
  • a plurality of antibodies can be administered in combination for treatment of a RSV infection or multiple viral infections, where at least one of the antibodies is an anti-RSV antibody or antigen-binding fragment thereof provided herein.
  • the anti-RSV antibodies provided can be administered in combination with one or more antiviral antibodies, which bind to and neutralize a virus, such as RSV.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided can be administered in combination with one or more antibodies, which can inhibit or alleviate one or more symptoms of a viral infection, such as a RSV infection.
  • two or more of the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered in combination.
  • the one or more additional agents can be administered simultaneously, sequentially or intermittently with the anti-RSV antibody or antigen-binding fragment thereof.
  • the agents can be co-administered with the anti-RSV antibody or antigen-binding fragment thereof, for example, as part of the same pharmaceutical composition or same method of delivery.
  • the agents can be co-administered with the anti-RSV antibody or antigen binding fragment thereof at the same time as the anti-RSV antibody or antigen-binding fragment thereof, but by a different means of delivery.
  • the agents also can be administered at a different time than administration of the anti-RSV antibody or antigen-binding fragment thereof, but close enough in time to the administration of the anti-RSV antibody or antigen binding fragment thereof to have a combined prophylactic or therapeutic effect.
  • the one or more additional agents are administered subsequent to or prior to the administration of the anti-RSV antibody or antigen-binding fragment thereof separated by a selected time period.
  • the time period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months.
  • the one or more additional agents are administered multiple times and/or the anti- RSV antibody or antigen-binding fragment thereof provided herein is administered multiple times.
  • administration of the combination inhibits the incidence of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the incidence of RSV infection in the absence of the combination.
  • administration of the combination decreases the severity of one or more symptoms of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the severity of the one or more symptoms of RSV infection in the absence of the combination.
  • the combination inhibits the binding of RSV to its host cell receptor by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the binding of RSV to its host cell receptor in the absence of the combination.
  • the combination inhibits RSV replication by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about
  • Any therapy which is known to be useful, or which is or has been used for the prevention, management, treatment, or amelioration of a RSV infection or one or more symptoms thereof can be used in combination with anti-RSV antibody or antigen-binding fragment thereof provided herein (see, e.g., Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis and Therapy, Berkow, M. D. et al.
  • therapies e.g., prophylactic or therapeutic agents which have been or are used for preventing, treating, managing, or ameliorating a RSV infection or one or more symptoms thereof).
  • agents include, but are not limited to, immunomodulatory agents, anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, non-steroidal anti-inflammatory drugs (e.g.
  • anti-inflammatory agents e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone
  • non-steroidal anti-inflammatory drugs e.g.
  • leukotriene antagonists e.g., montelukast, methyl xanthines, zafirlukast, and zileuton
  • bronchodilators such as b-agonists (e.g., bambuterol, bitolterol, clenbuterol, fenoterol, formoterol, indacaterol, isoetharine, metaproterenol, pirbuterol, procaterol, reproterol, rimiterol, salbutamol (Albuterol, Ventolin), levosalbutamol, salmeterol, tulobuterol and terbutaline) and anticholinergic agents (e.g., ipratropium bromide and oxitropium bromide), sulphasalazine, penicillamine, dapsone, antihist
  • b-agonists e.g., bambuterol, bitolterol, clen
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered in combination with one or more therapies for the treatment of a RSV infection, including but not limited to, administration of intravenous infusion of immunoglobulin, administration of supplemental oxygen and fluids or assisted breathing.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered in combination with one or more agents that regulate lung maturation and surfactant protein expression, such as, but not limited to, glucocorticoids, PPARy ligands, and vascular endothelial cell growth factor (VEGF).
  • glucocorticoids glucocorticoids
  • PPARy ligands vascular endothelial cell growth factor (VEGF).
  • VEGF vascular endothelial cell growth factor
  • antiviral agents that can be selected for combination therapy with an anti- RSV antibody or antigen-binding fragment thereof provided herein include, but are not limited to, antiviral compounds, antiviral proteins, antiviral peptides, antiviral protein conjugates and antiviral peptide conjugates, including, but not limited to, nucleoside analogs, nucleotide analogs, immunomodulators (e.g. interferons) and immunostimulants.
  • antiviral compounds include, but are not limited to, antiviral compounds, antiviral proteins, antiviral peptides, antiviral protein conjugates and antiviral peptide conjugates, including, but not limited to, nucleoside analogs, nucleotide analogs, immunomodulators (e.g. interferons) and immunostimulants.
  • Combination therapy using antibodies and/or anti-RSV antibodies and antigen-binding fragments provided herewith are contemplated as is combination with the antibodies and/or anti-RSV antibodies and antigen-binding fragments provided herein with other anti- RSV antibodies and anti-RSV antibodies and antigen-binding fragments.
  • antiviral agents for the treatment of virus infections that can be administered in combination with the anti-RSV antibodies or antigen-binding fragments thereof provided herein include, but are not limited to, acyclovir, famciclovir, ganciclovir, penciclovir, valacyclovir, valganciclovir, idoxuridine, trifluridine, brivudine, cidofovir, docosanol, fomivirsen, foscamet, tromantadine, imiquimod, podophyllotoxin, entecavir, lamivudine, telbivudine, clevudine, adefovir, tenofovir, boceprevir, telaprevir, pleconaril, arbidol, amantadine, rimantadine, oseltamivir, zanamivir, peramivir, inosine, interferon (e
  • antiviral agents for the prophylaxis and/or treatment of RSV infections that can be administered in combination with the anti-RSV antibodies or antigen-binding fragments thereof provided herein include, but are not limited to, ribavirin, NIH-351 (Gemini Technologies), recombinant RSV vaccine (Aviron), RSVf-2 (Intracel), F-50042 (Pierre Fabre), T-786 (Trimeris), VP-36676 (ViroPharma), RFI-641 (American Home Products), VP-14637 (ViroPharma), PFP-1 and PFP-2 (American Home Products), RSV vaccine (Avant Immunotherapeutics), F-50077 (Pierre Fabre), and other anti-RSV antibodies or antigen binding fragments thereof.
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered in combination with one or more agents capable of stimulating cellular immunity, such as cellular mucosal immunity.
  • agents capable of stimulating cellular immunity can be used.
  • immunostimulatory agents include, cytokines, such as, but not limited to, interferons (e.g., IFN-a, b, g, w), lymphokines and hematopoietic growth factors, such as, for example, GM-CSF (granulocyte macrophage colony stimulating factor), Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin- 10 (IL-10), Interleukin- 12 (IL-12), Interleukin- 14 (IL-14), and Tumor Necrosis Factor (TNF).
  • interferons e.g., IFN-a, b, g, w
  • dosages for the administration of such compounds are known in the art or can be determined by one skilled in the art according to known clinical factors (e.g., subject's species, size, body surface area, age, sex, immunocompetence, and general health, duration and route of administration, the kind and stage of the disease, and whether other treatments, such as other anti-pathogenic agents, are being administered concurrently).
  • the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered in combination with one or more additional antibodies or antigen-binding fragments thereof.
  • the one or more additional antibodies are antiviral antibodies.
  • the one or more additional antibodies bind to a viral antigen.
  • the one or more additional antibodies bind to a viral antigen that is a surface protein, such as a viral capsid protein or a viral envelope protein.
  • the one or more additional antibodies bind to a viral antigen that is expressed on the surface of an infected cell.
  • the one or more additional antibodies bind to a viral antigen that is expressed intracell ularly (i.e., within an infected cell).
  • the one or more additional antibodies binds to a virus that causes respiratory disease, such as, but not limited to, RSV, parainfluenza virus (PIV) or human metapneumovirus (hMPV).
  • a virus that causes respiratory disease such as, but not limited to, RSV, parainfluenza virus (PIV) or human metapneumovirus (hMPV).
  • Compositions containing the mixtures of antibodies also are provided herein.
  • the one or more additional antiviral antibodies are anti- RSV antibodies or antigen-binding fragments thereof.
  • an anti- RSV antibody or antigen-binding fragment thereof provided herein is administered in combination with the one or more additional anti-RSV antibodies or antigen-binding fragments thereof for the prophylaxis and/or treatment of a RSV infection.
  • Exemplary anti- RSV antibodies or antigen-binding fragments thereof for combination therapy with an anti- RSV antibody or antigen-binding fragment thereof provided herein include anti- RSV antibodies or antigen-binding fragments thereof that immunospecifically bind to and neutralize RSV.
  • the one or more additional anti-RSV antibodies or antigen-binding fragments thereof includes an antibody or antigen-binding fragment thereof that immunospecifically binds to RSV A subtype and/or RSV B subtype.
  • the one or more additional antiviral antibodies for combination therapy with an anti-RSV antibody or antigen-binding fragment thereof provided herein includes, but is not limited to, palivizumab (SYNAGIS®), motavizumab (NUMAX®),
  • the one or more additional antiviral antibodies for combination therapy includes an antibody or antigen binding fragment thereof containing a VH chain and/or VL chain having the amino acid sequence of a VH chain and/or VL chain of palivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P1212, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, IX- 493L1, FRH3-3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S, rsv6, rsvll, rs
  • the one or more additional antiviral antibodies for combination therapy includes an antibody or antigen-binding fragment thereof containing one or more CDRs of palivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P1212, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FRH3-3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, A4B4- F52S, rsv6, rsvll, rsvl3, rsvl9, rsv21, rsv22, rsv23, RF-1, or RF-2.
  • SYNAGIS® palivizumab
  • NUMAX® motavizumab
  • the one or more additional antiviral antibodies for combination therapy includes an antibody or antigen-binding fragment thereof containing one or more CDRs of from an anti-RSV mouse monoclonal antibody such as, but not limited to, MAbs 1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112, 1269, 1269, 1243 (Beeler et al. (1989) J. Virology 63(7):2841-2950), MAM51 (Mufson et al. (1987) J. Clin. Microbiol. 25:1635-1539), MAbs 43-1 and 13-1
  • Additional exemplary antibodies or antigen-binding fragments thereof that can be used for combination therapy with an anti-RSV antibody or antigen-binding fragment provided herein include, but are not limited to, anti-RSV antibodies or antigen-binding fragments thereof described in, for example, U.S. Pat. Nos.
  • Plasmid pEntry- SynagisHCK251T_Geneart_SEL was used by the vendor BaseClear (Netherlands) as a template for construction of site evaluation (SEL) library at positions 466-725 aa (counting from the Cbhl Met). An average number of mutant variants per aa position was around 17.
  • This expression vector contains the T. reesei cbhl promoter and terminator regions allowing for a strong inducible expression of a gene of interest and the T. reesei pyr2 selective marker conferring growth of transformants on minimal medium without supplementation with uridine.
  • the plasmid is maintained autonomously in fungal cells due to T. reesei derived telomere regions. Plasmids were propagated in commercially available Escherichia coli TOP10 cells (Invitrogen, US), purified, sequence verified, arrayed individually in 96 well MTPs and used for fungal transformation as described below.
  • pEntry-Synagis LC Geneart plasmid was constructed via the Gateway® BP recombination cloning and recombined further with pTrex6g destination vector in a similar way as described above resulting in the expression vector pTrex6g-Synagis_LC.
  • This vector served as a template to generate a PCR fragment expressing the light chain driven by the cbhl promoter and linked to the alS marker conferring resistance to chlorimuron ethyl to a fungal cell.
  • the expression cassette consists of a CBH1 promoter, CBH1 core, antibody HC and LC connected by CBH1 linker and kex2 for processing of CBH1, CBH1 terminator, and the alS marker conferring resistance to chlorimuron ethyl to a fungal cell.
  • the alS marker was used for making the host strains so that the pyr2 marker was available for the SEL variants.
  • the expression cassette was randomly integrated into the host T. reesei genome at multiple copies.
  • the full expression cassette was amplified by PCR. The PCR product was cleaned up and concentrated to 500-1000ng/pL.
  • the host T. reesei strain used for transformation was deleted for major cellulases and xylanases.
  • the strain was transformed using a standard PEG-protoplast transformation method. Transformation mixtures containing approximately 10 pg of DNA and 5x 10 6 protoplasts in a total volume of 250 pi were treated with 2 mL of 25% PEG solution, diluted with 2 volumes of 1.2M sorbitol/lOmM Tris, pH7.5/ lOmM CaC12 solution, and mixed with 26mL of 2% low melting agarose containing 1M sorbitol, 1 g/L uridine, 75 mg/L chlorimuron ethyl in minimal medium and distributed over four 10cm petri plates pre-poured containing 1.5% agarose, 1M sorbitol in minimal media.
  • Plasmids with variants were received from the vendor in a 96 well format arrayed according to a predetermined layout. Transformation mixtures containing approximately 1 mg of DNA and 5x 10 6 protoplasts in a total volume of 50 ml were treated with 200 m ⁇ of 25% PEG solution, diluted with 1 volumes of 1.2M sorbitol/lOmM Tris, pH7.5/ lOmM CaCh solution, rearranged robotically into 24 well MTPs and poured in 1 ml of 3% low melting agarose containing 1M sorbitol in minimal medium. After sufficient growth transformants from each well were pooled together and plated on fresh 24 well agar plates with minimal medium. Once sporulated, spores were harvested and used for inoculation of liquid cultures.
  • reesei trace elements (100%: 175 g/L citric acid (anhydrous), 200 g/L FeS04*7H20, 16 g/L ZnS04*7H20, 3.2 g/L CuS04*5H20, 1.4 g/L MnS04*H20, 0.8 g/L H3B03).
  • Plates were incubated in Infors shaker with a 50 mm throw at 200 rpm and 28C with 80% humidity. After 5-6 days of growth cultures were reformatted back to 96 well deep well MTPs and filtered using 96-well microtiter filter plates (0.2 pm hydrophilic PVDF membrane, Coming, Tewksbury MA). The plates were frozen in Axygen half-deep well plates (P-DW-ll-C).
  • the centrifuged plates were then moved to a robot to remove 20 pL of the crude material for Octet Protein A quantitation.
  • the 20 pL was added to 80 pL of IX PBS in a 384-well plate (Greiner Bio-One 781209).
  • Four library plates went into one 384-well plate and there was a separate 384-well plate for the duplicate growth of the four plates (plates Xa and Xb).
  • the filter plates were returned to the robot deck and the duplicate growth plates were added to the same filter plates. These plates were incubated and centrifuged as before. The resin was then washed with 880 pL of PBS buffer. The plates shook for 1 minute and then centrifuged at 1000 g for 2 minutes. The flow through was discarded, and the plates were returned to the robot for the second PBS washing. After the second washing, the plates were moved to a robot running the elution program.
  • the elution program handled four plates at a time. It added 11 pL of neutralization buffer (1 M Tris pH 9) to a clean half-deep well plate that the samples would be eluted into. The program then added 440 pL of elution buffer (100 mM glycine pH 2.7) to the filter plates. The plates then shook for 1 minute at setting 7 and then were filtered by centrifugation (lOOOg for 2 minutes) into the freshly prepped recovery plates. After centrifugation, the sample plates shook for 1 minute to ensure proper mixing of the neutralization buffer.
  • Example 2 Testing RSV variants for increased manufacturability, thermostability, and/or protease resistance
  • Protein A (Thermo Fisher Scientific 77674) was labeled with Alexa Fluor 546 NHS ester (Thermo Fisher Scientific A20102).
  • Protein L (Thermo Fisher Scientific 77680) was labeled with Alexa Fluor 488 NHS ester (Thermo Fisher Scientific A20100).
  • the labeled Protein A and Protein L were diluted with 107 mM KPi pH 7 and at a ratio that produced a FRET signal for the standard curve with the proper dynamic range.
  • the standard curve was commercial Synagis from AbbVie.
  • 40 pL of the Protein A and L solution was mixed with 10 pL of the purified antibody sample.
  • the FRET signal on the plate was read (ex: 485 nm em: 590 nm cutoff: 590 nm), and the concentration of the unknowns was determined from the Synagis standard curve. The samples were run in duplicate.
  • Unfolding of purified antiRSV polypeptide was measured as follows. To 10 pi of purified and protein normalized antibody at 120ppm in the 384 well plate (Roche Diagnostics, Indianapolis, IN), 5 pi of lOOmM Cellobiose in 200mM NaAc pH5 was added and mixed, then 5 pi of 250-fold diluted sypro orange (Thermo Fisher Scientific Fisher, Grand Island, NY) was added and mixed. The plate was sealed and placed in the Roche Lightcycler 480.
  • the FRET signal of the stressed samples was measured in duplicate (20 pL of antibody mixture and 30 pL of the labeled Protein A and L mixture). The differences in stability was determined by comparing the ratios of the stressed and unstressed FRET signal for each sample.
  • the ratio of labeled Protein A and L to antibody sample was optimized so that the unstress samples were at the top of the linear portion of the standard curve.
  • the stress plate was sealed with a Bio-Rad microseal B and place in an iEMS incubator at 30 °C for 2.5 hours. After the incubation, the FRET signal of the stressed samples was measured in duplicate (20 pL of antibody mixture and 30 pL of the labeled Protein A and L mixture). The differences in stability was determined by comparing the ratios of the stressed and unstressed FRET signal for each sample.
  • Variants were tested for thermal stability, Tm, and protease resistance.
  • Light chain positions with greater than 1.5 and 2.0 standard deviation improvement relative to the parent RSV antibody sequence are shown in Table 1 and Table 2.
  • Heavy chain positions with greater than 1.5 and 2.0 standard deviation improvement relative to the parent RSV antibody sequence are shown in Table 3 and Table 4.
  • Table 1 Light chain positions with greater than 1.5 standard deviation improvement relative to the parent antibody sequence
  • Table 2 Light chain positions with greater than 2 standard deviation improvement relative to the parent antibody sequence

Abstract

Provided herein, inter alia, are antibodies or antigen-binding fragments thereof that immunospecifically bind to Respiratory Syncytial Virus (RSV). Also provided are methods for of prevention, treatment and diagnosis of viral infection and/or the treatment of one more symptoms of RSV-mediated disease. Methods of generating antibodies that immunospecifically bind RSV also are provided.

Description

ANTI-RSV ANTIBODIES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/001,754, filed March 30, 2020, the disclosure of which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] Provided herein, inter alia, are improved antibodies and antigen-binding fragments thereof that immunospecifically bind to Respiratory Syncytial Virus (RSV) and/or neutralize RSV.
BACKGROUND
[0003] Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants and young children and is the major cause of infantile bronchiolitis (Welliver (2003) J Pediatr 143 : S 112). An estimated 64 million cases of respiratory illness and 160,000 deaths worldwide are attributable to RSV induced disease. In the United States alone, tens of thousands of infant hospitalizations are due to infections by paramyxoviruses, such as RSV and parainfluenza virus (PIV) (Shay et al. (1999) JAMA 282:1440-1446).
Severe RSV infection occurs most often in children and infants, especially in premature infants. Underlying health problems such as chronic lung disease or congenital heart disease can significantly increase the risk of serious illness. RSV infections also can cause serious illness in the elderly, individuals with chronic pulmonary disease and immunocompromised adults, such as bone marrow transplant recipients.
[0004] Several approaches to the prevention and treatment of RSV infection have been investigated, including vaccine development, antiviral compounds (ribavirin), antisense drugs, RNA interference technology, and antibody products, such as immunoglobulin or intravenous monoclonal antibodies. Intravenous immunoglobulin (RSV-IGIV; RespiGam®) isolated from donors and a monoclonal antibody, palivizumab (SYNAGIS™), have been approved for RSV prophylaxis in high risk children. Heard et al., Molecular Medicine, 5:35- 45, 1999 and WO 2008/106980 A2 also disclose anti-RSV antibodies. A vaccine or commercially available treatment for RSV, however, is not yet available. Only ribavirin is approved for treatment of RSV infection. In order to be effective for treatment of RSV infection, high doses, frequent administrations and/or volumes of antibody products, such as RSV-IG and palivizumab, are required due to low specificity. Further, the use of products, such as intravenous immunoglobulin, is dependent on donor availability. Accordingly, there exists a need for additional agents for the prevention or treatment of RSV infections.
[0005] The subject matter disclosed herein addresses these needs and provides additional benefits as well.
SUMMARY
[0006] Provided herein are isolated polypeptides, antibodies or antigen-binding fragments thereof for the prophylaxis and treatment of Respiratory syncytial virus (RSV) infection and RSV-mediated diseases or conditions as well as methods for making and using the same.
[0007] Accordingly, in some aspects, provided herein is an isolated anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising: (a) a heavy chain variable region which has at least 90% sequence identity to
QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RLTI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; X4 is A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 is A or G; and/or (b) a heavy chain constant region comprising
ASTKGPSVFPLAPSXsKXoTSXioGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSS GLYSLS S VVTVP S S SLGTQTYICNVNHKPSNTKVDTRXi IEPKSCDKTHTCPPC PAPEL1GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein Xs is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y, wherein (i) the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO: 3; and (ii) wherein said antibody exhibits one or more improved properties comprising increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or functional fragment thereof further comprises: (c) a light chain variable region which has at least 90% sequence identity to
DIQMX12X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21 LIYDTSKLASGVPSRFSGSGSX22X23EX24TLTISSLQPDDFATYYCFQGSGYPFTFGGGT KLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; Xi4 is P or F; X15 is T or H; Xi6 is A or S; X17 is C, M, V, or Y; Xis is G or L; X19 is F, N, or V; X20 is A, S, V, or W; X21 is L, S, or G; X22 is G or A; X23 is T or Y; and X24 is F or C; and/or (d) a light chain constant region comprising
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein optionally the antibody differs by at least one amino acid from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO:6.
[0008] In another aspect, provided herein is an isolated anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising: (a) a light chain variable region which has at least 90% sequence identity to
DIQMX12X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21 LIYDTSKLASGVPSRFSGSGSX22X23EX24TLTISSLQPDDFATYYCFQGSGYPFTFGGGT KLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; Xi4 is P or F; X15 is T or H; Xi6 is A or S; X17 is C, M, V, or Y; Xis is G or L; X19 is F, N, or V; X20 is A, S, V, or W; X21 is L, S, or G; X22 is G or A; X23 is T or Y; and X24 is F or C; and/or (b) a light chain constant region comprising
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein (i) the antibody differs by at least one amino acid from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO: 6; and (ii) wherein said antibody exhibits one or more improved properties comprising increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the light chain encoded by the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or functional fragment thereof further comprises: (a) a heavy chain variable region which has at least 90% sequence identity to QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RETI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, T, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; X41S A, G, S, or T; X5 is P or Y; Xe is A or S; and X71S A or G; and/or (b) a heavy chain constant region comprising
ASTKGPSVFPLAPSXsKXoTSXioGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSS GLYSLS S VVTVP S S SLGTQTYICNVNHKPSNTKVDTRXi IEPKSCDKTHTCPPC PAPEL1GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein Xs is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y, wherein optionally the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3.
