ISOLATED ANTIBODY CAPABLE OF BINDING SPECIFICALLY TO HEMAGGLUTININ (HA) PROTEIN OF INFLUENZA B, E VI

专利摘要:
isolated antibody or antigen-binding fragment thereof, nucleic acid molecule, and pharmaceutical composition. The present invention relates to binding molecules, such as human monoclonal antibodies, which bind to an epitope in the major hemagglutinin region of influenza a virus of phylogenetic group 1 and group 2, as well as influenza b viruses, and exhibit a wide neutralizing activity against such influenza viruses. the description provides nucleic acid molecules encoding the binding molecules, their sequences, and compositions comprising the binding molecules. the binding molecules can be used in the diagnosis, prophylaxis and/or treatment of influenza a viruses of phylogenetic group 1 and 2, as well as influenza b viruses.
公开号:BR112014000263B1
申请号:R112014000263-0
申请日:2012-07-12
公开日:2022-01-18
发明作者:Theodorus Hendrikus Jacobus Kwaks；David A. T. M. Zuijdgeest；Ronald Vogels；Robert Heinz Edward Friesen
申请人:Janssen Vaccines & Prevention B.V.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The invention relates to medicine. The invention particularly relates to human binding molecules capable of neutralizing influenza A viruses of both phylogenetic group 1 and phylogenetic group 2. In particular, the invention relates to binding molecules capable of neutralizing influenza A viruses of both phylogenetic group 1 and phylogenetic group 2, as well as influenza viruses B. The invention further relates to the diagnosis, prophylaxis and/or treatment of an infection caused by influenza A viruses of phylogenetic groups 1 and 2, and preferably also influenza viruses B. FUNDAMENTALS OF THE INVENTION
[0002] Influenza infection (also referred to as "influenza" or "the flu") is one of the most common diseases known to man, causing between three and five million cases of serious illness, and between 250,000 and 500,000 deaths every year around the world. Influenza spreads rapidly in seasonal epidemics, affecting 515% of the population and the burden of health care costs and low productivity is large (World Health Organization (WHO)).
[0003] There are 3 types of Influenza virus (types A, B and C) responsible for infectious pathologies in humans and animals. Type A and type B viruses are the agents responsible for the seasonal influenza epidemics and pandemics observed in humans.
[0004] Influenza A virus can be classified into Influenza virus subtypes based on variations in antigenic regions of two genes encoding the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cellular release. . Currently, sixteen HA subtypes (H1-H16) and nine NA (N1-N9) antigenic variants are known in the influenza A virus. Influenza virus subtypes can be further classified by reference to their phylogenetic group. Phylogenetic analysis (Fouchier et al, 2005) has demonstrated a subdivision of HAs comprising two main groups (Air, 1981): inter alia, subtypes H1, H2, H5 H9 in phylogenetic group 1 (herein also referred to as "group 1" ) and, inter alia, the H3, H4 and H7 subtypes in phylogenetic group 2 (or "group 2"). Only a few of the influenza A subtypes (ie, H1N1, H1N2 and H3N2) circulate among people, but all combinations of the 16 HA subtypes and 9 NA subtypes have been identified in animals, in particular avian species. Animals infected with influenza A often act as a reservoir for influenza viruses, and certain subtypes are shown to cross the species barrier in humans, such as the highly pathogenic strain influenza A H5N1.
[0005] Type B influenza virus strains are strictly human. The antigenic variations in HA in type B influenza virus strains are less virulent than those seen in type A strains. Two genetically and antigenically distinct strains of influenza B virus are circulating among humans, as represented by the B/Yamagata/16 strains. /88 (also referred to as B/Yamagata) and B Victoria/2/87 (B Victoria) (Ferguson et al, 2003). Although the spectrum of illness caused by influenza B virus is generally less virulent than that caused by influenza A virus, serious illness requiring hospitalization is still frequently seen in influenza B infection.
[0006] Current approaches to dealing with annual influenza epidemics include annual vaccination, preferably one that generates heterotypic cross-protection. However, influenza viruses that circulate among humans undergo permanent antigenic changes, which require annual adaptation of the influenza vaccine formulation to ensure the closest possible match between influenza vaccine strains and circulating influenza strains.
[0007] Although annual vaccination with influenza vaccines is the best way to prevent influenza, antiviral drugs such as oseltamivir (Tamiflu®) may be effective in preventing and treating influenza infection. However, the number of Influenza virus strains that show resistance against antiviral drugs, such as oseltamivir, is higher.
[0008] An alternative approach is the development of prophylactic or therapeutic antibody-based treatments to neutralize various seasonal and pandemic influenza viruses. The main target of most neutralizing antibodies that protect against Influenza virus infection is the globular head (HA1 part) of the HA viral protein, which contains the receptor binding site, but which undergoes continuous genetic evolution with amino acid substitutions at sites of antibody binding (antigenic movement).