[0009] In some embodiments of any of the embodiments disclosed herein, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, wherein Xi is R, T, I,
Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; X4is A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 is A or G. In some embodiments of any of the embodiments disclosed herein, the light chain variable region comprises the amino acid sequence of SEQ ID NO:4, wherein Xi2 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X2ois A, S, V, orW; X2i is L, S, or G; X22is G or A; X23 is T or Y ; and X24 is F or C. In some embodiments of any of the embodiments disclosed herein, said functional fragment is selected from the group consisting of Fab, Fab , F(ab')2 and Fv fragments. In some embodiments of any of the embodiments disclosed herein, said antibody is chimeric, humanized, or fully human. In some embodiments of any of the embodiments disclosed herein, said antibody competitively inhibits the binding of palivizumab to the surface of RSV.
[0010] In still other aspects, provided herein is a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 1 and/or the heavy chain constant region of SEQ ID NO:2. In other aspects, provided herein is a nucleic acid encoding the light chain variable region of SEQ ID NO:4 and/or the light chain constant region of SEQ ID NO:5. [0011] In yet another aspect, provided herein is a vector comprising any of the nucleic acids disclosed herein.
[0012] In further aspects, provided herein is a recombinant cell comprising any of the vectors disclosed herein. In some embodiments, the cell is a mammalian cell, a bacterial cell, or a fungal cell. In some embodiments, the fungal cell is T. reesei.
[0013] In other aspects, provided herein is a method for producing an anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising providing an isolated cell with a nucleic acid encoding said antibody or functional part thereof, wherein said antibody or functional part thereof comprises one or more of (a) a heavy chain variable region comprising
QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RLTI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, T, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; X41S A, G, S, or T; X5 is P or Y; Xe is A or S; and X71S A or G; (b) a heavy chain constant region comprising
ASTKGPSVFPLAPSXsKXoTSXioGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSS GLYSLS S VVTVP S S SLGTQTYICNVNHKPSNTKVDTRXi IEPKSCDKTHTCPPC PAPEL1GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein Xs is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y, (c) a light chain variable region comprising
DIQMX12X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21 LIYDTSKLASGVPSRFSGSGSX22X23EX24TLTISSLQPDDFATYYCFQGSGYPFTFGGGT KLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; Xi4 is P or F; X15 is T or H; Xi6 is A or S; X17 is C, M, V, or Y; Xis is G or L; X19 is F, N, or V; X20 is A, S, V, or W; X21 is L, S, or G; X22 is G or A; X23 is T or Y; and X24 is F or C; and/or (d) a light chain constant region comprising
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3 and/or a light chain encoded by the amino acid sequence of SEQ ID NO: 6. In some embodiments, the cell is a mammalian cell, a bacterial cell, or a fungal cell. In some embodiments, the fungal cell is T. reesei. In some embodiments of any of the embodiments disclosed herein, the antibody or functional fragment thereof exhibits one or more improved properties selected from the group consisting of increased manufacturability, thermostability, and protease resistance compared to an antibody that does not differ by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3 and/or a light chain encoded by the amino acid sequence of SEQ ID NO: 6.
[0014] In another aspect, provided herein is a method for treating or preventing a respiratory syncytial virus (RSV) infection in an individual in need thereof comprising administering a therapeutically effective amount of the antibody or functional fragment thereof of any of the isolated anti-respiratory syncytial virus (RSV) antibodies or functional fragments thereof disclosed herein to the individual. In some embodiments, the antibody or functional fragment thereof is administered parenterally or intravenously. In some embodiments of any of the embodiments disclosed herein, the individual is a human. In some embodiments, the human is a preterm infant (under 35 weeks gestation) infant, infant with congenital heart defects (CHD), infant with bronchopulmonary dysplasia (BPD), and/or infant with congenital malformations of the airway. In some embodiments of any of the embodiments disclosed herein, the individual is less than four years old. In some embodiments of any of the embodiments disclosed herein, the individual is further administered oxygen therapy.
[0015] In an additional aspect, provided herein is a pharmaceutical composition comprising any of the isolated anti-respiratory syncytial virus (RSV) antibodies or functional fragments thereof disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.
[0016] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.
[0017] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.
DETAILED DESCRIPTION
[0018] Provided are anti-RSV antibodies or antigen-binding fragments thereof that bind to and neutralize respiratory syncytial virus. The anti-RSV antibodies provided herein are neutralizing antibodies that recognize one or more epitopes on the surface of RSV. The antibodies provided herein can be used in prophylaxis therapies. The antibodies provided herein also can be used as therapeutics.
[0019] For example, the antibodies provided can be employed for the prevention and/or spread of pathogenic disease, including, but not limited to the inhibition of viral transmission between subjects, inhibition of establishment of viral infection in a host, and reduction of viral load in a subject. The antibodies also can be employed for preventing, treating, and/or alleviating one or more symptoms of a RSV infection or for reducing the duration of a RSV infection. Accordingly, treatment of patients with antibodies provided herein can decrease the mortality and/or morbidity rate associated with RSV infection.
[0020] RSV persistence is associated with the generation of escape mutants that cannot be neutralized by an antibody. Thus, the main challenges to development of therapeutic anti viral antibodies are the generation or identification of antibodies that have a neutralization epitope that is 1) conserved across various strains or serotypes and 2) is difficult for the evolving virus to generate escape mutants against. Antibodies provided herein also exhibit one or more improved properties including one or more of increased manufacturability, thermostability, and/or protease resistance compared to existing antibodies in the prior art. Thus, the provided anti-RSV antibodies, in addition to prophylaxis therapy, also are useful for the treatment of RSV infection.
[0021] Generally, the anti-RSV antibodies provided herein have the ability to inhibit or reduce one or more activities of the virus, such as, for example, association of the virus with a target cell membrane, fusion of the virus with the target cell membrane and/or cell entry, production of new viral particles, including inhibition of viral replication, or cell to cell fusion of an infected cell with another cell (i.e. syncytia formation). The provided anti- RSV antibodies also can be employed to increase the immune the response against a RSV infection. I. Definitions
[0022] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin. Hence, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). Antibodies include antibody fragments, such as anti-RSV antibody fragments. As used herein, the term antibody, thus, includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti- id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b).
[0023] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody that binds antigen (e.g. one or more CDRs and/or one or more antibody combining sites) and thus retains the binding specificity, and at least a portion of the specific binding ability of the full-length antibody. Hence, an antigen binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full- length antibodies, as well as synthetically, e.g. recombinantly produced derivatives. An antibody fragment is included among antibodies. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers. An antibody fragment generally contains at least or about 50 amino acids and typically at least or about 200 amino acids. An antigen-binding fragment includes any antibody fragment that when inserted into an antibody framework (such as by replacing a corresponding region) results in an antibody that immunospecifically binds (i.e. exhibits Ka of at least or at least about 107- 108 M 1) to the antigen.
[0024] As used herein, a "therapeutic antibody" refers to any antibody or antigen-binding fragment thereof that is administered for treatment of an animal, including a human. Such antibodies can be prepared by any known methods for the production of polypeptides, and hence, include, but are not limited to, recombinantly produced antibodies, synthetically produced antibodies, and therapeutic antibodies extracted from cells or tissues and other sources. As isolated from any sources or as produced, therapeutic antibodies can be heterogeneous in length or differ in post-translational modification, such as glycosylation (i.e. carbohydrate content). Heterogeneity of therapeutic antibodies also can differ depending on the source of the therapeutic antibodies. Hence, reference to therapeutic antibodies refers to the heterogeneous population as produced or isolated. When a homogeneous preparation is intended, it will be so-stated. References to therapeutic antibodies herein are to their monomeric, dimeric or other multimeric forms, as appropriate.
[0025] As used herein, a "neutralizing antibody" is any antibody or antigen-binding fragment thereof that binds to a pathogen and interferes with the ability of the pathogen to infect a cell and/or cause disease in a subject. Exemplary of neutralizing antibodies are neutralizing antibodies that bind to viruses, bacteria, and fungal pathogens. Typically, the neutralizing antibodies provide herein bind to the surface of the pathogen. In examples where the pathogen is a virus, a neutralizing antibody that binds to the virus typically binds to a protein on the surface of the virus. Depending on the class of the virus, the surface protein can be a capsid protein (e.g. a capsid protein of a non-enveloped virus) or a viral envelope protein (e.g., a viral envelope protein of an enveloped virus). In some examples, the protein is a glycoprotein. The ability of the virus to inhibit virus infectivity can be measure for example, by an in vitro neutralization assay, such as, for example, a plaque reduction assay using Vero host cells.
[0026] As used herein, a "conventional antibody" refers to an antibody that contains two heavy chains (which can be denoted H and H') and two light chains (which can be denoted L and L') and two antibody combining sites, where each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding capability ( e.g . heavy chains include, but are not limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or any functional region of (e.g. light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H') pairs with one light chain (L and L', respectively)
[0027] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g. VH-CH 1 -CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and hinge regions, such as human antibodies produced naturally by antibody secreting B cells and antibodies with the same domains that are synthetically produced.
[0028] As used herein, the term "derivative" refers to a polypeptide that contains an amino acid sequence of an anti-RSV antibody or a fragment thereof which has been modified, for example, by the introduction of amino acid residue substitutions, deletions or additions, by the covalent attachment of any type of molecule to the polypeptide (e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein). A derivative of an anti-RSV antibody or antigen-binding fragment thereof can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation. Further, a derivative of an anti-RSV antibody or antigen-binding fragment thereof can contain one or more non-classical amino acids. Typically, a polypeptide derivative possesses a similar or identical function as an anti-RSV antibody or antigen-binding fragment thereof provided herein (e.g., neutralization of RSV).
[0029] As used herein, the phrase "derived from" when referring to antibody fragments derived from another antibody, such as a monoclonal antibody, refers to the engineering of antibody fragments (e.g., Fab, F(ab'), F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments) that retain the binding specificity of the original antibody. Such fragments can be derived by a variety of methods known in the art, including, but not limited to, enzymatic cleavage, chemical crosslinking, recombinant means or combinations thereof. Generally, the derived antibody fragment shares the identical or substantially identical heavy chain variable region (VH) and light chain variable region (VL) of the parent antibody, such that the antibody fragment and the parent antibody bind the same epitope [0030] As used herein, a "parent antibody" or "source antibody" refers the to an antibody from which an antibody fragment (e.g., Fab, F(ab'), F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments) is derived.
[0031] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically contain chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics.
[0032] As used herein, a chimeric polypeptide refers to a polypeptide that contains portions from at least two different polypeptides or from two non-contiguous portions of a single polypeptide. Thus, a chimeric polypeptide generally includes a sequence of amino acid residues from all or part of one polypeptide and a sequence of amino acids from all or part of another different polypeptide. The two portions can be linked directly or indirectly and can be linked via peptide bonds, other covalent bonds or other non-covalent interactions of sufficient strength to maintain the integrity of a substantial portion of the chimeric polypeptide under equilibrium conditions and physiologic conditions, such as in isotonic pH 7 buffered saline. For purposes herein, chimeric polypeptides include those containing all or part of an anti- RSV antibody linked to another polypeptide, such as, for example, a multimerization domain, a heterologous immunoglobulin constant domain or framework region, or a diagnostic or therapeutic polypeptide.
[0033] As used herein, a fusion protein is a polypeptide engineered to contain sequences of amino acids corresponding to two distinct polypeptides, which are joined together, such as by expressing the fusion protein from a vector containing two nucleic acids, encoding the two polypeptides, in close proximity, e.g., adjacent, to one another along the length of the vector. Generally, a fusion protein provided herein refers to a polypeptide that contains a polypeptide having the amino acid sequence of an antibody or antigen-binding fragment thereof and a polypeptide or peptide having the amino acid sequence of a heterologous polypeptide or peptide, such as, for example, a diagnostic or therapeutic polypeptide. Accordingly, a fusion protein refers to a chimeric protein containing two, or portions from two, or more proteins or peptides that are linked directly or indirectly via peptide bonds. The two molecules can be adjacent in the construct or separated by a linker, or spacer polypeptide. The spacer can encode a polypeptide that alters the properties of the polypeptide, such as solubility or intracellular trafficking.
[0034] As used herein, "linker" or "spacer" peptide refers to short sequences of amino acids that join two polypeptide sequences (or nucleic acid encoding such an amino acid sequence). "Peptide linker" refers to the short sequence of amino acids joining the two polypeptide sequences. Exemplary of polypeptide linkers are linkers joining a peptide transduction domain to an antibody or linkers joining two antibody chains in a synthetic antibody fragment such as an scFv fragment. Linkers are well-known and any known linkers can be used in the provided methods. Exemplary of polypeptide linkers are (Gly-Ser) amino acid sequences, with some Glu or Lys residues dispersed throughout to increase solubility. Other exemplary linkers are described herein; any of these and other known linkers can be used with the provided compositions and methods.
[0035] As used herein, "antibody hinge region" or "hinge region" refers to a polypeptide region that exists naturally in the heavy chain of the gamma, delta and alpha antibody isotypes, between the CHI and CH2 domains that has no homology with the other antibody domains. This region is rich in proline residues and gives the IgG, IgD and IgA antibodies flexibility, allowing the two "arms" (each containing one antibody combining site) of the Fab portion to be mobile, assuming various angles with respect to one another as they bind antigen. This flexibility allows the Fab arms to move in order to align the antibody combining sites to interact with epitopes on cell surfaces or other antigens. Two interchain disulfide bonds within the hinge region stabilize the interaction between the two heavy chains. In some embodiments provided herein, the synthetically produced antibody fragments contain one or more hinge regions, for example, to promote stability via interactions between two antibody chains. Hinge regions are exemplary of dimerization domains.
[0036] As used herein, “humanized antibodies” refer to antibodies that are modified to include "human" sequences of amino acids so that administration to a human does not provoke an immune response. A humanized antibody typically contains complementarily determining regions (CDRs) derived from a non-human species immunoglobulin and the remainder of the antibody molecule derived mainly from a human immunoglobulin. Methods for preparation of such antibodies are known. For example, DNA encoding a monoclonal antibody can be altered by recombinant DNA techniques to encode an antibody in which the amino acid composition of the non-variable regions is based on human antibodies. Methods for identifying such regions are known, including computer programs, which are designed for identifying the variable and non-variable regions of immunoglobulins.
[0037] As used herein, an “Ig domain” is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
[0038] As used herein, a “variable domain” or “variable region” is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity, and thus are responsible for antigen recognition. Each variable region contains CDRs that are part of the antigen-binding site domain and framework regions (FRs).
[0039] As used herein, "antigen-binding domain," "antigen-binding site," "antigen combining site" and "antibody combining site" are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with cognate antigen. A native conventional full-length antibody molecule has two conventional antigen-binding sites, each containing portions of a heavy chain variable region and portions of a light chain variable region. A conventional antigen-binding site contains the loops that connect the anti-parallel beta strands within the variable region domains. The antigen combining sites can contain other portions of the variable region domains. Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions also are called complementarity-determining regions (CDRs).
[0040] As used herein, "hypervariable region," "HV," "complementarity-determining region" and "CDR" and "antibody CDR" are used interchangeably to refer to one of a plurality of portions within each variable region that together form an antigen-binding site of an antibody. Each variable region domain contains three CDRs, named CDR1, CDR2 and CDR3. The three CDRs are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide. The CDRs are located within the loops that join the parallel strands of the beta sheets of the variable domain. As described herein, one of skill in the art knows and can identify the CDRs based on Rabat or Chothia numbering (see e.g., Rabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
[0041] As used herein, “framework regions” (FRs) are the domains within the antibody variable region domains that are located within the beta sheets; the FR regions are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
[0042] As used herein, a "constant region" or “constant domain” is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved than that of the variable region domain. In conventional full-length antibody molecules, each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, which include, CHI, CH2, CH3 and CH4. Full-length IgA, IgD and IgG isotypes contain CHI, CH2, CH3 and a hinge region, while IgE and IgM contain CHI, CH2, CH3 and CH4. CHI and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms. Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g., through interactions with various cells, biomolecules and tissues.
[0043] As used herein, a functional region of an antibody is a portion of the antibody that contains at least a VH, VL, CH (e.g. CHI, CH2 or CH3), CL or hinge region domain of the antibody, or at least a functional region thereof.
[0044] As used herein, "specifically bind" or "immunospecifically bind" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of the antibody and the antigen. The antigen can be an isolated antigen or presented in a virus. Typically, an antibody that immunospecifically binds (or that specifically binds) to a virus antigen or virus is one that binds to the virus antigen (or to the antigen in the virus or to the virus) with an affinity constant Ka of about or 1 xlO7 M-l or lx 108 M-l or greater (or a dissociation constant (Kd) of lx 107 M or 1 xlO 8 M or less). Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of anti-RSV antibodies). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335). An antibody that immunospecifically binds to a virus antigen (or virus) can bind to other peptides, polypeptides, or proteins or viruses with equal or lower binding affinity. Typically, an antibody or antigen-binding fragment thereof provided herein that binds immunospecifically to a RSV protein (or RSV virus) does not cross-react with other antigens or cross reacts with substantially (at least 10-100 fold) lower affinity for such antigens. Antibodies or antigen-binding fragments that immunospecifically bind to a particular virus antigen (e.g. a RSV protein) can be identified, for example, by immunoassays, such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELIS As), surface plasmon resonance, or other techniques known to those of skill in the art. An antibody or antigen-binding fragment thereof that immunospecifically binds to an epitope on a RSV protein typically is one that binds to the epitope (presented in the protein or virus) with a higher binding affinity than to any cross-reactive epitope as determined using experimental techniques, such as, but not limited to, immunoassays, surface plasmon resonance, or other techniques known to those of skill in the art. Immunospecific binding to an isolated RSV protein (i.e., a recombinantly produced protein), such as RSV protein, does not necessarily mean that the antibody will exhibit the same immunospecific binding and/or neutralization of the virus. Such measurements and properties are distinct. The affinity for the antibody or antigen-binding fragments for virus or the antigen as presented in the virus can be determined. For purposes herein, when describing an affinity or related term, the target, such as the isolated protein or the virus, will be identified.
[0045] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide containing the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgE, or the last three constant region immunoglobulin domains of IgE and IgM. Optionally, an Fc domain can include all or part of the flexible hinge N- terminal to these domains. For IgA and IgM, Fc can include the J chain. For an exemplary Fc domain of IgG, Fc contains immunoglobulin domains Cy2 and Cy3, and optionally, all or part of the hinge between Cyl and Cy2. The boundaries of the Fc region can vary, but typically, include at least part of the hinge region. In addition, Fc also includes any allelic or species variant or any variant or modified form, such as any variant or modified form that alters the binding to an FcR or alters an Fc-mediated effector function.
[0046] As used herein, a "tag" or an "epitope tag" refers to a sequence of amino acids, typically added to the N- or C- terminus of a polypeptide, such as an antibody provided herein. The inclusion of tags fused to a polypeptide can facilitate polypeptide purification and/or detection. Typically, a tag or tag polypeptide refers to polypeptide that has enough residues to provide an epitope recognized by an antibody or can serve for detection or purification, yet is short enough such that it does not interfere with activity of chimeric polypeptide to which it is linked. The tag polypeptide typically is sufficiently unique so an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues and usually between about 8-50 amino acid residues, typically between 9-30 residues. The tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture. Such tags are well known and can be readily synthesized and designed. Exemplary tag polypeptides include those used for affinity purification and include, His tags, the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5, (Field et al. (1988) Mol. Cell. Biol. 8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (see, e.g., Evan et al. (1985) Molecular and Cellular Biology 5 :3610-3616); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. (1990) Protein Engineering 3:547- 553 (1990). An antibody used to detect an epitope-tagged antibody is typically referred to herein as a secondary antibody.
[0047] As used herein, "polypeptide" refers to two or more amino acids covalently joined.
The terms "polypeptide" and "protein" are used interchangeably herein.
[0048] As used herein, a "peptide" refers to a polypeptide that is from 2 to about or 40 amino acids in length.
[0049] As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids contained in the antibodies provided include the twenty naturally- occurring amino acids, non-natural amino acids, and amino acid analogs (e.g., amino acids wherein the a-carbon has a side chain). As used herein, the amino acids, which occur in the various amino acid sequences of polypeptides appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations. The nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard single-letter designations used routinely in the art.
[0050] As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NFh refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and adopted at 37 C.F.R. §§ 1.821 - 1.822, abbreviations for amino acid residues are used throughout. All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase "amino acid residue" is defined to include natural, modified, non-natural and unusual amino acids. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NFh or to a carboxyl- terminal group such as COOH. As used herein, "naturally occurring amino acids" refer to the 20 L-amino acids that occur in polypeptides. [0051] As used herein, an "activity" or a "functional activity" of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, ability to interact with a biomolecule, for example, through antigen-binding, DNA binding, ligand binding, or dimerization, enzymatic activity, for example, kinase activity or proteolytic activity. For an antibody (including antibody fragments), activities include, but are not limited to, the ability to specifically bind a particular antigen, affinity of antigen-binding (e.g. high or low affinity), avidity of antigen-binding (e.g. high or low avidity), on-rate, off-rate, effector functions, such as the ability to promote antigen neutralization or clearance, virus neutralization, and in vivo activities, such as the ability to prevent infection or invasion of a pathogen, or to promote clearance, or to penetrate a particular tissue or fluid or cell in the body or improved manufacturability, thermostability, or protease resistance. Activity can be assessed in vitro or in vivo using recognized assays, such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure on- or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry and binding assays (e.g., panning assays). For example, for an antibody polypeptide, activities can be assessed by measuring binding affinities, avidities, and/or binding coefficients (e.g., for on-/off-rates), and other activities in vitro or by measuring various effects in vivo, such as immune effects, e.g. antigen clearance, penetration or localization of the antibody into tissues, protection from disease, e.g. infection, serum or other fluid antibody titers, or other assays that are well known in the art. The results of such assays that indicate that a polypeptide exhibits an activity can be correlated to activity of the polypeptide in vivo, in which in vivo activity can be referred to as therapeutic activity, or biological activity. Activity of a modified polypeptide can be any level of percentage of activity of the unmodified polypeptide, including but not limited to, 1 % of the activity, 2 %, 3 %, 4 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 100 %, 200 %, 300 %, 400 %, 500 %, or more of activity compared to the unmodified polypeptide. Assays to determine functionality or activity of modified (e.g. variant) antibodies are well known in the art.