[0009] Recently, broad cross-neutralizing antibodies that recognize an epitope in the main conserved hemagglutinin region of influenza A virus of phylogenetic group 1 (including, for example, the H1 and H5 subtypes of influenza), have been identified (see, for example, e.g. WO2008/028946), as well as cross-neutralizing antibodies that recognize a highly conserved epitope in the HA major region of phylogenetic group 2 influenza A viruses (including, for example, subtypes H3 and H7) (WO 2010/130636 ). The neutralizing activity of these antibodies is restricted to both group 1 and group 2 influenza viruses. Furthermore, these antibodies are not able to bind and neutralize influenza B virus.
[00010] Furthermore, WO 2010/010466 describes a human F16 antibody that binds to hemagglutinin and is capable of binding and neutralizing influenza A subtypes of group 1 (including subtypes H1 and H5) and group 2 (including subtypes H3 and H7). This antibody also does not bind to the HA of the influenza B virus.
[00011] Furthermore, US 2009/0092620 describes a murine antibody that recognizes an antigenic structure present in the hemagglutinin of both the H1 and H3 subtypes, and in the hemagglutinin of the influenza B virus that belongs to the B/Victoria and B/Yamagata groups. Antibodies inhibit the hemagglutination activity of several H3N2 strains, implying that this antibody binds to an epitope on the globular main part of HA.
[00012] In view of the severity of respiratory illnesses caused by influenza A and influenza B viruses, as well as the high economic impact of seasonal epidemics, and the continuing risk of pandemics, there is a continuing need for efficient means of preventing and treating influenza subtypes A and B. Thus, there is a need for binding molecules, preferably human broadly neutralizing binding molecules, capable of cross-neutralizing influenza A viruses from both phylogenetic group 1 and phylogenetic group 2, and preferably also from influenza B virus. SUMMARY OF THE INVENTION
[00013] The invention provides binding molecules capable of specifically binding influenza A virus strains of both phylogenetic group 1 (including, for example, influenza viruses comprising H1 and H5 subtype HA) and influenza A virus strains of group phylogenetic 2 (including, for example, influenza viruses comprising HA subtype H3 and H7). In one embodiment, the binding molecules also exhibit neutralizing activity against strains of influenza A virus from either phylogenetic group 1 or phylogenetic group 2. In one embodiment, the binding molecules are, in addition, capable of specifically binding to influenza B virus strains including, for example, influenza B virus strains from the B/Yamagata and/or B Victoria strains. In one embodiment, the binding molecules are further capable of neutralizing influenza B virus strains including, for example, influenza B virus strains B/Yamagata and/or B Victoria strains. In one embodiment, the binding molecules are capable of neutralizing in vivo influenza A and/or B virus strains. In one embodiment, the binding molecules bind to a conserved epitope in the major region of the HA protein of influenza A and/or B viruses. B. In one embodiment, the binding molecules do not exhibit any hemagglutination-inhibiting (HI) activity.
[00014] The invention thus provides binding molecules that bind to an epitope in the main region of the hemagglutinin protein, which is shared between influenza A virus subtypes in phylogenetic group 1 and Influenza virus subtypes in phylogenetic group 2, as well as subtypes of influenza B virus, and therefore relates to binding molecules that cross-react between both group 1 and group 2 subtypes of influenza A viruses and influenza B viruses. The invention also relates to nucleic acid molecules which encode at least the binding region of the human binding molecules.
[00015] The binding molecules and/or nucleic acid molecules of the invention are suitable for use as a universal prophylactic, diagnostic and/or treatment agent for influenza A virus and influenza B virus, regardless of influenza subtype.
[00016] It can be assumed that the binding molecules according to the present invention bind so far to unknown and highly conserved epitopes, which are not or are much less prone to antigenic movement or displacement. In particular, this epitope is shared between influenza viruses that belong to both phylogenetic group 1 and phylogenetic group 2, and influenza B viruses. Also included is the use of binding molecules of the invention to identify and/or characterize these epitopes.
[00017] The invention further provides the use of the human binding molecules and/or the nucleic acid molecules of the invention in the diagnosis, prophylaxis and/or treatment of a subject who has, or is at risk of developing, an infection with the virus Influenza. Furthermore, the invention pertains to the use of the human binding molecules and/or nucleic acid molecules of the invention in the diagnosis/detection of such influenza infections. DESCRIPTION OF THE FIGURES
[00018] Figure 1 shows the blocking of conformational change of HAs H1, H5, H9, H3, and H7 by CR91 14. (A) Binding of CR9114 FACS to various conformations - precursor uncleaved (HAO); neutral pH, cleaved (HA); Melting pH, cleaved (melting pH) - of rHA expressed on the surface of A/New Caledonia/20/1999 (H1) A/Viet Nam/1203/2004 (H5), A/Hong Kong/1073/1999 (H9) ), A/Wisconsin/67/2005 (H3), and A/Netherlands/219/2003 (H7). Binding is expressed as the percentage of binding to untreated rHA (HAO). (B) FACS binding of CR9114 to HA expressed on the surface in the above manner, with the exception that mAb CR9114 was added prior to exposure to cleaved HAs at a pH of 4.9.
[00019] Figure 2 shows that MAb CR9114 competes with CR6261 and CR8020 to bind to H1 and H3, respectively. Additional degrees of binding of mAbs indicated in immobilized HA from A/New Caledonia/20/1999 (H1N1) saturated with 100 nM CR6261 or CR9114 (panels A and B), or in immobilized HA from A/Wisconsin/67/2005 (H3N2) saturated with 100 nM CR8020 or CR91 14 (panels C and D), measured using bilayer interferometry.
[00020] Figure 3 demonstrates the prophylactic efficiency of CR91 14 in the mouse model with lethal challenge with influenza B virus (B/Florida/04/2006). A. Kaplan-Meier survival curves of mice treated intravenously with either 15 mg/kg CR9114 or vehicle control on day 1 before challenge, followed by a challenge on day 0 of 25 LD B/Florida/04/2006. B. Mean change in body weight (%) from day 0. Bars represent 95% CI of mean. If a mouse is killed/euthanized during the study, the last observed body weight was taken into account. C. Median clinical scores. Bars represent interquartile ranges. Explanation of the clinical classification: 0=no clinical signs; l=rough coating; 2=rough coating, less reactive during handling; 3=rough coating, curled, forced breathing, less reactive during handling; 4=rough coating, curled, forced breathing, inactive response to handling/handling. DESCRIPTION OF THE INVENTION
[00021] Definitions of terms used in the present invention are given below.
[00022] The term "included" or "including" as used herein is deemed to be followed by the words "without limitation".
[00023] As used herein, the term "binding molecule" refers to an intact immunoglobulin, including monoclonal antibodies, such as human monoclonal, chimeric, or humanized antibodies, or to an antigen and/or variable binding domain that comprises the fragment of an immunoglobulin that competes with the intact immunoglobulin for specific binding to the immunoglobulin binding partner, e.g., HA. Regardless of structure, the antigen-binding fragment binds to the same antigen that is recognized by the intact immunoglobulin. An antigen-binding fragment may comprise a peptide or polypeptide comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 , 125, 150, 175, 200, or 250 contiguous amino acid residues of the binding molecule amino acid sequence.
[00024] The term "binding molecule" as used herein includes all immunoglobulin classes and subclasses known in the art. Depending on the amino acid sequence of the constant domain of their heavy chains, the binding molecules can be divided into the five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes) , for example, IgAl, IgA2, IgG1, IgG2, IgG3 and IgG4.
[00025] Antigen binding fragments include, inter alia, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single chain antibodies ( Fvsc), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, etc. The above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins, or may be genetically modified by recombinant DNA techniques. Production methods are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, which is incorporated herein by reference. A binding molecule or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other, or they may be different.
[00026] The binding molecule may be a naked or unconjugated binding molecule, but may also be part of an immunoconjugate. A naked or unconjugated binding molecule is intended to refer to a binding molecule that is unconjugated, operably linked or otherwise physically or functionally associated with an effector moiety or label, such as, inter alia, a toxic substance, a radioactive substance, a liposome, an enzyme. It will be understood that naked or unconjugated binding molecules do not exclude binding molecules that are stabilized, multimerized, humanized, or otherwise manipulated, other than by attaching an effector moiety or tag. Accordingly, all naked or unconjugated and post-translationally modified binding molecules are included herein, including where modifications are prepared in the cellular environment that produces natural binding molecule, by a cell that produces recombinant binding molecule, and are introduced manually after the initial preparation of the binding molecule. Of course, the term naked or unconjugated binding molecule does not exclude the ability of the binding molecule to form functional associations with cells and/or effector molecules after administration into the body, since such interactions are necessary in order to exert a biological effect. . The absence of the effector group or associated tag is therefore applied in the definition of the naked or unconjugated binding molecule in vitro, not in vivo.
[00027] As used herein, the term "biological sample" includes a variety of sample types, including blood samples and other fluids of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures, or cells derived from them and their progeny. The term also includes samples that have been manipulated in any way after their procedure, such as by treatment with reagents, solubilization, or enrichment with certain components, such as proteins or polynucleotides. The term includes various types of clinical samples obtained from any species, and also includes cells in culture, cell supernatants, and cell lysates.
[00028] The term "complementarity determining regions" (CDR), as used herein, means sequences in the variable regions of binding molecules, such as immunoglobulins, which in general contribute to a large extent to the binding site of antigen qe is complementary in shape and charge distribution with the epitope recognized on the antigen. CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, both present in the protein in its natural conformation and, in some cases, present in denatured proteins, for example, by solubilization in SDS . Epitopes can also consist of post-translational modifications of proteins.