[0052] As used herein, "exhibits at least one or more improved properties" refers to the activity exhibited by a modified polypeptide, such as a variant polypeptide produced according to the provided methods, such as a modified, e.g. variant antibody or other therapeutic polypeptide (e.g. a modified anti-RSV antibody or antigen-binding fragment thereof), compared to the target or unmodified polypeptide, that does not contain the modification. A modified, or variant, polypeptide that retains an activity of a target polypeptide can exhibit improved activity (for example one or more of improved manufacturability, thermostability, or protease resistance) or maintain the activity of the unmodified polypeptide. In some instances, a modified, or variant, polypeptide can retain an activity that is increased compared to an target or unmodified polypeptide. In some cases, a modified, or variant, polypeptide can retain an activity that is decreased compared to an unmodified or target polypeptide. Activity of a modified, or variant, polypeptide can be any level of percentage of activity of the unmodified or target polypeptide, including but not limited to, 1 % of the activity, 2 %, 3 %, 4 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 100 %, 200 %, 300 %, 400 %, 500 %, or more activity compared to the unmodified or target polypeptide. In other embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times,
6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times greater than unmodified or target polypeptide. Assays for retention of an activity depend on the activity to be retained. Such assays can be performed in vitro or in vivo. Activity can be measured, for example, using assays known in the art and described in the Examples below for activities such as but not limited to ELISA and panning assays. Activities of a modified, or variant, polypeptide compared to an unmodified or target polypeptide also can be assessed in terms of an in vivo therapeutic or biological activity or result following administration of the polypeptide.
[0053] As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages. Also included in the term "nucleic acid" are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid).
The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.
[0054] As used herein, “regulatory region” of a nucleic acid molecule means a cis-acting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.
[0055] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.
[0056] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.
[0057] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, nucleic acid encoding a leader peptide can be operably linked to nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.
[0058] As used herein, "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.
[0059] As used herein, “production by recombinant means by using recombinant DNA methods” means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.
[0060] As used herein, "expression" refers to the process by which polypeptides are produced by transcription and translation of polynucleotides. The level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.
[0061] As used herein, a "host cell" is a cell that is used in to receive, maintain, reproduce and amplify a vector. A host cell also can be used to express the polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids. In one example, the host cell is a genetic package, which can be induced to express the variant polypeptide on its surface. In another example, the host cell is infected with the genetic package. For example, the host cells can be phage- display compatible host cells, which can be transformed with phage or phagemid vectors and accommodate the packaging of phage expressing fusion proteins containing the variant polypeptides.
[0062] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation. Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression/display of the polypeptide encoded by the nucleic acid. The vectors typically remain episomal but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
[0063] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
[0064] As used herein, "similarity" between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. "Identity" refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).
[0065] As used herein, when a polypeptide or nucleic acid molecule or region thereof contains or has "identity" or "homology" to another polypeptide or nucleic acid molecule or region, the two molecules and/or regions share greater than or equal to at or about 40 % sequence identity, and typically greater than or equal to at or about 50 % sequence identity, such as at least or about 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % sequence identity; the precise percentage of identity can be specified if necessary. A nucleic acid molecule, or region thereof, that is identical or homologous to a second nucleic acid molecule or region can specifically hybridize to a nucleic acid molecule or region that is 100 % complementary to the second nucleic acid molecule or region. Identity alternatively can be compared between two theoretical nucleotide or amino acid sequences or between a nucleic acid or polypeptide molecule and a theoretical sequence.
[0066] Sequence "identity," per se, has an art-recognized meaning and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, e.g. : Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptides, the term "identity" is well known to skilled artisans (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).
[0067] Sequence identity compared along the full length of two polynucleotides or polypeptides refers to the percentage of identical nucleotide or amino acid residues along the full-length of the molecule. For example, if a polypeptide A has 100 amino acids and polypeptide B has 95 amino acids, which are identical to amino acids 1-95 of polypeptide A, then polypeptide B has 95 % identity when sequence identity is compared along the full length of a polypeptide A compared to full length of polypeptide B. Alternatively, sequence identity between polypeptide A and polypeptide B can be compared along a region, such as a 20 amino acid analogous region, of each polypeptide. In this case, if polypeptide A and B have 20 identical amino acids along that region, the sequence identity for the regions is 100 %. Alternatively, sequence identity can be compared along the length of a molecule, compared to a region of another molecule. Alternatively, sequence identity between polypeptide A and polypeptide B can be compared along the same length polypeptide but with amino acid replacements, such as conservative amino acid replacements or non conservative amino acid replacements. As discussed below, and known to those of skill in the art, various programs and methods for assessing identity are known to those of skill in the art. High levels of identity, such as 90 % or 95 % identity, readily can be determined without software.
[0068] Whether any two nucleic acid or polypeptide molecules have nucleotide sequences that are at least or about 60 %, 70 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 % or 99 % "identical" can be determined using known computer algorithms such as the "FASTA" program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J. et al. (1984) Nucleic Acids Research 12(I):387), BLASTP, BLASTN, FASTA (Altschul, S.F. et al. (1990) J. Molec. Biol. 215:403; Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carrillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar "MegAlign" program (Madison, WI) and the University of Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison WI)). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
[0069] As used herein, a "modification" is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.
[0070] As used herein, "substitution" refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule. Thus, one or more substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule. Substitution mutations compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence.
[0071] As used herein, the phase "having the same binding specificity" when used to describe an antibody in reference to another antibody, means that the antibody specifically binds (immunospecifically binds or specifically binds to the virus) to all or a part of the same antigenic epitope as the reference antibody. The epitope can be in the isolated protein, or in the protein in the virus. The ability of two antibodies to bind to the same epitope can be determined by known assays in the art such as, for example, surface plasmon resonance assays and antibody competition assays. Typically, antibodies that immunospecifically bind to the same epitope can compete for binding to the epitope, which can be measured, for example, by an in vitro binding competition assay (e.g. competition ELISA), using techniques known the art. Typically, a first antibody that immunospecifically binds to the same epitope as a second antibody can compete for binding to the epitope by about or 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 100 %, where the percentage competition is measured ability of the second antibody to displace binding of the first antibody to the epitope. In exemplary competition assays, the antigen is incubated in the presence a predetermined limiting dilution of a labeled antibody (e.g., 50- 70% saturation concentration), and serial dilutions of an unlabeled competing antibody. Competition is determined by measuring the binding of the labeled antibody to the antigen for any decreases in binding in the presence of the competing antibody. Variations of such assays, including various labeling techniques and detection methods including, for example, radiometric, fluorescent, enzymatic and colorimetric detection, are known in the art. The ability of a first antibody to bind to the same epitope as a second antibody also can be determined, for example, by virus neutralization assays using Monoclonal Antibody- Resistant Mutants (MARMs). For example, where a first anti-RSV antibody neutralizes wild- type RSV but not a particular mutant RSV, a second antibody that neutralizes the wild- type RSV but not the particular mutant RSV generally binds the same epitope on RSV as the first antibody. Where a first anti-RSV antibody neutralizes wild-type RSV but not a particular mutant RSV, a second antibody that neutralizes the wild-type RSV and the particular mutant RSV generally does not bind the same epitope on RSV as the first antibody.
[0072] As used herein, “disease or disorder” refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those involving RSV infection or those that increase the risk of a RSV infection.
[0073] As used herein, “infection” and “RSV infection” refer to all stages of a RSV life cycle in a host (including, but not limited to the invasion by and replication of RSV in a cell or body tissue), as well as the pathological state resulting from the invasion by and replication of a RSV. The invasion by and multiplication of a RSV includes, but is not limited to, the following steps: the docking of the RSV particle to a cell, fusion of a virus with a cell membrane, the introduction of viral genetic information into a cell, the expression of RSV proteins, the production of new RSV particles and the release of RSV particles from a cell. A RSV infection can be an upper respiratory tract RSV infection (URI), a lower respiratory tract RSV infection (LRI), or a combination thereof. In some examples, the pathological state resulting from the invasion by and replication of a RSV is an acute RSV disease.
[0074] As used herein, “acute RSV disease” refers to clinically significant disease in the lungs or lower respiratory tract as a result of a RSV infection, which can manifest as pneumonia and/or bronchiolitis, where such symptoms can include, for example, hypoxia, apnea, respiratory distress, rapid breathing, wheezing, and cyanosis. Acute RSV disease requires an affected individual to obtain medical intervention, such as hospitalization, administration of oxygen, intubation and/or ventilation.
[0075] As used herein, “treating” a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated or remain static following treatment. Hence treatment encompasses prophylaxis, therapy and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. Treatment also encompasses any pharmaceutical use of any antibody or antigen-binding fragment thereof provided or compositions provided herein.
[0076] As used herein, “prevention” or prophylaxis, and grammatically equivalent forms thereof, refers to methods in which the risk of developing disease or condition is reduced.
[0077] As used herein, a “pharmaceutically effective agent” includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.
[0078] As used herein, a “therapeutic effect” means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition.
[0079] As used herein, a “therapeutically effective amount” or a “therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder. [0080] As used herein, “therapeutic efficacy” refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the an agent, compound, material, or composition containing a compound has been administered.
[0081] As used herein, a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection. The full prophylactic effect does not necessarily occur by administration of one dose and can occur only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
[0082] As used herein, the terms “immunotherapeutically” or “immunotherapy” in conjunction with antibodies provided denotes prophylactic as well as therapeutic administration. Thus, the therapeutic antibodies provided can be administered to a subject at risk of contracting a virus infection (e.g. a RSV infection) in order to lessen the likelihood and/or severity of the disease or administered to subjects already evidencing active virus infection (e.g. a RSV infection).
[0083] As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.
[0084] As used herein, a “label” or “detectable moiety” is a detectable marker (e.g., a fluorescent molecule, chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be attached or linked directly or indirectly to a molecule (e.g., an anti -RSV antibody or antigen-binding fragment thereof provided herein) or associated therewith and can be detected in vivo and/or in vitro. The detection method can be any method known in the art, including known in vivo and/or in vitro methods of detection (e.g., imaging by visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signal, X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET) scanning, single-photon emission computed tomography (SPECT)), fluorescence spectroscopy or absorption). Indirect detection refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of an atom, molecule or composition that binds directly or indirectly to the detectable moiety (e.g., detection of a labeled secondary antibody or antigen-binding fragment thereof that binds to a primary antibody (e.g., an anti-RSV antibody or antigen-binding fragment thereof provided herein).
[0085] As used herein, the term “individual” refers to an animal, including a mammal, such as a human being.
[0086] As used herein, “animal” includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; pigs and other animals. Non-human animals exclude humans as the contemplated animal.
[0087] As used herein, a “human infant” refers to a human less than or about 24 months (e.g., less than or about 16 months, less than or about 12 months, less than or about 6 months, less than or about 3 months, less than or about 2 months, or less than or about 1 month of age). Typically, the human infant is bom at more than 38 weeks of gestational age.
[0088] As used herein, a “human infant bom prematurely” refers to a human bom at less than or about 40 weeks gestational age, typically, less than or about 38 weeks gestational age.
[0089] As used herein, an “isolated” or “purified” polypeptide or protein (e.g. an isolated antibody or antigen-binding fragment thereof) or biologically-active portion thereof (e.g. an isolated antigen-binding fragment) is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. As used herein, a “cellular extract” or “lysate” refers to a preparation or fraction which is made from a lysed or disrupted cell.
[0090] As used herein, “combination therapy” refers to administration of two or more different therapeutics, such as two or more different anti-RSV antibodies and/or anti- RSV antibodies and antigen-binding fragments thereof. The different therapeutic agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.
[0091] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of - 10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.
[0092] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.
[0093] As used herein, “optional” or “optionally” means that the subsequently circumstance or limitation on scope does or does not occur, and that the description includes instances where the circumstance or limitation on scope occurs and instances where it does not. For example, an a composition that optionally contains additional exogenous enzymes means that the enzymes can be present or not present in the composition.
[0094] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. [0095] It is also noted that the term “consisting essentially of,” as used herein refers to a composition wherein the component(s) after the term is in the presence of other known component(s) in a total amount that is less than 30% by weight of the total composition and do not contribute to or interferes with the actions or activities of the component(s).
[0096] It is further noted that the term "comprising,” as used herein, means including, but not limited to, the component(s) after the term “comprising.” The component(s) after the term “comprising” are required or mandatory, but the composition comprising the component(s) can further include other non-mandatory or optional component(s).
[0097] It is also noted that the term “consisting of,” as used herein, means including, and limited to, the component(s) after the term "consisting of.” The component(s) after the term “consisting of’ are therefore required or mandatory, and no other component(s) are present in the composition.
[0098] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[0099] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0100] Other definitions of terms may appear throughout the specification.
II. Compositions
A. Anti RSV -antibodies
[0101] Provided herein are anti -RSV antibodies or antigen-binding fragments thereof that can be employed for therapeutic, prophylactic and diagnostic use. The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be used, for example, for passive immunization of a subject against RSV or for treatment of a subject with a viral infection. In one example, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are used for prophylaxis, i. e.. the prevention of RSV infection. In another example, the anti- RSV antibodies or antigen-binding fragments thereof provided herein are used as therapeutic antibodies, i.e., for treatment of a RSV viral infection. In yet another example, the anti- RSV antibodies or antigen-binding fragments thereof provided herein are used for passive immunization of a subject against RSV. The provided anti-RSV antibodies or antigen-binding fragments thereof also can be used for detection of a RSV infection or for monitoring RSV infection in vitro and in vivo.
[0102] Full-length antibodies contain multiple chains, domains and regions. A full length conventional antibody contains two heavy chains and two light chains, each of which contains a plurality of immunoglobulin (Ig) domains. An Ig domain is characterized by a structure called the Ig fold, which contains two beta-pleated sheets, each containing anti parallel beta strands connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. The Ig domains in the antibody chains are variable (V) and constant (C) region domains. Each heavy chain is linked to a light chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. Linkage of the heavy chains is mediated by a flexible region of the heavy chain, known as the hinge region.
[0103] Each full-length conventional antibody light chain contains one variable region domain (VL) and one constant region domain (CL). Each full-length conventional heavy chain contains one variable region domain (VH) and three or four constant region domains (CH) and, in some cases, hinge region. Owing to recombination events discussed above, nucleic acid sequences encoding the variable region domains differ among antibodies and confer antigen-specificity to a particular antibody. The constant regions, on the other hand, are encoded by sequences that are more conserved among antibodies. These domains confer functional properties to antibodies, for example, the ability to interact with cells of the immune system and serum proteins in order to cause clearance of infectious agents. Different classes of antibodies, for example IgM, IgD, IgG, IgE and IgA, have different constant regions, allowing them to serve distinct effector functions.
[0104] Each variable region domain contains three portions called complementarity determining regions (CDRs) or hypervariable (HV) regions, which are encoded by highly variable nucleic acid sequences. The CDRs are located within the loops connecting the beta sheets of the variable region Ig domain. Together, the three heavy chain CDRs (CDR1,
CDR2 and CDR3) and three light chain CDRs (CDR1, CDR2 and CDR3) make up a conventional antigen-binding site (antibody combining site) of the antibody, which physically interacts with cognate antigen and provides the specificity of the antibody. A whole antibody contains two identical antibody combining sites, each made up of CDRs from one heavy and one light chain. Because they are contained within the loops connecting the beta strands, the three CDRs are non-contiguous along the linear amino acid sequence of the variable region. Upon folding of the antibody polypeptide, the CDR loops are in close proximity, making up the antigen combining site. The beta sheets of the variable region domains form the framework regions (FRs), which contain more conserved sequences that are important for other properties of the antibody, for example, stability.
[0105] The anti-RSV antibodies provided herein exhibit one or more properties that are advantageous or different from anti-RSV antibodies in the art (such as, for example, improved manufacturability, thermostability, and/or protease resistance).
[0106] In some embodiments, provided herein are isolated anti-respiratory syncytial virus (RSV) antibodies or functional fragments thereof having a heavy chain variable region which has at least about 90% (such as any of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to
QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RLTI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, T, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; XOs A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 1S A or G; and/or a heavy chain constant region comprising ASTKGPSVFPLAPSX8KX9TSX10GTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDTRXiiEPKSCDKTHTCPPCPAPELlGGPSVFLFPPKPKDTLMISRTPE VTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQD WLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein Xs is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y. The antibody can differ by at least one amino acid (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids) from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO: 3. Additionally, the antibody can exhibit one or more improved properties including, but not limited to, increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3.
[0107] The antibody or functional fragment thereof can comprise or further comprise a light chain variable region which has at least 90% (such as any of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to DIQMX12 X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21LIYDTSK LASGVPSRFSGSGSX22X23E
X24TLTIS SLQPDDF ATYY CFQGS GYPFTF GGGTKLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X201S A, S, V, orW; X211S L, S, or G; X221S G or A; X231S T or Y ; and X24 is F or C; and/or a light chain constant region comprising AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein optionally the antibody differs by at least one amino acid (such as any of 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids) from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO:6.
[0108] In some embodiments, the antibody exhibits increased or improved manufacturability compared to a parent antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and or the light chain encoded by the amino acid sequence of SEQ ID NO:6. For example, the antibody may display reduced aggregation-propensity, and/or increased productivity upon expression, relative to the parent antibody. For example, the ant-RSV antibody may display an increase in productivity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% relative to the parent antibody and/or a decrease in aggregation ( i.e . a reduction in the proportion of molecules in the native state ensemble which are aggregated) of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 90% or at least 99% relative to the parent antibody. The anti-RSV antibody can display a decrease in aggregation of up to 100% relative to the parent immunoglobulin (i.e. complete abolition of aggregation). [0109] Improvements in manufacturability may result fully or partially from reduced aggregation-propensity relative to the parent immunoglobulin. “Aggregation-propensity” relates to the tendency of the anti-RSV antibody to form insoluble aggregates after expression in a recombinant system. Reductions in aggregation propensity reduce the proportion of molecules in the native state ensemble of the immunoglobulin which exist in an aggregated form (Carpenter et al, 2009 J Pharm Sci. April; 98(4): 1201-5). In other words, the proportion of molecules within the native state ensemble of the anti-RSV antibody which exist in an aggregated or insoluble form is lower than the proportion within the native state ensemble of the parent antibody.
[0110] An anti-RSV antibody disclosed herein may show less self-association or aggregation compared to a parent antibody either under native conditions or at increased temperature (e.g. 60° C.) (i.e. conditions under which the antigen binding site of an immunoglobulin does not unfold). Preferably, the anti-RSV antibody shows less self-association or aggregation than parent antibody under native conditions (e.g. conditions which do not lead to unfolding of the antibody).
[0111] Aggregation propensity as described herein is distinct from thermal refolding efficiency (TRE), which relates to the ability of a protein to correctly refold after thermal denaturation and is typically measured using circular dichroism (CD) (Tanha et al Protein Eng Des Sel. 2006 November; 19(11):503-9). Reductions in the aggregation propensity of an antibody as described herein may have little or no effect on the thermal refolding efficiency of the immunoglobulin. Thermal refolding efficiency is therefore independent of aggregation propensity and does not have a significant impact on the manufacturability of an antibody.
[0112] Aggregation may be measured by conventional methods. Suitable techniques include GP-HPLC, HPLC and AUC (Gabrielson J P et al J Pharm Sci 2007 96(2): 268-79), protein loss after filtration; turbidity; fluorescent dye binding (e.g. Nile Red, thioflavin T or 8- anilino-l-napthalenesulfonic acid; see for example Hawe, A. et al Pharmaceutical Research 200825 (7) 1487-99 or Demeule, B et al 2007 Int J Pharm 329: 37-45), field-flow fractionation (FFF; Demeule, B et al. mAbs 2009 1(2): 142-150), and analytical ultracentrifugation (AU/AUC; Liu J et cal. AAPS J 2006 8: 580-9). Other suitable methods are described in Arvinte T. In “Methods for structural analysis of protein pharmaceuticals” AAPS Press, 2005: 661-6 and Kiese S et al J Pharm Sci 2008 97(10): 4347-66. [0113] Improvements in manufacturability may result fully or partially from increased productivity relative to the parent antibody. An anti-RSV antibody disclosed herein may display increased productivity than the parent antibody. For example, the variant immunoglobulin may show increased yields or titers compared to a parent immunoglobulin when expressed in a recombinant system, e.g. bacterial or mammalian cells. Productivity may be measured using standard techniques, such as the Bradford assay, spectrophotometry and ELISA.
[0114] An anti-RSV antibody disclosed herein may also display one or more of improved purification yields; reduced formulation problems; reduced immunogenicity and increased bioavailability relative to the parent antibody. In some embodiments, improvements in manufacturability may result from both reduced aggregation-propensity and increased productivity relative to the parent antibody. In some embodiments, an anti-RSV antibody disclosed herein displays the same or substantially the same activity as the parent antibody (i.e. antigen binding activity).
[0115] In some embodiments, the antibody exhibits increased or improved or enhanced thermostability compared to a parent antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and or the light chain encoded by the amino acid sequence of SEQ ID NO:6. As used herein, "thermostabilized" or "thermostability" refers to the quality of a protein or antibody to resist chemical or physical change as a result of increasing temperature. For the purposes of this invention, alterations to the amino acid sequence of an antibody may be made to increase the thermostability of said antibody compared to the parent antibody. Thermostability may be determined by any known method in the field, including the measurement of the antibody melting temperature (TM). Improvements in thermostability include increases in the TM by greater than or equal to 0.1 °C to greater than or equal to 10.0 °C. For example, the antibody may display increased thermostability, relative to the parent antibody. For example, the anti-RSV antibody may display an increase in thermostability of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% relative to the parent antibody.
[0116] In some embodiments, the antibody exhibits increased or improved or enhanced protease resistance compared to a parent antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and or the light chain encoded by the amino acid sequence of SEQ ID NO: 6. The term “protease resistant” refers to the ability of a molecule comprised of peptide bonds, to resist hydrolytic cleavage of one or more of its peptide bonds in the presence of a proteolytic enzyme. The resistance to proteolytic enzymes is a relative property and is compared to a molecule (such as a parent antibody) which is less able to withstand hydrolytic cleavage of one or more of its peptide bonds over a specified time period and under specified conditions, including the pH and or temperature at which the cleavage resistance is tested. One result of proteolytic cleavage indicative that cleavage has occurred is the generation of smaller fragments (lower molecular weight) as compared to the molecular weight of the intact, non-cleaved parent molecule. An anti-RSV antibody or a fragment thereof disclosed herein comprising a hinge, a CH2 domain and a CH3 domain is “protease resistant” or “resistant to proteolysis” or has “increased resistance to proteolysis” when more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a full length antibody (such as any of the anti-RSV antibodies disclosed herein) remains intact for a given period of time (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) when digested by a protease (such as, but not limited to, pepsin, matrix metalloprotease-3 (MMP-3), matrix metalloprotease-12 (MMP- 12), glutamyl endopeptidase V8 of Staphylococcus aureus (GluV8), or immunoglobulin degrading enzyme of Streptococcus pyogenes (IdeS)) in a given buffer (e.g., Tris-buffered saline) at a given temperature (e.g., at 37° C) at a given pH (e.g., at pH 7.5) at a given antibody concentration (e.g. of 0.5 mg/ml) with a given protease concentration (such as about approximately 1-2% (w/w) ratio to IgG). Amount of intact IgG can be assessed by SDS- PAGE.
B. Modification of Anti-RSV Antibodies
[0117] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be further modified. Modifications of an anti-RSV antibody or antigen-binding fragment can improve one or more properties of the antibody, including, but not limited to, decreasing the immunogenicity of the antibody or antigen-binding fragment, improving the half-life of the antibody or antigen-binding fragment, such as reducing the susceptibility to proteolysis and/or reducing susceptibility to oxidation, and altering or improving of the binding properties of the antibody or antigen-binding fragment thereof. Exemplary modifications include, but are not limited to, modifications of the primary amino acid sequence of the anti- RSV antibody or antigen-binding fragment thereof and alteration of the post-translational modification of the anti-RSV antibody or antigen-binding fragment thereof. Exemplary post- translational modifications include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with protecting/blocking group, proteolytic cleavage, linkage to a cellular ligand or other protein. Other exemplary modifications include attachment of one or more heterologous peptides to the anti-RSV antibody or antigen-binding fragment to alter or improve one or more properties of the antibody or antigen-binding fragment thereof.
[0118] Generally, the modifications do not result in increased immunogenicity of the antibody or antigen-binding fragment thereof or significantly negatively affect the binding of the antibody or antigen-binding fragment thereof to RSV. Methods of assessing the binding of the modified antibodies or antigen-binding fragments thereof to a RSV are provided herein and known in the art. For example, modified antibodies or antigen-binding fragments thereof can be assayed for binding to RSV by methods such as, but not limited to, ELISA, surface plasmon resonance (SPR), or through in vitro microneutralization assays.
[0119] Provided herein are methods of improving the half-life of any of the provided anti- RSV antibodies or antigen-binding fragments thereof. Increasing the half-life of the anti- RSV antibodies or antigen-binding fragments thereof provided herein can increase the therapeutic effectiveness of the anti-RSV antibodies or antigen-binding fragments thereof and allow for less frequent administration of the antibodies or antigen-binding fragments thereof for prophylaxis and/or treatment, such as preventing or treating a RSV infection, preventing, treating, and/or alleviating of one or more symptoms of a RSV infection, or reducing the duration of a RSV infection.
[0120] Modification of the anti-RSV antibodies or antigen-binding fragments thereof produced herein can include one or more amino acid substitutions, deletions or additions, either from natural mutation or human manipulation from the parent antibody. Methods for modification of polypeptides, such as antibodies, are known in the art and can be employed for the modification of any antibody or antigen-binding fragment thereof provided herein. In some examples, the pharmacokinetic properties of the anti-RSV antibodies or antigen binding fragments thereof provided herein can be enhanced through Fc modifications by techniques known to those skilled in the art. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide molecule encoding an antibody or an antigen-binding fragment provided herein in order to produce an polypeptide with one or more amino acid substitutions. Exemplary techniques for introducing mutations include, but are not limited to, site-directed mutagenesis and PCR-mediated mutagenesis.
[0121] The anti-RSV antibodies and antigen-binding fragments thereof provided herein can be modified by the attachment of a heterologous peptide to facilitate purification. Generally, such peptides are expressed as a fusion protein containing the antibody fused to the peptide at the C- or N-terminus of the antibody or antigen-binding fragment thereof. Exemplary peptides commonly used for purification include, but are not limited to, hexa-histidine peptides, hemagglutinin (HA) peptides, and flag tag peptides (see e.g., Wilson et al.
(1984) Cell 37:767; Witzgall et al. (1994) Anal Biochem 223:2, 291-8). The fusion does not necessarily need to be direct, but can occur through a linker peptide. In some examples, the linker peptide contains a protease cleavage site which allows for removal of the purification peptide following purification by cleavage with a protease that specifically recognizes the protease cleavage site.
[0122] The anti-RSV antibodies and antigen-binding fragments thereof provided herein also can be modified by the attachment of a heterologous polypeptide that targets the antibody or antigen-binding fragment to a particular cell type (e.g., respiratory epithelial cells), either in vitro or in vivo. In some examples an anti-RSV antibody or antigen-binding fragment thereof provided herein can be targeted to a particular cell type by fusing or conjugating the antibody or antigen-binding fragment thereof to an antibody specific for a particular cell surface receptor or other polypeptide that interacts with a specific cell receptor.
[0123] In some examples, an anti-RSV antibody or antigen-binding fragment thereof provided herein can be targeted to a target cell surface and/or taken up by the target cell by fusing or conjugating the antibody or antigen-binding fragment thereof to a peptide that binds to cell surface glycoproteins, such as a protein transduction domain (e.g., a TAT peptide). Exemplary protein transduction domains include, but are not limited to, PTDs derived from proteins such as human immunodeficiency virus 1 (HIV-1) TAT (Ruben et al. (1989) J.
Virol. 63:1-8; e.g., SEQ ID NOS:326-337, such as for example, GRKKRRQRRR (TAT 48- 57) SEQ ID NO:330)), the herpes virus tegument protein VP22 (Elliott and O'Hare (1997) Cell 88:223-233; e.g., SEQ ID NO:342), the homeotic protein of Drosophila melanogaster Antennapedia (Antp) protein (Penetratin PTD; Derossi et al. (1996) J. Biol. Chem. 271:18188-18193; e.g., SEQ ID NOS:311-314), the protegrin 1 (PG-1) anti-microbial peptide SynB (e.g., SynBl, SynB3, and Syn B4; Kokryakov et al. (1993) FEBS Lett. 327:231-236; e.g., SEQ ID NOS:323-325, respectively) and basic fibroblast growth factor (Jans (1994) FASEB J. 8:841-847; e.g., SEQ ID NO:307). PTDs also include synthetic PTDs, such as, but not limited to, polyarginine peptides (Futaki et al. (2003) J. Mol.
Recognit. 16:260-264; Suzuki et al. (2001) J. Biol. Chem. 276:5836-5840; e.g. SEQ ID NOS:315-316), transportan (Pooga et al. (1988) FASEB J. 12:67-77; Pooga et al.
(2001) FASEB J. 15:1451-1453; e.g, SEQ ID NOS:338-341), MAP (Oehlke et al.
(1998) Biochim. Biophys. Acta. 1414:127-139; e.g, SEQ ID NO:305), KALA (Wyman et al. (1997) Biochemistry 36:3008-3017; e.g., SEQ ID NO:303) and other cationic peptides, such as, for example, various b-cationic peptides (Akkarawongsa et al. (2008) Antimicrob. Agents and Chemother. 52(6):2120-2129).
[0124] The anti-RSV antibodies and antigen-binding fragments thereof provided herein can be modified by the attachment of diagnostic and/or therapeutic moiety to the antibody or antigen-binding fragment thereof. The anti-RSV antibodies and antigen-binding fragments thereof provided herein can be modified by the covalent attachment of any type of molecule, such as a diagnostic or therapeutic molecule, to the antibody or antigen-binding fragment thereof such that covalent attachment does not prevent the antibody or antigen-binding fragment thereof from binding to its corresponding epitope. For example, an anti- RSV antibody or antigen-binding fragment thereof provided herein can be further modified by covalent attachment of a molecule such that the covalent attachment does not prevent the antibody or antigen-binding fragment thereof from binding to RSV. In some examples, the antibodies or antigen-binding fragments thereof can be recombinantly fused to a heterologous polypeptide at the N terminus or C terminus or chemically conjugated, including covalent and non-covalent conjugation, to a heterologous polypeptide or other composition. For example, the heterologous polypeptide or composition can be a diagnostic polypeptide or other diagnostic moiety or a therapeutic polypeptide or other therapeutic moiety. Exemplary diagnostic and therapeutic moieties include, but are not limited to, drugs, radionucleotides, toxins, fluorescent molecules (see, e.g. International PCT Publication Nos. WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387). Diagnostic polypeptides or diagnostic moieties can be used, for example, as labels for in vivo or in vitro detection. Therapeutic polypeptides or therapeutic moieties can be used, for example, for therapy of a viral infection, such as RSV infection, or for treatment of one or more symptoms of a viral infection. [0125] Additional fusion proteins of the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be generated through the techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling can be employed to alter the activities of anti-RSV antibodies or antigen-binding fragments thereof provided herein, for example, to produce antibodies or antigen-binding fragments thereof with higher affinities and lower dissociation rates (see, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al. (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama (1998) Trends Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo and Blasco (1998) Biotechniques 24(2):308-13).
[0126] The provided anti-RSV antibodies or antigen-binding fragments thereof can also be attached to solid supports, which are useful for immunoassays or purification of the target antigen. Exemplary solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[0127] In some examples, the antibodies or antigen-binding fragments thereof provided herein can be further modified to reduce the immunogenicity in a subject, such as a human subject. For example, one or more amino acids in the antibody or antigen-binding fragment thereof can be modified to alter potential epitopes for human T-cells in order to eliminate or reduce the immunogenicity of the antibody or antigen-binding fragment thereof when exposed to the immune system of the subject. Exemplary modifications include substitutions, deletions and insertion of one or more amino acids, which eliminate or reduce the immunogenicity of the antibody or antigen-binding fragment thereof. Generally, such modifications do not alter the binding specificity of the antibody or antigen-binding fragment thereof for its respective antigen. Reducing the immunogenicity of the antibody or antigen binding fragment thereof can improve one or more properties of the antibody or antigen binding fragment thereof, such as, for example, improving the therapeutic efficacy of the antibody or antigen-binding fragment thereof and/or increasing the half-life of the antibody or antigen-binding fragment thereof in vivo.
[0128] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be conjugated to polymer molecules such as high molecular weight polyethylene glycol (PEG) to increase half-life and/or improve their pharmacokinetic profiles. Conjugation can be carried out by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function (see, e.g., Deckert et al., Int. J. Cancer 87: 382-390, 2000; Knight et al., Platelets 15: 409-418, 2004; Leong et al., Cytokine 16: 106-119, 2001; and Yang et al., Protein Eng. 16: 761-770, 2003). PEG can be attached to the antibodies or antigen binding fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or antigen-binding fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity can be used. The degree of conjugation can be monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography. PEG-derivatized antibodies or antigen-binding fragments thereof can be tested for binding activity to RSV antigens as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.
[0129] In some examples, the anti-RSV antibodies and antibody fragments provided herein can be further modified by conjugation to a detectable moiety. The detectable moieties can be detected directly or indirectly. Depending on the detectable moiety selected, the detectable moiety can be detected in vivo and/or in vitro. The detectable moieties can be employed, for example, in diagnostic methods for detecting exposure to RSV or localization of RSV or binding assays for determining the binding affinity of the anti-RSV antibody or antigen binding fragment thereof for RSV. The detectable moieties also can be employed in methods of preparation of the anti-RSV antibodies, such as, for example, purification of the antibody or antigen-binding fragment thereof. Typically, detectable moieties are selected such that conjugation of the detectable moiety does not interfere with the binding of the antibody or antigen-binding fragment thereof to the target epitope. Generally, the choice of the detectable moiety depends on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. One of skill in the art is familiar with labels and can identify a detectable label suitable for and compatible with the assay employed. Methods of labeling antibodies with detectable moieties are known in the art and include, for example, recombinant and chemical methods.
[0130] The detectable moiety can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied in the methods provided. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), in particular, gamma and positron emitting radioisotopes (e.g., 157Gd, 55Mn, 162Dy, 52Cr, and 56Fe), metallic ions, enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), electron transfer agents (e.g., including metal binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), magnetic beads (e.g., DYNABEADS™), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). For a review of various labeling or signal producing systems that can be used, see e.g. U.S. Pat. No. 4,391,904.
[0131] In some examples, the anti-RSV antibodies and antigen-binding fragments provided herein can be further modified by conjugation to a therapeutic moiety. Exemplary therapeutic moieties include, but are not limited to, a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive metal ion (e.g., alpha-emitters). Exemplary cytotoxin or cytotoxic agents include, but are not limited to, any agent that is detrimental to cells, such as, but not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Exemplary therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracy dines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), anti-mitotic agents (e.g., vincristine and vinblastine), and antivirals, such as, but not limited to, nucleoside analogs, such as zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin; foscamet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, and alpha-interferons. [0132] In some examples, the anti-RSV antibodies and antigen-binding fragments provided herein can be further modified by conjugation to a therapeutic moiety that is a therapeutic polypeptide. Exemplary therapeutic polypeptides include, but are not limited to, a toxin, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; or an immunostimulatory agent, such as a cytokine, such as, but not limited to, an interferon (e.g., IFN-a, b, g, w), a lymphokine, a hematopoietic growth factor, such as, for example, GM-CSF (granulocyte macrophage colony stimulating factor), Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin- 4 (IL-4), Interleukin-7 (IL-7), Interleukin- 10 (IL-10), Interleukin- 12 (IL-12), Interleukin- 14 (IL-14), and Tumor Necrosis Factor (TNF).
C. Pharmaceutical Compositions and Articles of Manufacture/Kits
1. Pharmaceutical Compositions
[0133] Provided herein are pharmaceutical compositions containing an anti-RSV antibody or antigen-binding fragment thereof provided herein. The pharmaceutical composition can be used for therapeutic, prophylactic, and/or diagnostic applications. The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be formulated with a pharmaceutical acceptable carrier or diluent. Generally, such pharmaceutical compositions utilize components which will not significantly impair the biological properties of the antibody or antigen-binding fragment thereof, such as the binding of to its specific epitope (e.g. binding to an epitope on a RSV protein). Each component is pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. The formulations can conveniently be presented in unit dosage form and can be prepared by methods well known in the art of pharmacy, including but not limited to, tablets, pills, powders, liquid solutions or suspensions (e.g., including injectable, ingestible and topical formulations (e.g., eye drops, gels or ointments), aerosols (e.g., nasal sprays), liposomes, suppositories, injectable and infusible solution and sustained release forms. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets, Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems, Dekker, NY. When administered systematically, the therapeutic composition is sterile, pyrogen-free, generally free of particulate matter, and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art. 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, e.g., “Remington: The Science and Practice of Pharmacy (Formerly Remington's Pharmaceutical Sciences),” 19th ed., Mack Publishing Company, Easton, Pa. (1995).
[0134] Pharmaceutical compositions provided herein can be in various forms, e.g., in solid, semi-solid, liquid, powder, aqueous, or lyophilized form. Examples of suitable pharmaceutical carriers are known in the art and include but are not limited to water, buffering agents, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, powders, among others. Pharmaceutical compositions provided herein can contain other additives including, for example, antioxidants, preservatives, antimicrobial agents, analgesic agents, binders, disintegrants, coloring, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents, flavoring agents, emulsions, such as oil/water emulsions, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and derivatives thereof, solvents, and miscellaneous ingredients such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, starch, among others (see, generally, Alfonso R. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins). Such carriers and/or additives can be formulated by conventional methods and can be administered to the subject at a suitable dose. Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can preserve the compositions from degradation within the body. [0135] Pharmaceutical compositions suitable for use include compositions wherein one or more anti-RSV antibodies are contained in an amount effective to achieve their intended purpose. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Therapeutically effective dosages can be determined by using in vitro and in vivo methods as described herein. Accordingly, an anti-RSV antibody or antigen binding fragment thereof provided herein, when in a pharmaceutical preparation, can be present in unit dose forms for administration.
[0136] An anti-RSV antibody or antigen-binding fragment thereof provided 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 protein preparations and art-known lyophilization and reconstitution techniques can be employed.
[0137] An anti-RSV antibody or antigen-binding fragment thereof provided herein can be provided as a controlled release or sustained release composition. Polymeric materials are known in the art for the formulation of pills and capsules which can achieve controlled or sustained release of the antibodies or antigen-binding fragments thereof provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983) I, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 7 1:105; U.S. Pat. Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326; PCT Publication Nos. WO 99/15154 and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and poly orthoesters. Generally, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. Any technique known in the art for the production of sustained release formulation can be used to produce a sustained release formulation containing one or more anti-RSV antibodies or antigen-binding fragments provided herein. [0138] In some examples, the pharmaceutical composition contains an anti-RSV antibody or antigen-binding fragment thereof provided herein and one or more additional antibodies. In some examples, the one or more additional antibodies includes, but is not limited to, palivizumab (SYNAGIS®), and derivatives thereof, such as, but not limited to, motavizumab (NUMAX®), AFFF, P12f2, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, IX- 493L1, FRH3-3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, and A4B4-F52S (see U.S. Pat. Nos. 5,824,307 and 6,818,216), rsv6, rsvll, rsvl3, rsvl9, rsv21, rsv22, rsv23 (see, e.g. U.S. Pat. Nos. 5,824,307, 6,685,942 and 6,818,216), ahuman anti-RSV antibody, such as, but not limited to, rsv6, rsvll, rsvl3, rsvl9 (i.e. Fab 19), rsv21, rsv22, rsv23, RF-1, RF-2 (see, e.g. U.S. Pat. Nos. 6,685,942 and 5,811,524), a humanized antibody derived from an anti-RSV mouse monoclonal antibody such as, but not limited to, MAbs 1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112, 1269, 1269, 1243 (Beeler et al. (1989) J. Virology 63(7):2841-2950), MAM51 (Mufson et al. (1987) J. Clin. Microbiol. 25:1635-1539), MAbs 43-1 and 13-1 (Femie et al. (1982) Proc. Soc. Exp. Biol. Med. 171:266-271), MAbs 1436C, 1302A, 1308F, and 1331H (Anderson et al. (1984) J. Clin. Microbiol. 19:934-936), or antigen-binding fragments thereof. Additional exemplary antibodies or antigen-binding fragments thereof that can be used in a pharmaceutical composition containing an anti-RSV antibody or antigen-binding fragment thereof provided herein include, but are not limited to, anti-RSV antibodies or antigen binding fragments thereof described in, for example, U.S. Pat. Nos. 6,413,771, 5,840,298,
5,811,524, 6,656,467, 6,537,809, 7,364,742, 7,070,786, 5,955,364, 7,488,477, 6,818,216, 5,824,307, 7,364,737, 6,685,942, and 5,762,905 and U.S. Patent Pub. Nos. 2007-0082002
2005-0175986, 2004-0234528, 2006-0198840, 2009-0110684, 2006-0159695, 2006- 0013824, 2005-0288491, 2005-0019758, 2008-0226630, 2009-0137003, and 2009-0092609.
2. Articles of Manufacture/Kits
[0139] Pharmaceutical compositions of anti-RSV antibodies or nucleic acids encoding anti- RSV antibodies, or a derivative or a biologically active portion thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for prophylaxis (i.e. vaccination, passive immunization) and/or treating the RSV- mediated disease or disorder, and a label that indicates that the antibody or nucleic acid molecule is to be used for vaccination and/or treating the disease or disorder. The pharmaceutical compositions can be packaged in unit dosage forms contain an amount of the pharmaceutical composition for a single dose or multiple doses. The packaged compositions can contain a lyophilized powder of the pharmaceutical compositions containing the anti- RSV antibodies or antigen-binding fragments thereof provided, which can be reconstituted (e.g. with water or saline) prior to administration.
[0140] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g, jet or ultrasonic nebulizers) and other single breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. The pharmaceutical composition also can be incorporated in, applied to or coated on a barrier or other protective device that is used for contraception from infection.
[0141] The anti-RSV antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies thereof, pharmaceutical compositions or combinations provided herein also can be provided as kits. Kits can optionally include one or more components such as instructions for use, devices and additional reagents (e.g, sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods. Exemplary kits can include the anti-RSV antibodies or antigen-binding fragments thereof provided herein, and can optionally include instructions for use, a device for administering the anti- RSV antibodies or antigen-binding fragments thereof to a subject, a device for detecting the anti-RSV antibodies or antigen-binding fragments thereof in a subject, a device for detecting the anti-RSV antibodies or antigen-binding fragments thereof in samples obtained from a subject, and a device for administering an additional therapeutic agent to a subject.
[0142] The kit can, optionally, include instructions. Instructions typically include a tangible expression describing the anti-RSV antibodies or antigen-binding fragments thereof and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, dosing regimens, and the proper administration method for administering the anti-RSV antibodies or antigen-binding fragments thereof. Instructions also can include guidance for monitoring the subject over the duration of the treatment time [0143] Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of the selected anti-RSV antibody or antigen-binding fragment thereof in a subject.
[0144] In some examples, the anti-RSV antibody or antigen-binding fragment thereof is provided in a diagnostic kit for the detection of RSV in an isolated biological sample (e.g., a fluid sample, such as blood, sputum, lavage, lung intubation sample, saliva, urine or lymph obtained from a subject). In some examples, the diagnostic kit contains a panel of one or more anti-RSV antibodies or antigen-binding fragments thereof and/or one or more control antibodies (i.e. non-RSV binding antibodies), where one or more antibodies in the panel is an anti-RSV antibody or antigen-binding fragment provided herein.