[00029] The term "deletion", as used herein, means a change in either the amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent, compared to the reference, often the molecule of naturally occurring.
[00030] The term "expression-regulating nucleic acid sequence", as used herein, refers to the polynucleotide sequence required and/or affecting the expression of an operably linked coding sequence in a particular host organism. Nucleic acid sequences which regulate expression, such as inter alia appropriate transcriptional initiation, termination, promoter, enhancer sequences; repressor or activator sequences; signals that process RNA efficiently, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., ribosome binding sites); sequences that improve protein stability and, where desired, sequences that improve protein secretion, can be any nucleic acid sequence that exhibits activity in the host organism of choice, and can be derived from genes encoding proteins, which are either homologous and heterologous to the host organism. The identification and use of sequences that regulate expression is routine for those skilled in the art.
[00031] The term "functional variant", as used herein, refers to a binding molecule that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids, compared to sequences of nucleotides and/or amino acids of the reference binding molecule which is capable of competing to bind the binding partner, i.e. the Influenza virus, with the reference binding molecule. In other words, modifications to the amino acid and/or nucleotide sequence of the reference binding molecule do not affect or significantly alter the binding characteristics of the binding molecule encoded by the nucleotide sequence, or who contains the amino acid sequence, i.e. , the binding molecule is still able to recognize and bind to its target. The functional variant may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications may be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as unnatural nucleotides and amino acids.
[00032] Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue with similar structural and chemical properties. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan). It will be apparent to those skilled in the art that also other classifications of amino acid residue families, in addition to those used above, may be employed. Furthermore, a variant may have non-conservative amino acid substitutions, for example, substitution of an amino acid for an amino acid residue that has different structural or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both. Guidelines for determining which amino acid residues can be substituted, inserted or deleted without immunological activity can be found using computer programs well known in the art.
[00033] A mutation in a nucleotide sequence can be a single change made at a locus (a point mutation), such as transition or transversion mutations, or alternatively, multiple nucleotides can be inserted, deleted, or altered at a single locus. In addition, one or more changes can be made at any number of loci in a nucleotide sequence. Mutations can be performed by any suitable method known in the art.
[00034] The term "Influenza virus subtype", as used herein in connection with influenza A virus, refers to variants of influenza A virus that are characterized by various combinations of the viral surface proteins hemagglutinin (H) and neuramidase ( N). In accordance with the present invention, Influenza virus subtypes may be referred to by the number H such as, for example, "Influenza virus comprising HA of the H1 or H3 subtype", or "Influenza virus H1", "Influenza virus H3", or by a combination of an H number and an N number such as, for example, "Influenza virus subtype H3N2" or "H3N2".
[00035] The term Influenza virus "subtype" specifically includes all the individual Influenza virus "strains" within each subtype, which generally result from mutations and show different pathogenic profiles. Such strains may also be referred to as multiple "isolates" of a viral subtype. Thus, as used herein, the terms "strains" and "isolates" can be used interchangeably. Current nomenclature for human Influenza virus strains or isolates includes the geographic location of the first isolate, strain number and year of isolation, usually with the antigenic description of HA and NA given in parentheses, e.g. A Moscow/10 /00 (H3N2). Non-human strains also include the host of origin in the nomenclature.
[00036] The term "neutralization", as used herein, in connection with the binding molecules of the invention refers to binding molecules that inhibit an Influenza virus from reproducibly infecting a target cell, regardless of the mechanism by which the neutralization is hit. Thus, neutralization can be achieved, for example, by inhibiting the attachment or adhesion of the virus to the cell surface, or by inhibiting the fusion of viral and cell membranes, after attachment of the virus to the target cell, and the like.
[00037] The term "cross-neutralization" or "cross-neutralization", as used herein in connection with the binding molecules of the invention, refers to the ability of the binding molecules of the invention to neutralize different subtypes of influenza A and/or B viruses. .
[00038] The term "host", as used herein, is intended to refer to an organism or cell into which a vector, such as a cloning vector or an expression vector, has been introduced. The organism or cell may be prokaryotic or eukaryote. Preferably, the isolated hosts are host cells, for example, host cells in culture. The term "host cells" only means that cells are modified to (super)-express the binding molecules of the invention, and include B cells that originally express these binding molecules and whose cells have been modified to over-express the binding molecule. binding by immortalization, amplification, expression enhancement, etc. It can be understood that the term host is intended to refer not only to the particular organism or cell, but also to the progeny of such an organism or cell. Because certain modifications may occur in successive generations due to both mutation and environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included in the scope of the term "host", as used here.
[00039] The term "human", when applied to binding molecules as defined herein, refers to molecules that are either directly derived from a human or based on a human germ-line sequence. When a binding molecule is derived from or based on a human sequence and subsequently modified, it is still considered human, in the manner used throughout the specification. In other words, the term human, when applied to binding molecules, is intended to include binding molecules with variable and constant regions derived from human germline immunoglobulin sequences, or based on the variable or constant regions that occur in a human or human. human lymphocyte, and are modified in some way. Thus, human binding molecules may include amino acid residues not encoded by human germ-line immunoglobulin sequences, comprise substitutions and/or deletions (e.g., mutations introduced, e.g., by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo). "Based on", as used herein, refers to the situation that a nucleic acid sequence can be copied exactly from a template, or with fewer mutations, such as by error-prone PCR methods, or performed synthetically by combining the mold exactly or with fewer modifications.
[00040] The term "insertion", also known as the term "addition", means a change in an amino acid or nucleotide sequence that results in the addition of one or more amino acid or nucleotide residues, respectively, compared to the parent sequence.
[00041] The term "isolated", when applied to binding molecules as defined herein, refers to binding molecules that are substantially free of other proteins or polypeptides, particularly free of other binding molecules with different antigenic specificities, and they are also substantially free of other cellular material and/or chemicals. For example, when the binding molecules are produced recombinantly, they are preferably substantially free of culture medium components, and when the binding molecules are produced by chemical synthesis, they are preferably substantially free of chemical precursors or other chemicals. , that is, they are separated from chemical precursors or other chemicals that are involved in protein synthesis. The term "isolated", when applied to nucleic acid molecules encoding binding molecules as defined herein, is intended to refer to nucleic acid molecules in which the nucleotide sequence encoding the binding molecules is free of another sequence. of nucleotides, particularly nucleotide sequences encoding binding molecules that bind to other binding partners. Furthermore, the term "isolated" refers to nucleic acid molecules that are substantially separate from other cellular components that naturally accompany the natural nucleic acid molecule in its natural host, for example, ribosomes, polymerases, or genomic sequences with to which they are naturally associated.
[00042] In addition, "isolated" nucleic acid molecules, such as cDNA molecules, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
[00043] The term "monoclonal antibody", as used herein, refers to a preparation of antibody molecules of single specificity. A monoclonal antibody exhibits a unique binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a binding specificity that has variable and constant regions derived from or based on human germ-line immunoglobulin sequences, derived from fully synthetic sequences. The method of preparing the monoclonal antibody is not relevant to binding specificity.
[00044] The term "naturally occurring", as used herein and applied to an object, refers to the fact that an object or compound can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature, and that has not been intentionally modified by humans in the laboratory, is naturally occurring.
[00045] The term "nucleic acid molecule", as used in the present invention, refers to a polymeric form of nucleotides and includes both sense and antisense strands of RNA, cDNA, genomic DNA, and polymers of synthetic and mixed forms of the previous. A nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of each type of nucleotide. The term also includes both single-stranded and double-stranded forms of DNA. In addition, a polynucleotide may include both naturally occurring and modified nucleotides linked by naturally occurring and/or non-naturally occurring nucleotide bonds. Nucleic acid molecules may be chemically or biochemically modified, or may contain unnatural or derived nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, tags, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), bonds charged (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified bonds (e.g., alpha anomeric nucleic acids , etc.). The above term is also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, looped, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a particular sequence through hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide bonds replace phosphate bonds in the main part of the molecule. A reference to a nucleic acid sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule with a particular sequence can be understood to include its complementary strand, with its complementary sequence. The complementary strand is also used, for example, for antisense therapy, hybridization probes and PCR oligonucleotide primers.
[00046] The term "operably linked" refers to two or more nucleic acid sequence elements that are generally physically linked, and are in a functional relationship with one another. For example, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating transcription or expression of a coding sequence, in which case the coding sequence can be understood to be "in control of" the promoter.
[00047] "Pharmaceutically acceptable excipient" means any inert substance that is combined with an active molecule, such as a drug, agent, or binding molecule to prepare a reliable or convenient dosage form. The "pharmaceutically acceptable excipient" is an excipient that is non-toxic to recipients at the dosages and concentrations used, and is compatible with other ingredients of the formulation comprising the drug, binding agent or molecule. Pharmaceutically acceptable excipients are widely applicable and known in the art.
[00048] The term "specifically binding", in the manner here used, in reference to the interaction of a binding molecule, e.g., an antibody, and its binding partner, e.g., an antigen, means that the interaction is dependent on the presence of a particular structure, for example an antigenic determinant or epitope on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms. Binding can be mediated by covalent or non-covalent interactions, or a combination of both. In still other words, the term "specifically binds" means that immunospecifically binds to an antigenic determinant or epitope and does not immunospecifically bind to other antigenic determinants or epitopes. A binding molecule that immunospecifically binds to an antigen may bind other peptides or polypeptides with lower affinity, in a manner determined, for example, by radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays. known in the art. Binding molecules or fragments thereof that immunospecifically bind to an antigen may cross-react with related antigens that carry the same epitope.