[0145] Kits provided herein also can include a device for administering the anti-RSV antibodies or antigen-binding fragments thereof to a subject. Any of a variety of devices known in the art for administering medications to a subject can be included in the kits provided herein. Exemplary devices include, but are not limited to, an inhaler (e.g., pressurized metered dose inhaler (MDI), dry powder inhaler (DPI), nebulizer (e.g., jet or ultrasonic nebulizers) and other single breath liquid system), a hypodermic needle, an intravenous needle, a catheter, and a liquid dispenser such as an eyedropper. Typically the device for administering the anti-RSV antibodies or antigen-binding fragments thereof of the kit will be compatible with the desired method of administration of the anti-RSV antibodies or antigen-binding fragments thereof. For example, an anti-RSV antibody or antigen-binding fragment thereof to be delivered by pulmonary administration can be included in a kit with or contained in an inhaler or a nebulizer.
III. Methods
A. Methods of Producing Anti-RSV Antibodies and Modified or Variant Forms Thereof and Nucleic Acids Encoding Antibodies
[0146] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be generated by any suitable method known in the art for the preparation of antibodies, including chemical synthesis and recombinant expression techniques. Various combinations of host cells and vectors can be used to receive, maintain, reproduce and amplify nucleic acids (e.g. nucleic acids encoding antibodies such as the anti-RSV antibodies or antigen- binding fragments thereof provided), and to express polypeptides encoded by the nucleic acids. In general, the choice of host cell and vector depends on whether amplification, polypeptide expression, and/or display on a genetic package, such as a phage, is desired. Methods for transforming host cells are well known. Any known transformation method (e.g., transformation, transfection, infection, electroporation and sonoporation) can be used to transform the host cell with nucleic acids. Procedures for the production of antibodies, such as monoclonal antibodies and antibody fragments, such as, but not limited to, Fab fragments and single chain antibodies are well known in the art.
[0147] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, but not limited to, the use of hybridoma, recombinant expression, phage display technologies or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et ak, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, Monoclonal Antibodies and T-Cell Hybridomas 5630681 (Elsevier N.Y. 1981).
[0148] Polypeptides, such as any set forth herein, including the anti-RSV antibodies or antigen-binding fragments thereof provided herein, can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired polypeptides can be expressed in any organism suitable to produce the required amounts and forms of the proteins, such as for example, needed for analysis, administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals (e.g., rabbits, mice, rats, and livestock, such as, but not limited to, goats, sheep, and cattle), including production in serum, milk and eggs. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.
1. Nucleic Acids
[0149] Provided herein are isolated nucleic acid molecules encoding an anti-RSV antibody or antigen-binding fragment thereof provided herein. In some examples, the isolated nucleic acid molecule provided encodes an antibody or antigen-binding fragment having an amino acid sequence set forth in SEQ ID NOs:l-2 and/or 4-5.
[0150] Nucleic acid molecules encoding the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be prepared using well-known recombinant techniques for manipulation of nucleic acid molecules (see, e.g., techniques described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology, John Wiley & Sons, NY). In some examples, methods, such as, but not limited to, recombinant DNA techniques, site directed mutagenesis, and polymerase chain reaction (PCR) can be used to generate modified antibodies or antigen-binding fragments thereof having a different amino acid sequence, for example, to create amino acid substitutions, deletions, and/or insertions.
[0151] In some examples, one or more of the CDRs of an anti-RSV antibody or antigen binding fragment thereof provided herein is inserted within framework regions using routine recombinant DNA techniques. The framework regions can be selected from naturally occurring or consensus framework regions, including human framework regions (see, e.g., Chothia et al. (1998) J. Mol. Biol. 278: 457-479 for exemplary framework regions). Generally, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody or antigen-binding fragment thereof that maintains the antigen binding specificity of the parent anti-RSV antibody or antigen-binding fragment thereof. Alterations to the polynucleotide can be made to improve one or more properties of the encoded antibody or antigen-binding fragment thereof and within the skill of the art. In some examples, one or more modifications of the polynucleotide can be made to produce amino acid substitutions within the framework regions, which, for example, improve binding of the antibody or antigen-binding fragment thereof to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
2. Vectors
[0152] Provided herein are vectors that contain nucleic acid or nucleic acids (encoding the heavy and light chain) encoding the anti-RSV antibodies or antigen-binding fragments thereof. Generally, nucleic acid encoding the heavy chain of an antibody is cloned into a vector and the nucleic acid encoding the light chain of an antibody is cloned into the vector. The genes can be cloned into a single vector for dual expression thereof, or into separate vectors. If desired, the vectors also can contain further sequences encoding additional constant region(s) or hinge regions to generate other antibody forms.
[0153] Many expression vectors are available and known to those of skill in the art and can be used for expression of polypeptides. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.
[0154] Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g. a hexa-his tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association.
[0155] Expression of the antibodies or antigen-binding fragments thereof can be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bemoist and Chambon, (1981) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., (1981) Proc. Natl. Acad. Sci.
USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., (1982) Nature 296:39-42); prokaryotic expression vectors such as the b-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful Proteins from Recombinant Bacteria”: in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera-Estrella et al., (1984) Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., (1981) Nucleic Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., (1984) Nature 310:115-120); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., (1984) Cell 38:639-646; Omitz et al., (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., (1985) Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., (1984) Cell 38:647-658; Adams et al., (1985) Nature 318:533-538; Alexander et al.,
(1987) Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., (1986) Cell 45:485-495), albumin gene control region which is active in liver (Pinckert et al., (1987) Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., (1985) Mol. Cell. Biol. 5:1639-403); Hammer et al., (1987) Science 235:53-58), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., (1987) Genes and Devel. 1:161-171), betaglobin gene control region which is active in myeloid cells (Magram et al., (1985) Nature 315:338-340); Kollias et al., (1986) Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., (1987) Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Shani (1985) Nature 314:283-286), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al.,
(1986) Science 234:1372-1378).
[0156] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the antibody, or portion thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the antibody chain and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.
[0157] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter.
[0158] For purposes herein, vectors are provided that contain a sequence of nucleotides that encodes the heavy chain and/or light chain variable region of an anti-RSV antibody. In some examples, vectors provided herein contain a sequence of nucleotides that encodes the constant region of an antibody operably linked to the nucleic acid sequence encoding the variable region of the antibody. The vector can include the sequence for one or all of a CHI, CH2, hinge, CH3 or CH4 and/or CL. Generally, such as for expression of Fabs, the vector contains the sequence for a CHI or CL (kappa or lambda light chains). The sequences of constant regions or hinge regions are known to one of skill in the art (see e.g. U.S. Published Application No. 20080248028) and described herein.
[0159] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding an antibody or antigen-binding fragment thereof provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.
[0160] Exemplary plasmid vectors useful to produce the antibodies or antigen-binding fragments provided herein contain a strong promoter, such as the HCMV immediate early enhancer/promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 polyA signal. The plasmid can be multi cistronic to enable expression of the full-length heavy and light chains of the antibody, a single chain Fv fragment or other immunoglobulin fragments.
3. Cell Expression Systems
[0161] Nucleic acids encoding the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be expressed in a suitable host. Cells containing the vectors and nucleic acids encoding the anti-RSV antibodies or antigen-binding fragments thereof provided herein are provided. Generally, any cell type that can be engineered to express heterologous DNA and has a secretory pathway is suitable. Expression hosts include prokaryotic and eukaryotic organisms, such as bacterial cells ( e.g . E. coli ), yeast cells, fungal cells, Archae, plant cells, insect cells and animal cells including human cells. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. Further, the choice of expression host is often related to the choice of vector and transcription and translation elements used. For example, the choice of expression host is often, but not always, dependent on the choice of precursor sequence utilized. For example, many heterologous signal sequences can only be expressed in a host cell of the same species (i.e., an insect cell signal sequence is optimally expressed in an insect cell). In contrast, other signal sequences can be used in heterologous hosts such as, for example, the human serum albumin (hHSA) signal sequence which works well in yeast, insect, or mammalian host cells and the tissue plasminogen activator pre/pro sequence which has been demonstrated to be functional in insect and mammalian cells (Tan et ak,
(2002) Protein Eng. 15:337). The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification. Thus, the vector system must be compatible with the host cell used.
[0162] Expression in eukaryotic hosts can include expression in yeasts such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as Drosophila cells and lepidopteran cells, plants and plant cells such as tobacco, com, rice, algae, and lemna, or fungal cells such as T. reesei. Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs. [0163] Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated. Generally, standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large quantity of antibody chains, which is then purified using standard techniques (see e.g., Colley et al.
(1989) J. Biol. Chem., 264:17619-17622; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed.), 1990). Transformations of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison (1977) J.
Bact. 132:349-351; Clark-Curtiss and Curtiss (1983) Methods in Enzymology, 101, 347-362). For example, any of the well-known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors (e.g., baculovirus, vaccinia virus, adenovirus and other viruses), and any other the other well known methods for introducing cloned genomic DNA, cDNA, plasmid DNA, cosmid DNA, synthetic DNA or other foreign genetic material into a host cell.
[0164] Generally, for purposes herein, host cells are transfected with a first vector encoding at least a VH chain or heavy chain of an anti-RSV antibody and a second vector encoding at least a VL chain or light chain of the anti-RSV antibody. Host cells also can be transfected with a single vector encoding both the heavy and light chain or portion thereof.
[0165] In one example, nucleic acid encoding the heavy chain of an antibody is ligated into a first expression vector and nucleic acid encoding the light chain of an antibody is ligated into a second expression vector. The expression vectors can be the same or different, although generally they are sufficiently compatible to allow comparable expression of proteins (heavy and light chain) therefrom. The first and second expression vectors are generally co transfected into host cells, typically at a 1:1 ratio. Exemplary of vectors include, but are not limited to, p01HC and pKLC (Tiller et al. (2008) Journal of Immunological Methods, 329:112-24). Other expression vectors include the L chain expression vector pAG4622 and the heavy chain expression vector pAH4604 (Coloma et al. (1992) J. Immunol. Methods, 152:89-104). The pAG4622 vector contains the genomic sequence encoding the C- region domain of the human K L chain and the gpt selectable marker. The pAH4604 vectors contain the hisD selectable marker and sequences encoding the human H chain gΐ C-region domain. [0166] In another example, the heavy and light chain can be cloned into a single vector that has expression cassettes for both the heavy and light chain. In one example, genes encoding the heavy and light chains can be cloned into the mammalian expression vector pTT5 (NRC Biotechnology Research). In another example, genes encoding the heavy and light chains, or portions thereof, can be cloned into pCALM (SEQ ID NO: 102).
[0167] For expression of a full-length Ig, sequences encoding the VH-CHl-hinge-CH2-Cn3 can be cloned into a first expression vector and sequences encoding the VL-CL domains can be cloned into a second expression vector. To generate a Fab, sequences encoding the VH- CH1 can be cloned into a first expression vector and sequences encoding the VL-CL domains can be cloned into a second expression vector. For a conventional antibody, a heavy chain pairs with a light chain and a Fab monomer is generated.
[0168] Expression can be in any cell expression system known to one of skill in the art. Exemplary cells for expression include, but are not limited to, 293FS cells, HEK293-6E cells or CHO cells. Other expression vectors and host cells are described below. Upon expression, antibody heavy and light chains pair by disulfide bond to form a full-length antibody or fragments thereof.
4. Purification of Antibodies
[0169] Methods for purification of polypeptides, including the anti-RSV antibodies or antigen-binding fragments thereof provided herein, from host cells will depend on the chosen host cells and expression systems. For secreted molecules, proteins generally are purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. In one example, polypeptides are isolated from the host cells by centrifugation and cell lysis (e.g. by repeated freeze-thaw in a dry ice/ethanol bath), followed by centrifugation and retention of the supernatant containing the polypeptides. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary further the proteins can be extracted and further purified using standard methods in the art.
[0170] Proteins, such as the anti-RSV antibodies or antigen-binding fragments thereof provided herein, can be purified, for example, from lysed cell extracts, using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation and ionic exchange chromatography, such as anion exchange. Affinity purification techniques also can be utilized to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind proteases can be used in affinity purification. Expression constructs also can be engineered to add an affinity tag to a protein such as a myc epitope, GST fusion or His6 and affinity purified with myc antibody, glutathione resin and Ni- resin, respectively. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques.
[0171] Typically, antibodies and portions thereof are purified by any procedure known to one of skill in the art. The antibodies can be purified to substantial purity using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography or column chromatography. For example, antibodies can be purified by column chromatography. Exemplary of a method to purify antibodies is by using column chromatography, wherein a solid support column material is linked to Protein G, a cell surface-associated protein from Streptococcus, that binds immunoglobulins with high affinity. The antibodies can be purified to 60%, 70%, 80% purity and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity.
Purity can be assessed by standard methods such as by SDS-PAGE and coomassie staining.
[0172] The isolated polypeptides then can be analyzed, for example, by separation on a gel ( e.g . SDS-Page gel), size fractionation (e.g. separation on a Sephacryl™ S-200 HiPrep™ 16x60 size exclusion column (Amersham from GE Healthcare Life Sciences, Piscataway, N.J.). Isolated polypeptides also can be analyzed in binding assays, typically binding assays using a binding partner bound to a solid support, for example, to a plate ( e.g. ELISA-based binding assays) or a bead, to determine their ability to bind desired binding partners. The binding assays described in the sections below, which are used to assess binding of precipitated phage displaying the polypeptides, also can be used to assess polypeptides isolated directly from host cell lysates. For example, binding assays can be carried out to determine whether antibody polypeptides bind to one or more antigens, for example, by coating the antigen on a solid support, such as a well of an assay plate and incubating the isolated polypeptides on the solid support, followed by washing and detection with secondary reagents, e.g. enzyme-labeled antibodies and substrates.
B. Assessing Anti-RSV Antibody Properties and Activities
[0173] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be characterized in a variety of ways well-known to one of skill in the art. For example, the anti- RSV antibodies or antigen-binding fragments thereof provided herein can be assayed for the ability to immunospecifically bind to a protein of human Respiratory Syncytial Virus (RSV). Such assays can be performed, for example, in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), on beads (Lam (1991) Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378- 6382; and Felici (1991) J. Mol. Biol. 222:301-310). Antibodies or antigen-binding fragments thereof that have been identified to immunospecifically bind to a RSV antigen or a fragment thereof also can be assayed for their specificity and affinity for a RSV antigen. The binding specificity, or epitope, can be determined, for example, by competition assays with other anti- RSV antibodies and/or virus neutralization assays using Monoclonal Antibody-Resistant Mutants (MARMs). In addition, in vitro assays and in vivo animal models using the anti- RSV antibodies or antigen-binding fragments thereof provided herein can be employed for measuring the level of RSV neutralization effected by contact or administration of the anti- RSV antibodies or antigen-binding fragments thereof.
1. Binding Assays
[0174] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be assessed for their ability to bind a selected target (e.g., RSV virus) and the specificity for such targets by any method known to one of skill in the art. Exemplary assays are provided in the Examples and described herein below. Binding assays can be performed in solution, suspension or on a solid support. For example, target antigens can be immobilized to a solid support (e.g. a carbon or plastic surface, a tissue culture dish or chip) and contacted with antibody or antigen-binding fragment thereof. Unbound antibody or target protein can be washed away and bound complexes can then be detected. Binding assays can be performed under conditions to reduce nonspecific binding, such as by using a high ionic strength buffer (e.g., 0.3-0.4 M NaCl) with nonionic detergent (e.g. 0.1% TRITON X®-100 or TWEEN®
20) and/or blocking proteins (e.g. bovine serum albumin or gelatin). Negative controls also can be included in such assays as a measure of background binding. Binding affinities can be determined using Scatchard analysis (Munson et ak, (1980) Anal. Biochem., 107:220), surface plasmon resonance, isothermal calorimetry, or other methods known to one of skill in the art.
[0175] Exemplary immunoassays which can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as, but not limited to, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), Meso Scale Discovery (MSD, Gaithersburg, Md.), “sandwich” immunoassays, immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., (1986) Amer. Clin. Prod. Rev. 5:34-41). Exemplary immunoassays not intended by way of limitation are described briefly below.
[0176] Immunoprecipitation protocols generally involve lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or TRITON® X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody or antigen-binding fragment thereof of interest to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody or antigen-binding fragment thereof of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art is knowledgeable as to the parameters that can be modified to increase the binding of the antibody or antigen-binding fragment thereof to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0177] Western blot analysis generally involves preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody or antigen-binding fragment thereof (i.e., the antibody or antigen-binding fragment thereof of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti -human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art is knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
[0178] ELISAs involve preparing antigen, coating the well of a 96-well microtiter plate with the antigen, adding the antibody or antigen-binding fragment thereof of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs, the antibody or antigen-binding fragment thereof of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound can be added to the well. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated well. One of skill in the art is knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
[0179] The binding affinity of an antibody or antigen-binding fragment thereof to an antigen and the off-rate of an antibody-antigen interaction can be determined, for example, by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) with the antibody or antigen-binding fragment thereof of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody or antigen-binding fragment thereof bound to the labeled antigen. The affinity of an anti-RSV antibody or antigen-binding fragment thereof provided herein for a RSV antigen and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, a RSV antigen is incubated with an anti-RSV antibody or antigen-binding fragment thereof provided herein conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody. In some examples, surface plasmon resonance (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335) kinetic analysis can be used to determine the binding on and off rates of antibodies or antigen-binding fragments thereof to a RSV antigen. Surface plasmon resonance kinetic analysis involves analyzing the binding and dissociation of a RSV antigen from chips with immobilized antibodies or fragments thereof on their surface.
[0180] The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be assayed for their ability to inhibit the binding of RSV to its host cell receptor using techniques known to those of skill in the art. For example, cells expressing the receptor for RSV can be contacted with RSV in the presence or absence of an antibody or antigen binding fragment thereof and the ability of the antibody or fragment thereof to inhibit RSV's binding can measured by, for example, flow cytometry or a scintillation assay. RSV (e.g., a RSV antigen such as F glycoprotein or G glycoprotein) or the antibody or antibody fragment can be labeled with a detectable compound such as a radioactive label (e.g., 32P, 35S, and 1251) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between RSV and its host cell receptor. [0181] The ability of antibodies or antigen-binding fragments thereof to inhibit RSV from binding to its receptor also can be determined in cell-free assays. For example, RSV or a RSV antigen such as F glycoprotein can be contacted with an antibody or fragment thereof and the ability of the antibody or antibody fragment to inhibit RSV or the RSV antigen from binding to its host cell receptor can be determined. In some examples, the antibody or the antigen-binding fragment is immobilized on a solid support and RSV or a RSV antigen is labeled with a detectable compound. In some examples, RSV or a RSV antigen is immobilized on a solid support and the antibody or fragment thereof is labeled with a detectable compound. The RSV or RSV antigen can be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate. In some examples, a RSV antigen can be a fusion protein comprising the RSV antigen and a domain such as glutathionine-5 -transferase. In some examples, a RSV antigen can be biotinylated using techniques well known to those of skill in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).
2. Binding Specificity
[0182] The binding specificity, or epitope, of the anti-RSV antibodies or antigen binding fragments thereof provided herein can be determined by any assay known to one of skill in the art, including, but not limited to surface plasmon resonance assays, competition assays and virus neutralization assays using Monoclonal Antibody-Resistant Mutants (MARMs). The epitope can be in the isolated protein, or in the protein in the virus. The ability of two antibodies to bind to the same epitope can be determined by known assays in the art such as, for example, surface plasmon resonance assays and antibody competition assays. Typically, antibodies that immunospecifically bind to the same epitope can compete for binding to the epitope, which can be measured, for example, by an in vitro binding competition assay (e.g. competition ELISA), using techniques known the art. Typically, a first antibody that immunospecifically binds to the same epitope as a second antibody can compete for binding to the epitope by about or 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, where the percentage competition is measured ability of the second antibody to displace binding of the first antibody to the epitope. In exemplary competition assays, the antigen is incubated in the presence a predetermined limiting dilution of a labeled antibody (e.g., 50-70% saturation concentration), and serial dilutions of an unlabeled competing antibody. Competition is determined by measuring the binding of the labeled antibody to the antigen for any decreases in binding in the presence of the competing antibody. Variations of such assays, including various labeling techniques and detection methods including, for example, radiometric, fluorescent, enzymatic and colorimetric detection, are known in the art. The ability of a first antibody to bind to the same epitope as a second antibody also can be determined, for example, by virus neutralization assays using Monoclonal Antibody -Resistant Mutants. A MARM is a mutant respiratory syncytial virus (RSV) that not neutralized by a monoclonal antibody that neutralizes the wildtype RSV virus, i. e.. a MARM is an RSV escape mutant. MARMs are generated by culturing wildtype RSV in the presence of a monoclonal antibody for successive rounds of viral replication in the presence of the antibody such that after each successive round of virus replication, cytopathic effects (CPE) are observed in the presence of increasing concentrations of antibodies until a mutant virus results that is not neutralized by the antibody. If a first antibody can neutralize a MARM generated against a second antibody, one can conclude that the antibodies specifically bind to or interact with different epitopes. For example, where a first anti- RSV antibody neutralizes wild-type RSV but not a particular mutant RSV (i.e., MARM), a second antibody that neutralizes the wild-type RSV but not the particular mutant RSV generally binds the same epitope on RSV as the first antibody. Where a first anti-RSV antibody neutralizes wild-type RSV but not a particular mutant RSV, a second antibody that neutralizes the wild-type RSV and the particular mutant RSV generally does not bind the same epitope on RSV as the first antibody.
3. In vitro Assays for Analyzing Virus Neutralization Effects of Antibodies
[0183] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be analyzed by any suitable method known in the art for the detection of viral neutralization. Methods for detection of viral neutralization include, but are not limited to, plaque assays and assays for inhibition of syncytium formation. Such assays can be employed to assess, for example, inhibition of viral attachment, viral entry and cell-to-cell spread of the virus (see, e.g. Burioni et al., (1994 ) Proc. Natl. Acad. Sci. U.S.A. 91:355-359; Sanna et al.
(2000) Virology 270:386-3961; and De Logu et al., (1998) J Clin Microbiol 36:3198-3204). One of skill in the art can identify any assay capable of measuring viral neutralization.
[0184] Standard plaque assays include, for example, plaque reduction assays, plaque size reduction assays, neutralization assays and neutralization kinetic assays. These assays measure the formation of viral plaques (i.e. areas of lysed cells) following infection of target cell monolayers by a virus. Exemplary target cell lines that can be used in plaque reduction assays include, but are not limited to, Vero cells, MRC-5 cells, RC-37 cells, BHK-21/C13 cells and HEp-2 cells. One of skill in the art can identify appropriate target cell lines for use in a plaque assay. Selection of an appropriate cell line for a plaque assay can depend on known factors, such as, for example, cell infectivity and the ability of the virus to propagate in and lyse the target cell. Examples 6 and 9 exemplify in vitro neutralization assays.