[00049] Preferably, binding molecules or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigens.
[00050] A "substitution" as used herein means the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively.
[00051] The term "therapeutically effective amount" refers to an amount of the binding molecule as defined herein that is effective to prevent, ameliorate and/or treat a condition resulting from infection with an influenza B virus. as used herein, may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of influenza infection.
[00052] The term "treatment" refers to therapeutic treatment, as well as prophylactic or preventive measures to cure, or halt, or at least slow the progress of the disease. Those in need of treatment include those who already suffer from a condition that results from infection with the Influenza virus, as well as those in which infection with the Influenza virus must be prevented. Individuals partially or fully recovered from Influenza virus infection may also need treatment. Prevention includes inhibiting or reducing the spread of Influenza virus or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with Influenza virus infection.
[00053] The term "vector" means a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will be replicated, and in some cases expressed. In other words, a vector is capable of carrying a nucleic acid molecule to which it is bound. Cloning as well as expression vectors are contemplated by the term "vector" as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC), and vectors derived from bacteriophage, or plant, or animal (including human) viruses. Vectors comprise an origin of replication recognized by the proposed host, and in the case of expression vectors, promoter and other regulatory regions recognized by the host. A vector that contains a second nucleic acid molecule is introduced into a cell by transformation, transfection, or by preparing to use viral entry mechanisms. Certain vectors are capable of autonomous replication in a host into which they are introduced (eg vectors with a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into a host's genome upon introduction into the host, and are thereby replicated along with the host's genome. DETAILED DESCRIPTION
[00054] In a first aspect, the present invention includes binding molecules capable of specifically binding to hemagglutinin (HA) of influenza A virus phylogenetic group 1 subtypes and influenza A virus subtypes of phylogenetic group 2. binding are capable of neutralizing influenza A virus subtypes of both phylogenetic group 1 and phylogenetic group 2. The binding molecules of the invention are thus unique in that they are capable of cross-neutralizing with influenza A virus group 1 strains and group 2 strains. 2 of the influenza A virus. In one embodiment, the binding molecules are capable of neutralizing at least one or more, preferably two or more, preferably three or more, preferably four or more, even more preferably five or more subtypes of group 1 of the influenza A viruses selected from the group consisting of subtype H1, H2, H5, H6, H8, H9 and H11, and at least one or more, preferably two or more, preferably between three or more group 2 influenza A virus subtypes selected from the group consisting of subtype H3, H4, H7 and H10. In one embodiment, the binding molecules are capable of specifically binding the hemagglutinin (HA) of influenza B virus subtypes. In another embodiment, the binding molecules are capable of neutralizing influenza B viruses. binding are capable of neutralizing influenza A and/or B virus in vivo. Influenza virus A and B strains can be either human or non-human influenza virus strains (i.e., obtained from non-human animals, for example birds).
[00055] Preferably, the binding molecules are human binding molecules. In a preferred embodiment, the binding molecules are human antibodies, or antigen-binding fragments thereof.
[00056] In one embodiment, the binding molecules are derived from the VH1-69 germline gene. Thus, the binding molecules use all the structure encoded by the VH1-69 germ line.
[00057] In one embodiment, the binding interaction of the binding molecules, preferably the antibody, and HA is mediated exclusively by the variable heavy chain sequences.
[00058] In one embodiment, the binding molecules comprise a CDR1 heavy chain comprising the amino acid sequence of SEQ ID NO: 133 or SEQ ID NO: 139, a CDR2 heavy chain comprising the amino acid sequence of SEQ ID NO: 134, SEQ ID NO: 140 or SEQ ID NO: 151, and a CDR3 heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 135, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 152, SEQ ID NO: 161, and SEQ ID NO: 162. The CDR regions of binding molecules of the invention are shown in Table 7. The CDR regions are according to Kabat et al. (1991), in the manner described in Sequences of Proteins of Immunological Interest.
[00059] Influenza viruses infect cells by binding to sialic acid residues on the cell surface of target cells, and then transferring to endosomes, fusing their membranes with the endosomal membranes and releasing the genome-transcriptase complex into the cell. Both receptor binding and membrane fusion processes are mediated by the HA glycoprotein. Influenza A virus HA comprises two structurally distinct regions, i.e., a major globular region that contains a receptor binding site, which is responsible for attaching the virus to the target cell and is involved in the hemagglutination activity of HA, and a core region that contains a fusion peptide that is required for membrane fusion between the viral envelope and the endosomal membrane of the cell. The HA protein is a trimer in which each monomer consists of two disulfide-linked glycopolypeptides, HA1 and HA2, which are produced during infection by proteolytic cleavage of a precursor (HA0). Cleavage is necessary for virus infectivity as it is required to prepare HA in membrane fusion to allow conformational change.