[0185] Plaque reduction assays can be used to measure the ability of the anti-RSV antibody or antigen-binding fragment thereof to effect viral neutralization in solution. In exemplary plaque reduction assays, the antibody or antigen-binding fragment thereof and the virus are pre-incubated prior to the addition of target cells. Target cells are then infected with the antibody/virus mixture and a plaque assay is performed following a predetermined infection period. One of skill in the art can determine the incubation times required based on known examples in the art. A reduction in the number of virus plaques produced following infection of the target cells indicates the ability of the antibody or antigen-binding fragment thereof to prevent binding of the virus to the target cells independent of antibody or antigen-binding fragment thereof attachment to the target cell and/or antibody, or antigen-binding fragment thereof, internalization.
[0186] Plaque size reduction assays can be used to measure the ability of the anti- RSV antibody or antigen-binding fragment thereof to inhibit of viral cell-to-cell spread. In exemplary plaque size reduction assays, the target cells are first infected with the virus for a predetermined infection period and then the antibody or antigen-binding fragment thereof is added to the infected cell. One of skill in the art can determine the incubation times required based on known examples in the art. A reduction in the size (i.e. diameter) of the virus plaques indicates that the antibody or antigen-binding fragment thereof is capable of preventing viral cell-to-cell spread.
[0187] Virus neutralization assays can be used to measure the ability of the anti- RSV antibody or antigen-binding fragment thereof to effect viral neutralization at the target cell surface by association of the antibody or antigen-binding fragment thereof with the target cell prior to virus exposure. In exemplary virus neutralization assays, the antibody or antigen binding fragment thereof and target cells are pre-incubated for a predetermined period of time to allow for binding of the antibody or antigen-binding fragment thereof to the targeted cell. Following the pre-incubation period, the unbound antibody is removed and the target cells are infected with the virus. A reduction in the number of plaques in this assay indicates the ability of the antibody or antigen-binding fragment thereof to prevent viral infection dependent upon attachment to the target cell and/or internalization of the antibody or antigen binding fragment thereof. This assay also can be used to measure neutralization kinetics by varying antibody or antigen-binding fragment concentrations and pre-incubation times.
[0188] Exemplary assays for inhibition of syncytium formation can be employed to measure antibody-mediated inhibition of viral cytopathic effects by blocking the formation of syncytia when using a fusogenic viral strain. One of skill in the art can identify an appropriate fusogenic viral strain for use in the assay.
[0189] The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be assayed for their ability to inhibit or downregulate RSV replication using techniques known to those of skill in the art. For example, RSV replication can be assayed by a plaque assay such as described, e.g., by Johnson et al. (1997) Journal of Infectious Diseases 176:1215-1224. The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be assayed for their ability to inhibit or downregulate the expression of RSV polypeptides. Techniques known to those of skill in the art, including, but not limited to, Western blot analysis, Northern blot analysis, and RT-PCR can be used to measure the expression of RSV polypeptides.
4. In vivo Animal Models for Assessing Efficacy of the Anti- RSV Antibodies
[0190] In vivo studies using animal models can be performed to assess the efficacy of the anti-RSV antibodies or antigen-binding fragments thereof provided herein. In vivo studies using animal models can be performed to assess any toxicity of administration of such antibodies or antigen-binding fragments thereof. A variety of assays, such as those employing in vivo animal models, are available to those of skill in the art for evaluating the ability of the anti-RSV antibodies to inhibit or treat RSV virus infection and for assaying any toxicity. The therapeutic effect of the anti-RSV antibodies can be assessed using animal models of the pathogenic infection, including animal models of viral infection. Such animal models are known in the art, and include, but are not limited to, animal models for RSV infection, such as but not limited to cotton rat, inbred mouse, calf, ferret, hamster, guinea pig, chimpanzee, owl monkey, rhesus monkey, African green monkey, cebus monkey, squirrel monkey, bonnet monkey, baboon, (see, e.g., Prince et al. (1978 )Am. J. Pathol. 93:771-791; Prince et al. (1979) Infect. Immunol. 26:764-766; Byrd and Prince (1997) Clinical Infectious Diseases 25:1363-1368, including references cited therein, for exemplary models of RSV infection). For in vivo testing of an antibody or antigen-binding fragment or composition's toxicity, any animal model system known in the art can be used, including, but not limited to, rats, mice, cows, monkeys, and rabbits.
5. In vitro and In vivo Assays for Measuring Antibody Efficacy
[0191] Efficacy in treating or preventing viral infection can be demonstrated by detecting the ability of a anti-RSV antibody or antigen-binding fragment thereof provided herein to inhibit the replication of the virus, to inhibit transmission or prevent the virus from establishing itself in its host, to reduce the incidence of RSV infection, or to prevent, ameliorate or alleviate one or more symptoms associated with RSV infection. The treatment is considered therapeutic if there is, for example, a reduction is viral load, amelioration of one or more symptoms, a reduction in the duration of a RSV infection, or a decrease in mortality and/or morbidity following administration of an antibody or composition provided herein. Further, the treatment is considered therapeutic if there is an increase in the immune response following the administration of one or more antibodies or antigen-binding fragments thereof which immunospecifically bind to one or more RSV antigens.
[0192] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be tested in vitro and in vivo for the ability to induce the expression of cytokines such as IFN-a, IFN-b, IFN-g, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-15. Techniques known to those of skill in the art can be used to measure the level of expression of cytokines. For example, the level of expression of cytokines can be measured by analyzing the level of RNA of cytokines by, for example, RT-PCR and Northern blot analysis, and by analyzing the level of cytokines by, for example, immunoprecipitation followed by Western blot analysis or ELISA.
[0193] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be tested in vitro and in vivo for their ability to modulate the biological activity of immune cells, including human immune cells (e.g., T-cells, B-cells, and Natural Killer cells). The ability of an anti-RSV antibody or antigen-binding fragment to modulate the biological activity of immune cells can be assessed by detecting the expression of antigens, detecting the proliferation of immune cells, detecting the activation of signaling molecules, detecting the effector function of immune cells, or detecting the differentiation of immune cells. Techniques known to those of skill in the art can be used for measuring these activities. For example, cellular proliferation can be assayed by 3H-thymidine incorporation assays and trypan blue cell counts. Antigen expression can be assayed, for example, by immunoassays including, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, immunohistochemistry radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and FACS analysis. The activation of signaling molecules can be assayed, for example, by kinase assays and electrophoretic shift assays (EMSAs).
[0194] The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be tested for their ability to inhibit viral replication or reduce viral load in in vitro, ex vivo and in vivo assays. The anti-RSV antibodies or antigen-binding fragments thereof also can be assayed for their ability to decrease the time course of RSV infection. The anti- RSV antibodies or antigen-binding fragments thereof also can be assayed for their ability to increase the survival period of humans suffering from RSV infection by at least or about 25%, at least or about 50%, at least or about 60%, at least or about 75%, at least or about 85%, at least or about 95%, or at least or about 99%. Further, anti-RSV antibodies or antigen binding fragments thereof can be assayed for their ability reduce the hospitalization period of humans suffering from RSV infection by at least or about 60%, at least or about 75%, at least or about 85%, at least or about 95%, or at least or about 99%. Techniques known to those of skill in the art can be used to analyze the function of the anti-RSV antibodies or antigen binding fragments thereof provided herein in vivo.
[0195] In accordance with the methods and uses provided herein, clinical trials with human subjects need not be performed in order to demonstrate the prophylactic and/or therapeutic efficacy of the anti-RSV antibodies or antigen-binding fragments thereof provided herein. In vitro and animal model studies using the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be extrapolated to humans and are sufficient for demonstrating the prophylactic and/or therapeutic utility of the anti-RSV antibodies or antigen-binding fragments. C. Diagnostic Uses
[0196] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be used in diagnostic assays for the detection, purification, and/or neutralization of RSV. Exemplary diagnostic assays include in vitro and in vivo detection of RSV. For example, assays using the anti-RSV antibodies or antigen-binding fragments thereof provided herein for qualitatively and quantitatively measuring levels of RSV in an isolated biological sample (e.g., sputum) or in vivo are provided.
[0197] As described herein, the anti-RSV antibodies or antigen-binding fragments thereof can be conjugated to a detectable moiety for in vitro or in vivo detection. Such antibodies can be employed, for example, to evaluate the localization and/or persistence of the anti- RSV antibody or antigen-binding fragment thereof at an in vivo site, such as, for example, a mucosal site. The anti-RSV antibodies or antigen-binding fragments thereof which are coupled to a detectable moiety can be detected in vivo by any suitable method known in the art. The anti-RSV antibodies or antigen-binding fragments thereof which are coupled to a detectable moiety also can be detected in isolated biological samples, such as tissue or fluid samples obtained from the subject following administration of the antibody or antigen binding fragment thereof.
1. In vitro Detection of Pathogenic Infection
[0198] In general, RSV can be detected in a subject or patient based on the presence of one or more RSV proteins and/or polynucleotides encoding such proteins in a biological sample (e.g., blood, sera, sputum urine and/or other appropriate cells or tissues) obtained from a subject or patient. Such proteins can be used as markers to indicate the presence or absence of RSV in a subject or patient. The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be employed for detection of the level of antigen and/or epitope that binds to the agent in the biological sample.
[0199] A variety of assay formats are known to those of ordinary skill in the art for using a anti-RSV antibody or antigen-binding fragment thereof to detect polypeptide markers in a sample (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of RSV in a subject or patient can be determined by contacting a biological sample obtained from a subject or patient with an anti- RSV antibody or antigen-binding fragment thereof provided herein and detecting in the sample a level of polypeptide that binds to the anti-RSV antibody or antigen-binding fragment thereof.
[0200] In some examples, the assay involves the use of an anti-RSV antibody or antigen binding fragment thereof provided herein immobilized on a solid support to bind to and remove the target polypeptide from the remainder of the sample. The bound polypeptide can then be detected using a detection reagent that contains a reporter group and specifically binds to the antibody/polypeptide complex. Such detection reagents can contain, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent.
[0201] In some examples, a competitive assay can be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized anti-RSV antibody or antigen-binding fragment thereof after incubation of the anti-RSV antibody or antigen binding fragment thereof with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the anti-RSV antibody or antigen-binding fragment thereof is indicative of the reactivity of the sample with the immobilized anti- RSV antibody or antigen-binding fragment thereof. Suitable polypeptides for use within such assays include full length RSV proteins and portions thereof, to which an anti-RSV antibody or antigen-binding fragment thereof binds, as described above.
[0202] The solid support can be any material known to those of ordinary skill in the art to which the protein can be attached. For example, the solid support can be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. The support also can be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support also can be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The anti-RSV antibody or antigen binding fragment thereof can be immobilized on the solid support using a variety of techniques known to those of skill in the art. The anti-RSV antibody or antigen-binding fragment thereof can be immobilized by adsorption to a well in a microtiter plate or to a membrane. In such cases, adsorption can be achieved by contacting the anti-RSV antibody or antigen-binding fragment thereof, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of anti-RSV antibody or antigen-binding fragment thereof ranging from about 10 ng to about 10 jag, and typically about 100 ng to about 1 jag, is sufficient to immobilize an adequate amount of anti-RSV antibody or antigen binding fragment thereof.
[0203] Covalent attachment of anti-RSV antibody or antigen-binding fragment thereof to a solid support can generally be achieved by first reacting the support with a bifunctional reagent that will react with the support and a functional group, such as a hydroxyl or amino group, on the anti-RSV antibody or antigen-binding fragment thereof. For example, the anti- RSV antibody or antigen-binding fragment thereof can be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
[0204] In some examples, the assay is performed in a flow-through or strip test format, wherein the anti-RSV antibody or antigen-binding fragment thereof is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized anti-RSV antibody or antigen-binding fragment thereof as the sample passes through the membrane. A second, labeled binding agent then binds to the anti- RSV antibody or antigen-binding fragment thereof-polypeptide complex as a solution containing the second binding agent flows through the membrane.
[0205] Additional assay protocols exist in the art that are suitable for use with the RSV proteins or anti-RSV antibodies or antigen-binding fragments thereof provided. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols can be readily modified to use RSV polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such protein-specific antibodies can allow for the identification of RSV infection.
[0206] To improve sensitivity, multiple RSV protein markers can be assayed within a given sample. It will be apparent that anti-RSV antibodies or antigen-binding fragments thereof specific for different RSV polypeptides can be combined within a single assay. Further, multiple primers or probes can be used concurrently. The selection of RSV protein markers can be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for RSV proteins provided herein can be combined with assays for other known RSV antigens.
2. In vivo Detection of Pathogenic Infection
[0207] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be employed as an in vivo diagnostic agent. For example, the anti-RSV antibodies or antigen binding fragments thereof can provide an image of infected tissues (e.g., RSV infection in the lungs) using detection methods such as, for example, magnetic resonance imaging, X-ray imaging, computerized emission tomography and other imaging technologies. For the imaging of RSV infected tissues, for example, the antibody portion of the anti-RSV antibody generally will bind to RSV (e.g., binding a RSV protein epitope), and the imaging agent will be an agent detectable upon imaging, such as a paramagnetic, radioactive or fluorescent agent that is coupled to the anti-RSV antibody or antigen-binding fragment thereof. Generally, for use as a diagnostic agent, the anti-RSV antibody or antigen-binding fragment thereof is coupled directly or indirectly to the imaging agent.
[0208] Many appropriate imaging agents are known in the art, as are methods for their attachment to the anti-RSV antibodies or antigen-binding fragments (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509). Exemplary attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the antibody or antigen-binding fragment thereof (U.S. Pat. No. 4,472,509). The antibodies also can be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of such coupling agents or by reaction with an isothiocyanate.
[0209] For in vivo diagnostic imaging, the type of detection instrument available is considered when selecting a given radioisotope. The radioisotope selected has a type of decay which is detectable for a given type of instrument. Another factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized Typically, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which can be readily detected by conventional gamma cameras. [0210] For in vivo diagnosis, radioisotopes can be bound to the antibodies or antigen-binding fragments thereof provided herein either directly or indirectly by using an intermediate functional group. Exemplary intermediate functional groups which can be used to bind radioisotopes, which exist as metallic ions, to antibodies include bifunctional chelating agents, such as diethylene-triamine pentaacetic acid (DTP A) and ethylenediaminetetraacetic acid (EDTA) and similar molecules. Examples of metallic ions which can be bound to the anti-RSV antibodies or antigen-binding fragments thereof provided include, but are not limited to, 72Arsenic, 211Astatine, 14Carbon, 51Chromium, 36Chlorine, 57Cobalt, 58Cobalt, 67Copper, 15 2Europium, 67Gallium, 68Gallium, 3Hydrogen, 123Iodine, 125Iodine, 131Iodine, mIndium, 59 Iron
Iron, 32Phosphorus, 188Rhenium, 97Ruthenium, 75Selenium, 35Sulphur, 99mTechnicium, 261Thal ium, 90Yttrium and 89Zirconium.
[0211] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Generally, gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include, but are not limited to, 157Gd, 55Mn, 162Dy, 52Cr, and 56Fe.
[0212] Exemplary paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III). Ions useful, for example, in X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and bismuth (III).
[0213] The concentration of detectably labeled anti-RSV antibody or antigen-binding fragment thereof which is administered is sufficient such that the binding to RSV is detectable compared to the background. Further, it is desirable that the detectably labeled anti-RSV antibody or antigen-binding fragment thereof be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio. [0214] The dosage of detectably labeled anti-RSV antibody or antigen-binding fragment thereof for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of a human monoclonal antibody can vary, for example, from about 0.01 mg/m2 to about 500 mg/m2, 0.1 mg/m2 to about 200 mg/m2, or about 0.1 mg/m2 to about 10 mg/m2. Such dosages can vary, for example, depending on whether multiple injections are given, tissue, and other factors known to those of skill in the art.
D. Prophylactic and Therapeutic Uses
[0215] The anti-RSV antibodies or antigen-binding fragments thereof provided herein and pharmaceutical compositions containing anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject for prophylaxis and therapy. For example, the antibodies or antigen-binding fragments thereof provided can be administered for treatment of a disease or condition, such as a RSV infection. In some examples, the antibodies or antigen-binding fragments thereof provided can be administered to a subject for prophylactic uses, such as the prevention and/or spread of RSV infection, including, but not limited to the inhibition of establishment of RSV infection in a host or inhibition of RSV transmission between subjects. In some examples, the antibodies or antigen-binding fragments thereof provided can be administered to a subject for the reduction of RSV viral load in the subject. The antibodies or antigen-binding fragments thereof also can be administered to a subject for preventing, treating, and/or alleviating of one or more symptoms of a RSV infection or reduce the duration of a RSV infection.
[0216] In some examples, administration of an anti-RSV antibody or antigen-binding fragment thereof provided herein inhibits the incidence of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about
80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about
60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about
40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about
20%, at least or about 15%, or at least or about 10% relative to the incidence of RSV infection in the absence of the anti-RSV antibody or antigen-binding fragment. In some examples, administration of an anti-RSV antibody or antigen-binding fragment provided herein decreases the severity of one or more symptoms of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the severity of the one or more symptoms of RSV infection in the absence of the anti-RSV antibody or antigen binding fragment.
1. Subjects for Therapy
[0217] A subject or candidate for therapy with an anti-RSV antibody or antigen-binding fragment thereof provided herein includes, but is not limited to, a subject, such as a human patient, that has been exposed to a RSV virus, a subject, such as a human patient, who exhibits one or more symptoms of a RSV infection and a subject, such as a human patient, who is at risk of a RSV infection. Exemplary RSV virus infections include those caused by RSV viruses, such as, but not limited to, acute RSV disease, RSV upper respiratory tract infection (URI) and/or RSV lower respiratory tract infection (LRI), including, for example, bronchiolitis and pneumonia.
[0218] In some examples, the subject for therapy with an anti-RSV antibody or antigen binding fragment thereof provided herein is a mammal. In some examples, the subject for therapy with an anti-RSV antibody or antigen-binding fragment thereof provided herein is a primate. In particular examples, the subject for therapy with an anti-RSV antibody or antigen binding fragment thereof provided herein is a human.
[0219] The provided anti-RSV antibodies or antigen-binding fragments thereof can be administered to a subject, such as a human patient, for the treatment of any RSV-mediated disease. For example, the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject to alleviate one or more symptoms or conditions associated with a RSV virus infection, including, but not limited to, asthma, wheezing, reactive airway disease (RAD), and chronic obstructive pulmonary disease (COPD). Such diseases and condition are well known and readily diagnosed by physicians or ordinary skill.
[0220] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject, such a human patient, having a RSV virus infection for the maintenance or suppression therapy of recurring RSV virus-mediated disease. [0221] The provided anti-RSV antibodies or antigen-binding fragments thereof can be administered to a subject, such as a human patient, at risk of a RSV virus infection, including, but not limited to, a prematurely bom (pre-term) infant (e.g. , a human infant bom less than 38 weeks of gestational age, such as, for example, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, or 37 weeks gestational age); an infant (e.g., a human infant bom more than 37 weeks gestational age), a subject having cystic fibrosis, bronchopulmonary dysplasia, congenital heart disease, congenital immunodeficiency, or acquired immunodeficiency (e.g, an AIDS patient), leukemia, non-Hodgkin lymphoma, an immunosuppressed patient, such as, for example, a recipient of a transplant (e.g. a bone marrow transplant or a kidney transplant), or elderly subjects, including individuals in nursing homes or rehabilitation centers. In some examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject, such as a pre-term infant or infant exposed to one or more environmental risk factors, such as, but not limited to attending daycare, having school aged siblings, exposure to environmental air pollutants, congenital airway abnormalities, and/or severe neuromuscular disease. In some examples, the provided anti-RSV antibodies or antigen-binding fragments thereof can be administered to a subject, such an infant or child who is younger than two years, having chronic lung disease or congenital heart disease, including congestive heart failure, pulmonary hypertension, and cyanotic heart disease.
[0222] Tests for various pathogens and pathogenic infection are known in the art and can be employed for the assessing whether a subject is a candidate for therapy with an anti- RSV antibody or antigen-binding fragment thereof provided herein. For example, tests for RSV virus infection, are known and include for example, viral culture plaque assays, antigen detection test, polymerase chain reaction (PCR) tests, and various antibody serological tests. Tests for viral infection can be performed on samples obtained from tissue or fluid samples, such as spinal fluid, blood, or urine. Additional tests include, but are not limited to chest X-rays, which can show signs of pneumonia, other blood tests, such as a chemistry screening, a complete blood count, or arterial blood gases (ABGs) analysis, and oximetry, to measure the amount of oxygen in the blood.
[0223] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject, who is at an increased risk of RSV infection during particular times of the year. RSV season typically extends from October through May. Subjects, who exhibit increased susceptibility to virus infection during this time, such as infants the elderly or immunocompromised patients, can be administered an anti-RSV antibody or antigen-binding fragment thereof provided herein for the prophylaxis and/or treatment of RSV infection just prior to and/or during RSV season. In some examples, the anti-RSV antibody or antigen binding fragment thereof provided herein is administered one time, two times, three times, four times or five times during RSV season. In some examples, the anti-RSV antibody or antigen-binding fragment thereof provided herein is administered one time, two times, three times, four times or five times within one month, two months or three months, prior to a RSV season.
2. Dosages
[0224] The anti-RSV antibody or antigen-binding fragment thereof provided herein is administered in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration of an anti-RSV antibody or antigen-binding fragment thereof can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by using the assays provided herein or known in the art.
[0225] An effective amount of antibody or antigen-binding fragment thereof to be administered therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. In addition, the attending physician takes into consideration various factors known to modify the action of drugs, including severity and type of disease, patient's health, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Accordingly, it will be necessary for the therapist to titer the dosage of the antibody or antigen-binding fragment thereof and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer the antibody or antigen-binding fragment thereof until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays. Exemplary assays for monitoring treatment of a viral infection are know in the art and include for example, viral titer assays.
[0226] Generally, the dosage ranges for the administration of the anti-RSV antibodies or antigen-binding fragments thereof provided herein are those large enough to produce the desired effect in which the symptom(s) of the pathogen-mediated disease ( e.g . viral disease) are ameliorated or the likelihood of virus infection is decreased. In some examples, the anti- RSV antibodies or antigen-binding fragments thereof provided herein are administered in an amount effective for inducing an immune response in the subject. The dosage is not so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema or congestive heart failure. Generally, the dosage will vary with the age, condition, sex and the extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of the appearance of any adverse side effect. Exemplary dosages for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection include, but are not limited to, about or 0.01 mg/kg to about or 300 mg/kg, such as for example, about or 0.01 mg/kg, about or 0.1 mg/kg, about or 0.5 mg/kg, about or 1 mg/kg, about or 5 mg/kg, about or 10 mg/kg, about or 15 mg/kg, about or 20 mg/kg, about or 25 mg/kg, about or 30 mg/kg, about or 35 mg/kg, about or 40 mg/kg, about or 45 mg/kg, about or 50 mg/kg, about or 100 mg/kg, about or 150 mg/kg, about or 200 mg/kg, about or 250 mg/kg, or about or 300 mg/kg.
[0227] In some examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered to a subject at a dosage effective to achieve a desired serum titer. In particular examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a serum titer of at least or about 1 pg/ml, at least or about 2 pg/ml, at least or about 3 pg/ml, at least or about 4 pg/ml, at least or about 5 pg/ml, at least or about 6 pg/ml, at least or about 7 pg/ml, at least or about 8 pg/ml, at least or about 9 pg/ml, at least or about 10 pg/ml, at least or about 15 pg/ml, at least or about 20 pg/ml, at least or about 25 pg/ml, at least or about 30 pg/ml, at least or about 40 pg/ml, at least or about 50 pg/ml, at least or about 60 pg/ml, at least or about 70 pg/ml, at least or about 80 pg/ml, at least or about 90 pg/ml, at least or about 100 pg/ml, at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days or 40 days following administration of a first dose of the antibody or antigen-binding fragment thereof and prior to a subsequent dose of the antibody or antigen-binding fragment thereof.
[0228] In some examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered by pulmonary delivery to a subject at a dosage effective to achieve a desired titer in an intubation sample, sputum or lavage from the lungs. In particular examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a titer of 10 pg/mg (ng anti-RSV antibody or antigen-binding fragment thereof per mg lung protein) or about 10 pg/mg, 15 pg/mg or about 15 pg/mg, 20 pg/mg or about 20 pg/mg, 25 pg/mg or about 25 pg/mg, 30 pg/mg or about 30 pg/mg, 40 pg/mg or about 40 pg/mg, 50 pg/mg or about 50 pg/mg, 60 pg/mg or about 60 pg/mg, 70 pg/mg or about 70 pg/mg, 80 pg/mg or about 80 pg/mg, 90 pg/mg or about 90 pg/mg, 100 pg/mg or about 100 pg/mg, 110 pg/mg or about
110 pg/mg, 120 pg/mg or about 120 pg/mg, 130 pg/mg or about 130 pg/mg, 140 pg/mg or about 140 pg/mg, or 150 pg/mg or about 150 pg/mg in an intubation sample or lavage from the lungs at or about 10 days, 15 days, 20 days, 25 days, 30 days, 35 days or 40 days following administration of a first dose of the antibody or antigen-binding fragment thereof and prior to a subsequent dose of the antibody or antigen-binding fragment thereof.
[0229] For treatment of a viral infection, the dosage of the anti-RSV antibodies or antigen binding fragments thereof can vary depending on the type and severity of the disease. The anti-RSV antibodies or antigen-binding fragments thereof can be administered single dose, in multiple separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs or the desired improvement in the patient's condition is achieved. Repeated administrations can include increased or decreased amounts of the anti-RSV antibody or antigen-binding fragment thereof depending on the progress of the treatment. Other dosage regimens also are contemplated.
[0230] In some examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered one time, two times, three times, four times, five times, six time, seven times, eight times, nine times, ten times or more per day or over several days. In particular examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered one time, two times, three times, four times, five times, six time, seven times, eight times, nine times, ten times or more for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a serum titer of at least or about 1 pg/ml, at least or about 2 pg/ml, at least or about 3 pg/ml, at least or about 4 pg/ml, at least or about 5 pg/ml, at least or about 6 pg/ml, at least or about 7 pg/ml, at least or about 8 pg/ml, at least or about 9 pg/ml, at least or about 10 mg/ml, at least or about 15 pg/ml, at least or about 20 pg/ml, at least or about 25 pg/ml. at least or about 30 pg/ml, at least or about 40 pg/ml, at least or about 50 pg/ml. at least or about 60 pg/ml, at least or about 70 pg/ml, at least or about 80 pg/ml, at least or about 90 pg/ml, at least or about 100 pg/ml, at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days or 40 days following administration of a first dose, second dose, third dose, fourth dose, fifth dose, sixth dose, seventh dose, eighth dose, ninth dose, tenth dose of the antibody or antigen-binding fragment thereof and prior to a subsequent dose of the antibody or antigen-binding fragment thereof. In a particular example, the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered four times for the prevention or treatment of a RSV infection and/or amelioration of one or more symptoms of a RSV infection at an amount effective to achieve a serum titer of at least or about 72 pg/ml at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days or 40 days following administration of the fourth dose of the antibody or antigen-binding fragment thereof and prior to a subsequent dose of the antibody or antigen-binding fragment thereof.
[0231] In some examples, the anti-RSV antibodies or antigen-binding fragments thereof are administered in a sequence of two or more administrations, where the administrations are separated by a selected time period. In some examples, the selected time period is at least or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months.
[0232] In some examples, a prophylactically effective amount of an anti-RSV antibody or antigen-binding fragment thereof provided herein is administered one or more times just prior to RSV season. In some examples, a prophylactically effective amount of an anti- RSV antibody or antigen-binding fragment thereof provided herein is administered one or more times just prior to RSV season and/or one or more times during RSV season.
[0233] Therapeutic efficacy of a particular dosage or dosage regimen also can be assessed, for example, by measurement of viral titer in the subject prior to and following administration of one or more doses of the anti-RSV antibody or antigen-binding fragment thereof. Dosage amounts and/or frequency of administration can be modified depending on the desired rate of clearance of the virus in the subject. [0234] As will be understood by one of skill in the art, the optimal treatment regimen will vary and it is within the scope of the treatment methods to evaluate the status of the disease under treatment and the general health of the patient prior to, and following one or more cycles of therapy in order to determine the optimal therapeutic dosage and frequency of administration. It is to be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the pharmaceutical formulations, and that the dosages set forth herein are exemplary only and are not intended to limit the scope thereof. The amount of an anti-RSV antibody or antigen-binding fragment thereof to be administered for the treatment of a disease or condition, for example a viral infection ( e.g . a RSV virus infection), can be determined by standard clinical techniques (e.g. viral titer or antigen detection assays). In addition, in vitro assays and animal models can be employed to help identify optimal dosage ranges. Such assays can provide dosages ranges that can be extrapolated to administration to subjects, such as humans. Methods of identifying optimal dosage ranges based on animal models are well known by those of skill in the art.
3. Routes of Administration
[0235] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered to a subject by any method known in the art for the administration of polypeptides, including for example systemic or local administration. The anti- RSV antibodies or antigen-binding fragments thereof can be administered by routes, such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavity), topical, epidural, or mucosal (e.g. intranasal or oral). The anti-RSV antibodies or antigen-binding fragments thereof can be administered externally to a subject, at the site of the disease for exertion of local or transdermal action. Compositions containing anti- RSV antibodies or antigen-binding fragments thereof can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa). Compositions containing anti-RSV antibodies or antigen-binding fragments can be administered together with other biologically active agents. The mode of administration can include topical or other administration of a composition on, in or around areas of the body that may come on contact with fluid, cells, or tissues that are infected, contaminated or have associated therewith a virus, such as a RSV virus. The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered by topical or aerosol routes for delivery directly to target organs, such as the lungs (e.g. by pulmonary aerosol). In some examples, the provided anti- RSV antibodies or antigen-binding fragments thereof can be administered as a controlled release formulation as such as by apump (see, e.g., Langer (1990) Science 249:1527-1533; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al. (1980) Surgery 88:507; and Saudek et al. (1989) N. Engl. J. Med. 321:574) or via the use of various polymers known in the art and described elsewhere herein. In some examples, a controlled or sustained release system can be placed in proximity of the therapeutic target, for examples, the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
[0236] In particular examples, the provided anti-RSV antibodies or antigen-binding fragments thereof are administered by pulmonary delivery (see, e.g., U.S. Pat. Nos.
6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and
WO 99/66903). Exemplary methods of pulmonary delivery are known in the art and include, but are not limited to, aerosol methods, such as inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems), intratracheal instillation and insufflation. In some examples, pulmonary delivery can be enhanced by co-administration of or administration of a co-formulation containing the anti-RSV antibodies or antigen-binding fragments thereof provided herein and a permeation enhancer, such as, for example, surfactants, fatty acids, saccharides, chelating agents and enzyme inhibitors, such as protease inhibitors.
[0237] Appropriate methods for delivery, such as pulmonary delivery, can be selected by one of skill in the art based on the properties of the dosage amount of the anti-RSV antibody or antigen-binding fragment thereof or the pharmaceutical composition containing the antibody or antigen-binding fragment thereof. Such properties include, but are not limited to, solubility, hygroscopicity, crystallization properties, melting point, density, viscosity, flow, stability and degradation profile.
[0238] In some examples, the anti-RSV antibodies or antigen-binding fragments thereof provided herein increase the efficacy mucosal immunization against a virus. Thus, in particular examples the anti-RSV antibodies or antigen-binding fragments thereof are administered to a mucosal surface. For example, the anti-RSV antibodies or antigen-binding fragments thereof can be delivered via routes such as oral (e.g., buccal, sublingual), ocular (e.g., comeal, conjunctival, intravitreally, intra-aqueous injection), intranasal, genital (e.g., vaginal), rectal, pulmonary, stomachic, or intestinal. The anti-RSV antibodies or antigen binding fragments thereof provided herein can be administered systemically, such as parenterally, for example, by injection or by gradual infusion over time or enterally (i.e., digestive tract). The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered topically, such as for example, by topical installation or application (e.g., intratracheal instillation and insufflation using a bronchoscope or other artificial airway) of liquid solutions, gels, ointments, powders or by inhalation (e.g., nasal sprays, inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems)). Administration can be effected prior to exposure to the virus or subsequent to exposure to the virus.
4. Combination Therapies
[0239] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered alone or in combination with one or more therapeutic agents or therapies for the prophylaxis and/or treatment of a disease or condition. For example, the provided anti- RSV antibodies or antigen-binding fragments thereof can be administered in combination with one or more antiviral agents for prophylaxis. In another example, the provided anti- RSV antibodies or antigen-binding fragments thereof can be administered in combination with one or more antiviral agents treatment of a viral infection, such as a respiratory viral infection. In some examples, the respiratory viral infection is a RSV infection. The antiviral agents can include agents to decrease and/or eliminate the pathogenic infection or agents to alleviate one or more symptoms of a pathogenic infection. In some examples, a plurality of antibodies or antigen-binding fragments thereof (e.g., one or more antiviral antibodies) also can be administered in combination, where at least one of the antibodies is an anti- RSV antibody or antigen-binding fragment thereof provided herein. In some examples, a plurality of antibodies can be administered in combination for the prophylaxis, where at least one of the antibodies is an anti-RSV antibody or antigen-binding fragment thereof provided herein. In other examples, a plurality of antibodies can be administered in combination for treatment of a RSV infection or multiple viral infections, where at least one of the antibodies is an anti-RSV antibody or antigen-binding fragment thereof provided herein. In some examples, the anti-RSV antibodies provided can be administered in combination with one or more antiviral antibodies, which bind to and neutralize a virus, such as RSV. In some examples, the anti-RSV antibodies or antigen-binding fragments thereof provided can be administered in combination with one or more antibodies, which can inhibit or alleviate one or more symptoms of a viral infection, such as a RSV infection. In some examples, two or more of the anti-RSV antibodies or antigen-binding fragments thereof provided herein are administered in combination.
[0240] The one or more additional agents can be administered simultaneously, sequentially or intermittently with the anti-RSV antibody or antigen-binding fragment thereof. The agents can be co-administered with the anti-RSV antibody or antigen-binding fragment thereof, for example, as part of the same pharmaceutical composition or same method of delivery. In some examples, the agents can be co-administered with the anti-RSV antibody or antigen binding fragment thereof at the same time as the anti-RSV antibody or antigen-binding fragment thereof, but by a different means of delivery. The agents also can be administered at a different time than administration of the anti-RSV antibody or antigen-binding fragment thereof, but close enough in time to the administration of the anti-RSV antibody or antigen binding fragment thereof to have a combined prophylactic or therapeutic effect. In some examples, the one or more additional agents are administered subsequent to or prior to the administration of the anti-RSV antibody or antigen-binding fragment thereof separated by a selected time period. In some examples, the time period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. In some examples, the one or more additional agents are administered multiple times and/or the anti- RSV antibody or antigen-binding fragment thereof provided herein is administered multiple times.
[0241] In some examples, administration of the combination inhibits the incidence of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the incidence of RSV infection in the absence of the combination. In some examples, administration of the combination decreases the severity of one or more symptoms of RSV infection by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the severity of the one or more symptoms of RSV infection in the absence of the combination.
[0242] In some examples, the combination inhibits the binding of RSV to its host cell receptor by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% relative to the binding of RSV to its host cell receptor in the absence of the combination. In some examples, the combination inhibits RSV replication by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about
75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about
55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about
35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about
15%, or at least or about 10% relative to RSV replication in the absence of the combination.
[0243] Any therapy which is known to be useful, or which is or has been used for the prevention, management, treatment, or amelioration of a RSV infection or one or more symptoms thereof can be used in combination with anti-RSV antibody or antigen-binding fragment thereof provided herein (see, e.g., Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis and Therapy, Berkow, M. D. et al. (eds.), 17th Ed., Merck Sharp & Dohme Research Laboratories, Rahway, N.J., 1999; Cecil Textbook of Medicine, 20th Ed., Bennett and Plum (eds.), W. B. Saunders, Philadelphia, 1996, for information regarding therapies (e.g., prophylactic or therapeutic agents) which have been or are used for preventing, treating, managing, or ameliorating a RSV infection or one or more symptoms thereof). Examples of such agents include, but are not limited to, immunomodulatory agents, anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, non-steroidal anti-inflammatory drugs (e.g. aspirin, ibuprofen, diclofenac, and COX-2 inhibitors)), pain relievers, leukotriene antagonists (e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), bronchodilators, such as b-agonists (e.g., bambuterol, bitolterol, clenbuterol, fenoterol, formoterol, indacaterol, isoetharine, metaproterenol, pirbuterol, procaterol, reproterol, rimiterol, salbutamol (Albuterol, Ventolin), levosalbutamol, salmeterol, tulobuterol and terbutaline) and anticholinergic agents (e.g., ipratropium bromide and oxitropium bromide), sulphasalazine, penicillamine, dapsone, antihistamines, anti-malarial agents (e.g., hydroxychloroquine), and antiviral agents. The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered in combination with one or more therapies for the treatment of a RSV infection, including but not limited to, administration of intravenous infusion of immunoglobulin, administration of supplemental oxygen and fluids or assisted breathing. The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered in combination with one or more agents that regulate lung maturation and surfactant protein expression, such as, but not limited to, glucocorticoids, PPARy ligands, and vascular endothelial cell growth factor (VEGF).
[0244] Exemplary antiviral agents that can be selected for combination therapy with an anti- RSV antibody or antigen-binding fragment thereof provided herein include, but are not limited to, antiviral compounds, antiviral proteins, antiviral peptides, antiviral protein conjugates and antiviral peptide conjugates, including, but not limited to, nucleoside analogs, nucleotide analogs, immunomodulators (e.g. interferons) and immunostimulants.
Combination therapy using antibodies and/or anti-RSV antibodies and antigen-binding fragments provided herewith are contemplated as is combination with the antibodies and/or anti-RSV antibodies and antigen-binding fragments provided herein with other anti- RSV antibodies and anti-RSV antibodies and antigen-binding fragments.
[0245] Exemplary antiviral agents for the treatment of virus infections that can be administered in combination with the anti-RSV antibodies or antigen-binding fragments thereof provided herein include, but are not limited to, acyclovir, famciclovir, ganciclovir, penciclovir, valacyclovir, valganciclovir, idoxuridine, trifluridine, brivudine, cidofovir, docosanol, fomivirsen, foscamet, tromantadine, imiquimod, podophyllotoxin, entecavir, lamivudine, telbivudine, clevudine, adefovir, tenofovir, boceprevir, telaprevir, pleconaril, arbidol, amantadine, rimantadine, oseltamivir, zanamivir, peramivir, inosine, interferon (e.g., Interferon alfa-2b, Peginterferon alfa-2a), ribavirin/taribavirin, abacavir, emtricitabine, lamivudine, didanosine, zidovudine, apricitabine, stampidine, elvucitabine, racivir, amdoxovir, stavudine, zalcitabine, tenofovir, efavirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine, atazanavir, fosamprenavir, lopinavir, darunavir, nelfmavir, ritonavir, saquinavir, tipranavir, amprenavir, indinavir, enfuvirtide, maraviroc, vicriviroc, PRO 140, ibalizumab, raltegravir, elvitegravir, bevirimat, vivecon, including tautomeric forms, analogs, isomers, polymorphs, solvates, derivatives, or salts thereof.
[0246] Exemplary antiviral agents for the prophylaxis and/or treatment of RSV infections that can be administered in combination with the anti-RSV antibodies or antigen-binding fragments thereof provided herein include, but are not limited to, ribavirin, NIH-351 (Gemini Technologies), recombinant RSV vaccine (Aviron), RSVf-2 (Intracel), F-50042 (Pierre Fabre), T-786 (Trimeris), VP-36676 (ViroPharma), RFI-641 (American Home Products), VP-14637 (ViroPharma), PFP-1 and PFP-2 (American Home Products), RSV vaccine (Avant Immunotherapeutics), F-50077 (Pierre Fabre), and other anti-RSV antibodies or antigen binding fragments thereof.
[0247] The anti-RSV antibodies or antigen-binding fragments thereof provided herein also can be administered in combination with one or more agents capable of stimulating cellular immunity, such as cellular mucosal immunity. Any agent capable of stimulatory cellular immunity can be used. Exemplary immunostimulatory agents include, cytokines, such as, but not limited to, interferons (e.g., IFN-a, b, g, w), lymphokines and hematopoietic growth factors, such as, for example, GM-CSF (granulocyte macrophage colony stimulating factor), Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin- 10 (IL-10), Interleukin- 12 (IL-12), Interleukin- 14 (IL-14), and Tumor Necrosis Factor (TNF).
[0248] For combination therapies with anti-pathogenic agents, dosages for the administration of such compounds are known in the art or can be determined by one skilled in the art according to known clinical factors (e.g., subject's species, size, body surface area, age, sex, immunocompetence, and general health, duration and route of administration, the kind and stage of the disease, and whether other treatments, such as other anti-pathogenic agents, are being administered concurrently).
[0249] The anti-RSV antibodies or antigen-binding fragments thereof provided herein can be administered in combination with one or more additional antibodies or antigen-binding fragments thereof. In some examples, the one or more additional antibodies are antiviral antibodies. In some examples, the one or more additional antibodies bind to a viral antigen. In some examples, the one or more additional antibodies bind to a viral antigen that is a surface protein, such as a viral capsid protein or a viral envelope protein. In some examples, the one or more additional antibodies bind to a viral antigen that is expressed on the surface of an infected cell. In some examples, the one or more additional antibodies bind to a viral antigen that is expressed intracell ularly (i.e., within an infected cell). In some examples, the one or more additional antibodies binds to a virus that causes respiratory disease, such as, but not limited to, RSV, parainfluenza virus (PIV) or human metapneumovirus (hMPV). Compositions containing the mixtures of antibodies also are provided herein.
[0250] In some examples, the one or more additional antiviral antibodies are anti- RSV antibodies or antigen-binding fragments thereof. In some examples, an anti- RSV antibody or antigen-binding fragment thereof provided herein is administered in combination with the one or more additional anti-RSV antibodies or antigen-binding fragments thereof for the prophylaxis and/or treatment of a RSV infection. Exemplary anti- RSV antibodies or antigen-binding fragments thereof for combination therapy with an anti- RSV antibody or antigen-binding fragment thereof provided herein include anti- RSV antibodies or antigen-binding fragments thereof that immunospecifically bind to and neutralize RSV. In some examples, the one or more additional anti-RSV antibodies or antigen-binding fragments thereof includes an antibody or antigen-binding fragment thereof that immunospecifically binds to RSV A subtype and/or RSV B subtype.
[0251] In some examples, the one or more additional antiviral antibodies for combination therapy with an anti-RSV antibody or antigen-binding fragment thereof provided herein includes, but is not limited to, palivizumab (SYNAGIS®), motavizumab (NUMAX®),
AFFF, P1212, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3- 3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S, (see U.S. Pat. Nos. 5,824,307 and 6,818,216), rsv6, rsvll, rsvl3, rsvl9, rsv21, rsv22, rsv23 (see U.S. Pat. No. 6,685,942), RF-1, RF-2 (see U.S. Pat. No. 5,811,524), or antigen-binding fragments thereof. In some examples, the one or more additional antiviral antibodies for combination therapy includes an antibody or antigen binding fragment thereof containing a VH chain and/or VL chain having the amino acid sequence of a VH chain and/or VL chain of palivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P1212, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, IX- 493L1, FRH3-3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S, rsv6, rsvll, rsvl3, rsvl9, rsv21, rsv22, rsv23, RF- 1, or RF-2. In some examples, the one or more additional antiviral antibodies for combination therapy includes an antibody or antigen-binding fragment thereof containing one or more CDRs of palivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P1212, P12f4, Plld4, Ale9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FRH3-3F4, M3H9, Y10H6, DG, AFFF(l), 6H8, L1-7E5, L2-15B10, A13all, Alh5, A4B4(1), A4B4L1FR-S28R, A4B4- F52S, rsv6, rsvll, rsvl3, rsvl9, rsv21, rsv22, rsv23, RF-1, or RF-2. In some examples, the one or more additional antiviral antibodies for combination therapy includes an antibody or antigen-binding fragment thereof containing one or more CDRs of from an anti-RSV mouse monoclonal antibody such as, but not limited to, MAbs 1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112, 1269, 1269, 1243 (Beeler et al. (1989) J. Virology 63(7):2841-2950), MAM51 (Mufson et al. (1987) J. Clin. Microbiol. 25:1635-1539), MAbs 43-1 and 13-1
(Femie et al. (1982) Proc. Soc. Exp. Biol. Med. 171:266-271), MAbs 1436C, 1302A, 1308F, and 1331H (Anderson et al. (1984) J. Clin. Microbiol. 19:934-936). Additional exemplary antibodies or antigen-binding fragments thereof that can be used for combination therapy with an anti-RSV antibody or antigen-binding fragment provided herein include, but are not limited to, anti-RSV antibodies or antigen-binding fragments thereof described in, for example, U.S. Pat. Nos. 6,413,771, 5,840,298, 5,811,524, 6,656,467, 6,537,809, 7,364,742, 7,070,786, 5,955,364, 7,488,477, 6,818,216, 5,824,307, 7,364,737, 6,685,942, and 5,762,905 and U.S. Patent Pub. Nos. 2007-0082002, 2005-0175986, 2004-0234528, 2006-0198840, 2009-0110684, 2006-0159695, 2006-0013824, 2005-0288491, 2005-0019758, 2008- 0226630, 2009-0137003, and 2009-0092609.