[00060] Activation of the prepared molecule occurs at low pH in endosomes, between pH 5 and pH 6, and requires extensive changes in the HA structure. Each of the stages in the preparation and activation of HA for its participation in the membrane fusion process presents a different target for inhibition, for example by monoclonal antibodies. In one embodiment, the binding molecules are capable of blocking the pH-induced conformational changes in HA associated with membrane fusion.
[00061] The binding molecules of the invention may be capable of specifically binding the HAO, HA1 and/or HA2 subunit of the HA protein. They may be able to specifically bind linear, or structural and/or conformational epitopes on HA0, HA1 and/or HA2 of the HA protein. The HA molecule can be purified from the virus or produced recombinantly, and optionally isolated before use. Alternatively, HA can be expressed on the surface of cells. In one embodiment, the binding molecules of the invention are capable of specifically binding an epitope in the major region of HA. In one embodiment, the binding molecules bind to an epitope that is accessible in the pre-fusion conformation of HA.
[00062] The binding molecules of the invention may be capable of specifically binding influenza viruses that are viable, live and/or infective, or that are in inactivated/attenuated form. Methods for inactivating/attenuating viruses, e.g., influenza viruses, are well known in the art and include, but are not limited to, treatment with formalin, β-propiolactone (BPL), merthiolate and/or ultraviolet light.
[00063] The binding molecules of the invention may also be capable of specifically binding one or more fragments of influenza viruses, such as inter alia a preparation of one or more proteins and/or (poly)peptides derived from influenza virus subtypes A and/or B, or one or more recombinantly produced influenza A and/or B virus proteins and/or polypeptides. The nucleotide and/or amino acid sequence of proteins from various strains of influenza A and B can be found in the GenBank database, NCBI Virus Influenza Sequence database, Influenza Sequence (ISD) database, EMBL database and/or or other databases. It is also within the skill of the art to find such sequences in the respective databases.
[00064] In another embodiment, the binding molecules of the invention are capable of specifically binding a fragment of the aforementioned proteins and/or polypeptides, wherein the fragment at least comprises an epitope recognized by the binding molecules of the invention. An "epitope" as used herein is a moiety that is capable of binding a binding molecule of the invention with sufficient high affinity to form a detectable antigen-binding molecule complex.
[00065] The binding molecules of the invention may or may not be able to specifically bind to the extracellular part of HA (also called soluble HAs (HAs) herein).
[00066] The binding molecules of the invention may be intact immunoglobulin molecules, such as polyclonal or monoclonal antibodies, or the binding molecules may be antigen-binding fragments thereof, including, but not limited to, heavy and light chain variable regions, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single chain antibodies (Fvsc), bivalent single chain antibodies, phage phage antibodies single chain, diabodies, triabodies, tetrabodies, and (polypeptides that contain at least a fragment of an immunoglobulin, which is sufficient to confer specific antigen binding to Influenza virus strains or a fragment thereof. In a preferred embodiment, the binding molecules of the invention are human monoclonal antibodies, and/or antigen-binding fragments thereof. The binding molecules can also be Nanobodies, alphabodies, affibodies, FN3 domain structures and other structures s domain-based in (human) repeat proteins such as adnectins, anticalines, darpins, etc., or other structures comprising epitope binding sequences.
[00067] The binding molecules of the invention can be used in non-isolated or isolated form. Furthermore, the binding molecules of the invention may be used alone or in a mixture comprising at least one binding molecule (or variant or fragment thereof) of the invention, and/or with other binding molecules that bind influenza and have the effect of inhibiting the Influenza virus. In other words, the binding molecules can be used in combination, for example, as a pharmaceutical composition comprising two or more binding molecules of the invention, variants or fragments thereof. For example, binding molecules with different but complementary activities can be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect, but alternatively binding molecules with identical activities can also be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect. Optionally, the mixture optionally comprises at least one other therapeutic agent. Preferably, the therapeutic agent such as, for example, M2 inhibitors (e.g. amantidine, rimantadine) and/or neuraminidase inhibitors (e.g. zanamivir, oseltamivir) are used in the prophylaxis and/or treatment of an Influenza virus infection. .
[00068] Typically, binding molecules according to the invention can bind to their binding partners, i.e. a group 1 influenza A virus (such as H1N1) and a group 2 influenza A virus (such as H3N2 ), and/or an influenza B virus, and/or fragments thereof, with an affinity constant (Kd value) that is less than 0.2 x 10-4 M, 1.0 x 10-5 M, 1 .0 x 10 -6 M, 1.0 x 10 -7 M, preferably less than 1.0 x 10 -8 M, more preferably less than 1.0 x 10 -9 M, more preferably less than 1.0 x 10-10, even more preferably less than 1.0 x 10 -11 M, 12 and in particular less than 1.0 x 10 -12 M. Affinity constants may vary between antibody isotypes. For example, binding affinity for an IgM isotype refers to a binding affinity of at least about 1.0 x 10 -7 M. Affinity constants, for example, can be measured using plasma surface resonance. , for example, using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden).