[0252] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.
EXAMPLES
Example 1: Generation and Evaluation of Site Evaluation Libraries
A. Plasmids and site evaluation construction for anti-RSV HC and LC [0253] Sequences for the heavy chain of monoclonal antibodies against respiratory syncytial virus (palivizumab or Synagis) were codon optimized and synthesized by GeneArt GmH (Germany). To prevent from potential degradation by Kex2 furin-like protease during expression in a fungal cell, a lysine at position 251 in the heavy chain was mutated to threonine (K251T). Initially synthetic sequences of the anti-RSV HC were cloned individually behind a catalytical core of the Trichoderma reesei native cellobiohydrolase I (CBH1) together with its linker region (1-479 aa). To release mature antibody chains from the carrier partner a Kex2 cleavage site was introduced between the linker and HC.
[0254] A fusion construct of cbhl-HC Synagis was then amplified by PCR with gene specific primers extended with the attBl and attB2 sites to allow for the Gateway® BP recombination cloning into pDonor221 vector (Invitrogen, USA). Plasmid pEntry- SynagisHCK251T_Geneart_SEL was used by the vendor BaseClear (Netherlands) as a template for construction of site evaluation (SEL) library at positions 466-725 aa (counting from the Cbhl Met). An average number of mutant variants per aa position was around 17. Mutated sequences were further cloned via the Gateway® LR recombination technique into pTTTpyr2-ISceI destination vector resulting in the final expression plasmids pTTTpyr2- ISceI-SynagisHC_ Geneart SEL.
[0255] This expression vector contains the T. reesei cbhl promoter and terminator regions allowing for a strong inducible expression of a gene of interest and the T. reesei pyr2 selective marker conferring growth of transformants on minimal medium without supplementation with uridine. The plasmid is maintained autonomously in fungal cells due to T. reesei derived telomere regions. Plasmids were propagated in commercially available Escherichia coli TOP10 cells (Invitrogen, US), purified, sequence verified, arrayed individually in 96 well MTPs and used for fungal transformation as described below.
[0256] pEntry-Synagis LC Geneart plasmid was constructed via the Gateway® BP recombination cloning and recombined further with pTrex6g destination vector in a similar way as described above resulting in the expression vector pTrex6g-Synagis_LC. This vector served as a template to generate a PCR fragment expressing the light chain driven by the cbhl promoter and linked to the alS marker conferring resistance to chlorimuron ethyl to a fungal cell.
B. Fungal host strain construction and transformation
[0257] The expression cassette consists of a CBH1 promoter, CBH1 core, antibody HC and LC connected by CBH1 linker and kex2 for processing of CBH1, CBH1 terminator, and the alS marker conferring resistance to chlorimuron ethyl to a fungal cell. The alS marker was used for making the host strains so that the pyr2 marker was available for the SEL variants. The expression cassette was randomly integrated into the host T. reesei genome at multiple copies. The full expression cassette was amplified by PCR. The PCR product was cleaned up and concentrated to 500-1000ng/pL.
[0258] The host T. reesei strain used for transformation was deleted for major cellulases and xylanases. The strain was transformed using a standard PEG-protoplast transformation method. Transformation mixtures containing approximately 10 pg of DNA and 5x 106 protoplasts in a total volume of 250 pi were treated with 2 mL of 25% PEG solution, diluted with 2 volumes of 1.2M sorbitol/lOmM Tris, pH7.5/ lOmM CaC12 solution, and mixed with 26mL of 2% low melting agarose containing 1M sorbitol, 1 g/L uridine, 75 mg/L chlorimuron ethyl in minimal medium and distributed over four 10cm petri plates pre-poured containing 1.5% agarose, 1M sorbitol in minimal media. After sufficient growth transformants from each plate were observed, individual colonies were picked onto fresh 10cm petri plates containing 1.5% agar, lg/L uridine, 75 mg/L chlorimuron ethyl, 4 per plate to allow room for assessing stability. The stable colony phenotype is concentric circular growth with smooth edges. Once stable transformants were observed and well sporulated, spores were harvested and used for inoculation of liquid cultures.
[0259] All high throughput transformations with Synagis HC variants were performed robotically in a 24 well MTP format using Biomek robots (Beckman Coulter, USA).
Plasmids with variants were received from the vendor in a 96 well format arrayed according to a predetermined layout. Transformation mixtures containing approximately 1 mg of DNA and 5x 106 protoplasts in a total volume of 50 ml were treated with 200 mΐ of 25% PEG solution, diluted with 1 volumes of 1.2M sorbitol/lOmM Tris, pH7.5/ lOmM CaCh solution, rearranged robotically into 24 well MTPs and poured in 1 ml of 3% low melting agarose containing 1M sorbitol in minimal medium. After sufficient growth transformants from each well were pooled together and plated on fresh 24 well agar plates with minimal medium. Once sporulated, spores were harvested and used for inoculation of liquid cultures.
C. Fungal fermentations in slow release 24 well MTPs
[0260] To generate sufficiently high antibody titers 105-106 T. reesei spores were inoculated in customer made 24 well MTPs composed of the Sylgard 170 elastomer (from Dow Coming, USA) premixed with lactose which was slowly released in the medium during fermentation to ensure continuous production. Cultures were grown in 1.25 ml of medium containing: 16 g/L glucose, 9 g/L casamino acids, 10 g/L (NH4)2S04, 4.5 g/L KH2P04, 1 g/L MgS04*7H20, 1 g/L CaC12*2H20, 33 g/L PIPPS buffer [pH 5.5], 0.25% T. reesei trace elements (100%: 175 g/L citric acid (anhydrous), 200 g/L FeS04*7H20, 16 g/L ZnS04*7H20, 3.2 g/L CuS04*5H20, 1.4 g/L MnS04*H20, 0.8 g/L H3B03).
[0261] Plates were incubated in Infors shaker with a 50 mm throw at 200 rpm and 28C with 80% humidity. After 5-6 days of growth cultures were reformatted back to 96 well deep well MTPs and filtered using 96-well microtiter filter plates (0.2 pm hydrophilic PVDF membrane, Coming, Tewksbury MA). The plates were frozen in Axygen half-deep well plates (P-DW-ll-C).
D. Purification
[0262] Plates were moved from the freezer to the cold room to allow the samples to gradually thaw overnight at 4 °C. Before purification, grown WT samples were removed from the plates and these samples were pooled. One mL per well of pooled WT, pooled low binding control, pooled high binding control, and pooled vector only (vector expressing CBH1 in same strain) samples were added to designated wells. The library plates were grown in duplicate and these controls were added to both plates. The plates gently shook for 2 minutes to homogenize the fluid in the wells followed by centrifugation for 1 minute to pellet any precipitate.
[0263] The centrifuged plates were then moved to a robot to remove 20 pL of the crude material for Octet Protein A quantitation. The 20 pL was added to 80 pL of IX PBS in a 384-well plate (Greiner Bio-One 781209). Four library plates went into one 384-well plate and there was a separate 384-well plate for the duplicate growth of the four plates (plates Xa and Xb).
[0264] After samples were removed for the Octet quantitation, the plates were then purified. The robot handled four library plates at a time. The robot added 50 pL of 1 M KPi pH 7 to pH up the supernatant to improve the antibody binding to the Protein A resin. The robot then transferred the crude material (max 880 pL per well) from the four plates to 2 mL filter plates (Pall 8275) filled previously with 220 pL of Protein A resin in PBS. These filter plates then shook for 5 minutes on a shaker. The plates were then filtered by centrifugation at lOOOg for 2 minutes, and the flow through was collected in the empty harvest plate that the samples were transferred from. This material was stored until after quantitation. The filter plates were returned to the robot deck and the duplicate growth plates were added to the same filter plates. These plates were incubated and centrifuged as before. The resin was then washed with 880 pL of PBS buffer. The plates shook for 1 minute and then centrifuged at 1000 g for 2 minutes. The flow through was discarded, and the plates were returned to the robot for the second PBS washing. After the second washing, the plates were moved to a robot running the elution program.
[0265] The elution program handled four plates at a time. It added 11 pL of neutralization buffer (1 M Tris pH 9) to a clean half-deep well plate that the samples would be eluted into. The program then added 440 pL of elution buffer (100 mM glycine pH 2.7) to the filter plates. The plates then shook for 1 minute at setting 7 and then were filtered by centrifugation (lOOOg for 2 minutes) into the freshly prepped recovery plates. After centrifugation, the sample plates shook for 1 minute to ensure proper mixing of the neutralization buffer.
Example 2: Testing RSV variants for increased manufacturability, thermostability, and/or protease resistance
A. FRET Quantitation Assay and Normalization
[0266] Protein A (Thermo Fisher Scientific 77674) was labeled with Alexa Fluor 546 NHS ester (Thermo Fisher Scientific A20102). Protein L (Thermo Fisher Scientific 77680) was labeled with Alexa Fluor 488 NHS ester (Thermo Fisher Scientific A20100). The labeled Protein A and Protein L were diluted with 107 mM KPi pH 7 and at a ratio that produced a FRET signal for the standard curve with the proper dynamic range. The standard curve was commercial Synagis from AbbVie. In a Coming 3605 plate, 40 pL of the Protein A and L solution was mixed with 10 pL of the purified antibody sample. The FRET signal on the plate was read (ex: 485 nm em: 590 nm cutoff: 590 nm), and the concentration of the unknowns was determined from the Synagis standard curve. The samples were run in duplicate.
[0267] After analyzing the data, the plates were normalized to 120 ppm. The dilution buffer was Tris-Gly buffer that was at the same pH and concentration is in the purified samples. For the wells that were less than 120 ppm, they were not diluted and were used as is. B. Protein thermal shift (“Tm”) assay using SYPRO® Orange and PCR thermocycler
[0268] Unfolding of purified antiRSV polypeptide (including wild type and variants) was measured as follows. To 10 pi of purified and protein normalized antibody at 120ppm in the 384 well plate (Roche Diagnostics, Indianapolis, IN), 5 pi of lOOmM Cellobiose in 200mM NaAc pH5 was added and mixed, then 5 pi of 250-fold diluted sypro orange (Thermo Fisher Scientific Fisher, Grand Island, NY) was added and mixed. The plate was sealed and placed in the Roche Lightcycler 480. A program with 5 minutes at 37°C, a gradient from 37°C to 97°C with a heating ramp rate of 0.02°C/sec and 38 acquisitions/°C was run, the relative fluorescence change at EX 472nm/EM 570nm was recorded. The data was exported and was processed using R scripts. The first derivative of the fluorescence change vs. temperature is calculated, the transition temperatures corresponding to Fc, and Fab unfolding were reported.
C. Thermal Stability
[0269] In a 96-well PCR plate, 30 pL of 150 mM Na Acetate pH 5 was mixed with 70 pL of normalized antibody sample. For the unstressed FRET read, 20 pL of this antibody mixture was added to 30 pL of the labeled Protein A and L mixture in a 3605 plate. This unstressed FRET measurement was performed in duplicate. The ratio of labeled Protein A and L to antibody sample was optimized so that the unstress samples were at the top of the linear portion of the standard curve. The PCR plate was sealed with a Bio-Rad microseal B and place in a tetrad thermocycler to heat stress the plates for 10 minutes. After the temperature stress, the FRET signal of the stressed samples was measured in duplicate (20 pL of antibody mixture and 30 pL of the labeled Protein A and L mixture). The differences in stability was determined by comparing the ratios of the stressed and unstressed FRET signal for each sample.
D. Protease Stability
[0270] In a 3605 plate, 30 pL of concentrated T. reesei broth in 150 mM Na Acetate pH 5 was mixed with 70 pL of normalized antibody sample. The concentrated T. reesei broth was made by concentrating the broth using a 10 kDa MWCO membrane 10-fold. The T. reesei used to make the broth was an empty strain not expressing an antibody but did express background proteases. For the unstressed FRET read, 20 pL of this antibody mixture was added to 30 pL of the labeled Protein A and L mixture in a 3605 plate. This unstressed FRET measurement was performed in duplicate. The ratio of labeled Protein A and L to antibody sample was optimized so that the unstress samples were at the top of the linear portion of the standard curve. The stress plate was sealed with a Bio-Rad microseal B and place in an iEMS incubator at 30 °C for 2.5 hours. After the incubation, the FRET signal of the stressed samples was measured in duplicate (20 pL of antibody mixture and 30 pL of the labeled Protein A and L mixture). The differences in stability was determined by comparing the ratios of the stressed and unstressed FRET signal for each sample.
E. Results
[0271] Variants were tested for thermal stability, Tm, and protease resistance. Light chain positions with greater than 1.5 and 2.0 standard deviation improvement relative to the parent RSV antibody sequence are shown in Table 1 and Table 2. Heavy chain positions with greater than 1.5 and 2.0 standard deviation improvement relative to the parent RSV antibody sequence are shown in Table 3 and Table 4.
Table 1: Light chain positions with greater than 1.5 standard deviation improvement relative to the parent antibody sequence
Figure imgf000096_0001
Table 2: Light chain positions with greater than 2 standard deviation improvement relative to the parent antibody sequence
Figure imgf000097_0001
Table 3: Heavy chain positions with greater than 1.5 standard deviation improvement relative to the parent antibody sequence
Figure imgf000097_0002
Table 4: Heavy chain positions with greater than 2 standard deviation improvement relative to the parent antibody sequence
Figure imgf000097_0003
SEQUENCES
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPS1KSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGA GTTVTV S S ASTKGP SVFPLAPSSKSTS GGT AALGCL VKD YFPEP VTV S WN S GALTS G VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDTRVEPKSCDKTH TCPPCPAPEL1GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:3)
DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASG VPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:6)

Claims

CLAIMS We claim:
1. An isolated anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising:
(a) a heavy chain variable region which has at least 90% sequence identity to QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RLTI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; X4 is A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 is A or G; and/or
(b) a heavy chain constant region comprising ASTKGPSVFPLAPSXsKXoTSXioGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDTRXiiEPKSCDKTHTCPPCPAPELlGGPSVFLFPPKPKDTLMISRTPE VTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQD WLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein Xs is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y, wherein (i) the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO: 3; and (ii) wherein said antibody exhibits one or more improved properties comprising increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the heavy chain encoded by the amino acid sequence of SEQ ID NO: 3.
2. The antibody or functional fragment thereof of claim 1, further comprising:
(c) a light chain variable region which has at least 90% sequence identity to DIQMX12 X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21LIYDTSK LASGVPSRFSGSGSX22X23E
X24TLTIS SLQPDDF ATYY CFQGS GYPFTF GGGTKLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X201S A, S, V, orW; X211S L, S, or G; X221S G or A; X231S T or Y ; and X24 is F or C; and/or (d) a light chain constant region comprising
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein optionally the antibody differs by at least one amino acid from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO:6.
3. An isolated anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising:
(a) a light chain variable region which has at least 90% sequence identity to DIQMX12 X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21LIYDTSK LASGVPSRFSGSGSX22X23E
X24TLTIS SLQPDDF ATYY CFQGS GYPFTF GGGTKLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X201S A, S, V, orW; X211S L, S, or G; X221S G or A; X231S T or Y ; and X24 is F or C; and/or
(b) a light chain constant region comprising
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein (i) the antibody differs by at least one amino acid from an antibody comprising a light chain encoded by the amino acid sequence of SEQ ID NO: 6; and (ii) wherein said antibody exhibits one or more improved properties comprising increased manufacturability, thermostability, and/or protease resistance compared to an antibody comprising the light chain encoded by the amino acid sequence of SEQ ID NO: 6.
4. The antibody or functional fragment thereof of claim 3, further comprising:
(a) a heavy chain variable region which has at least 90% sequence identity to
QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RLTI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, T, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; XQs A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 1S A or G; and/or
(b) a heavy chain constant region comprising ASTKGPSVFPLAPSX8KX9TSX10GTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDTRXiiEPKSCDKTHTCPPCPAPELlGGPSVFLFPPKPKDTLMISRTPE VTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein X8 is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y, wherein optionally the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO: 3.
5. The antibody or functional fragment thereof of claim 1 or claim 4, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, wherein Xi is R, T, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; X4 is A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 is A or G.
6. The antibody or functional fragment thereof of claim 1 or claim 4, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:4, wherein X12 is T or P; X13 is Q or S; X14 is P or F; X15 is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xisis G or L; X191S F, N, or V; X201S A, S, V, or W; X21 is L, S, or G; X221S G or A; X23 1S T or Y; and X24 is F or C.
7. The antibody or functional fragment thereof of any one of claims 1-6, wherein said functional fragment is selected from the group consisting of Fab, Fab , F(ab )2 and Fv fragments.
8. The antibody or functional fragment thereof of any one of claims 1-6, wherein said antibody is chimeric, humanized, or fully human.
9. The antibody or functional fragment thereof of any one of claims 1-8, wherein said antibody competitively inhibits the binding of palivizumab to the surface of RSV.
10. A nucleic acid encoding the heavy chain variable region of SEQ ID NO: 1 and/or the heavy chain constant region of SEQ ID NO: 2.
11. A nucleic acid encoding the light chain variable region of SEQ ID NO:4 and/or the light chain constant region of SEQ ID NO:5.
12. A vector comprising the nucleic acid of claim 10 or claim 11.
13. The vector of claim 12, comprising the nucleic acid of claim 10 and claim 11.
14. A recombinant cell comprising the vector of claim 12 or claim 13.
15. The recombinant cell of claim 14, wherein the cell is a mammalian cell, a bacterial cell, or a fungal cell.
16. The recombinant cell of claim 15, wherein the fungal cell is T. reesei.
17. A method for producing an anti-respiratory syncytial virus (RSV) antibody or functional fragment thereof comprising providing an isolated cell with a nucleic acid encoding said antibody or functional part thereof, wherein said antibody or functional part thereof comprises one or more of:
(a) a heavy chain variable region comprising
QVTLX1ESGPALVKPTQTLX2LTCTFX3GFSLSTSGMSVGWIRQPPGKX4LEWLADIW WDDKKD YNP S IKS RLTI SKDTSKNQ V VLKVTNMDX5X6DTX7TYY CAR SMITNWYFDV W GAGTTVTV S S (SEQ ID NO: 1), wherein Xi is R, T, I, Y, W, C, or S; X2 is T, N, or V; X3 is S, V, L, or W; XOs A, G, S, or T; X5 is P or Y; Xe is A or S; and X7 1S A or G;
(b) a heavy chain constant region comprising ASTKGPSVFPLAPSX8KX9TSX10GTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDTRXiiEPKSCDKTHTCPPCPAPELlGGPSVFLFPPKPKDTLMISRTPE VTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein Xs is S or Y; X9 is S or Q; X10 is G or M; and X11 is V or Y, (c) a light chain variable region comprising DIQMX12
X13SX14SX15LSX16SVGDRVTITCKX17QLSVGYMHWYQQKPX18X19X20PKX21LIYDTSK
LASGVPSRFSGSGSX22X23E
X24TLTIS SLQPDDF ATYY CFQGS GYPFTF GGGTKLEAKRTV (SEQ ID NO:4), wherein X12 is T or P; X13 is Q or S; X14 is P or F; Xis is T or H; Xi6is A or S; Xnis C, M, V, or Y; Xi8is G or L; X191S F, N, orV; X201S A, S, V, orW; X211S L, S, or G; X221S G or A; X231S T or Y ; and X24 is F or C; and/or
(d) a light chain constant region comprising
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPX25EAKVQWKVDNALQSGNX26QESVTE QDSKDSTYSLSSTLTLSKADYEX27HKVYACEVTHQGLSSX28VTX29SFNRGEC (SEQ ID NO:5, wherein X25 is R or M; X26 is S or E; X27 is K or T; X28 is P or M; and X29 is K or G, wherein the antibody differs by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3 and/or a light chain encoded by the amino acid sequence of SEQ ID NO: 6.
18. The method of claim 17, wherein the cell is a mammalian cell, a bacterial cell, or a fungal cell.
19. The method of claim 18, wherein the fungal cell is T. reesei.
20. The method of any one of claims 17-19, wherein the antibody or functional fragment thereof exhibits one or more improved properties selected from the group consisting of increased manufacturability, thermostability, and protease resistance compared to an antibody that does not differ by at least one amino acid from an antibody comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:3 and/or a light chain encoded by the amino acid sequence of SEQ ID NO: 6.
21. A method for treating or preventing a respiratory syncytial virus (RSV) infection in an individual in need thereof comprising administering a therapeutically effective amount of the antibody or functional fragment thereof of any one of claims 1-9 to the individual.
22. The method of claim 21, wherein they antibody or functional fragment thereof is administered parenterally or intravenously.
23. The method of claim 21 or claim 22, wherein the individual is a human.
24. The method of claim 23, wherein the human is a preterm infant (under 35 weeks gestation) infant, infant with congenital heart defects (CHD), infant with bronchopulmonary dysplasia (BPD), and/or infant with congenital malformations of the airway.
25. The method of claim 23 or claim 24, wherein the individual is less than four years old.
26. The method of any one of claims 21-25, wherein the individual is further administered oxygen therapy.
27. A pharmaceutical composition comprising the antibody or functional fragment thereof of any one of claims 1-9 and a pharmaceutically acceptable carrier, diluent, or excipient.
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