[00069] The binding molecules of the invention exhibit neutralizing activity. Neutralizing activity, for example, can be measured in the manner described herein. Alternative assays that measure neutralization activity are described, for example, in WHO Manual on Animal Influenza Diagnsis and Surveillance, Geneva: World Health Organization, 2005, version 2002.5.
[00070] Typically, the binding molecules according to the invention show a neutralizing activity of 50 µg/ml or less, preferably 20 µg/ml or less, more preferably a neutralizing activity of 10 or less, even more preferably 5 or less, in the manner determined in an in vitro virus neutralization assay (VNA), in the manner described in example 6. The binding molecules according to the invention can bind the Influenza virus or a fragment thereof, in soluble form such as, for example, in a sample or in suspension, or they can bind to influenza viruses or fragments thereof attached or attached to a carrier or substrate, e.g. microtiter plates, membranes and microspheres, etc. Carriers or substrates may consist of glass, plastic (e.g. polystyrene), polysaccharides, nylon, nitrocellulose or teflon, etc. The surface of such supports may be solid or porous and conveniently. Furthermore, the binding molecules can bind to Influenza viruses in purified/isolated or non-purified/unisolated form.
[00071] In the manner discussed above, the present invention relates to isolated human binding molecules which are capable of recognizing and binding to an epitope on the influenza hemagglutinin (HA) protein, wherein said binding molecules exhibit binding activity. neutralization against influenza A viruses of phylogenetic group 1 and influenza A viruses of phylogenetic group 2. According to the invention, it can be seen that the binding molecules of the present invention cross-neutralize Influenza virus subtypes belonging to both phylogenetic groups. Those skilled in the art, based on the present disclosures, can determine whether an antibody actually cross-reacts with HA proteins of different subtypes, and can also determine whether they are capable of neutralizing influenza viruses of different subtypes in vitro and/or in vitro. alive.
[00072] In one embodiment, the binding molecule according to the present invention is selected from the group consisting of: a) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 133, a CDR2 region of heavy chain of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 135,b) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 140, and a heavy chain CDR3 region of SEQ ID NO: 141, c) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145,d) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO : 151, and a heavy chain CDR3 region of SEQ ID NO: 152, e) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152,f) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO :139, a heavy chain CDR2 region of SEQ ID NO: 151, and a heavy chain CDR3 region of SEQ ID NO: 161,g) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139 , a heavy chain CDR2 region of SEQ ID NO: 151, and a heavy chain CDR3 region of SEQ ID NO: 162, and h) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 141.
[00073] In a preferred embodiment, the binding molecule comprises a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 139, a heavy chain CDR2 region comprising an amino acid sequence of SEQ ID NO: 134 , and a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 145 or SEQ ID NO: 152.
[00074] In another embodiment, the human binding molecules according to the invention are selected from the group consisting of: a) a binding molecule having a heavy chain CDR1 region of SEQ ID NO: 133, a CDR2 region chain CDR3 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 135, a light chain CDR1 region that has the amino acid sequence of SEQ ID NO: 136, a heavy chain CDR2 region light chain which has the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 region which has the amino acid sequence of SEQ ID NO: 138,b) a binding molecule which comprises a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 140, and a heavy chain CDR3 region of SEQ ID NO: 141, a light chain CDR1 region showing the amino acid sequence of SEQ ID NO : 142, a region of light chain CDR2 showing the amino acid sequence of SEQ ID NO: 143 , and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 144, c) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, a light chain CDR1 region that displays the amino acid sequence of SEQ ID NO: 146, a light chain CDR2 region that displays the amino acid sequence of SEQ ID NO: 174, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 147,d) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, a light chain CDR1 region showing the amino acid sequence of SEQ ID NO: 148, a light chain CDR2 region which shows the amino acid sequence of SEQ ID NO: 149, and a 1 chain CDR3 region eve which shows the amino acid sequence of SEQ ID NO: 150, e) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 151, and a Heavy chain CDR3 of SEQ ID NO: 152, a region of light chain CDR1 that has the amino acid sequence of SEQ ID NO: 153, a region of light chain CDR2 that has the amino acid sequence of SEQ ID NO: 154 , and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 155, f) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region displaying the amino acid sequence of SEQ ID NO: 148, a light chain CDR2 region displaying the sequence amino acid sequence of SEQ ID NO: 149, and a light chain CDR3 region that displays the sequence of a amino acids of SEQ ID NO: 150, g) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a region of light chain CDR1 which has the amino acid sequence of SEQ ID NO: 156, a region of CDR2 light chain which has the amino acid sequence of SEQ ID NO: 157, and a region of CDR3 chain having the amino acid sequence of SEQ ID NO: 158, h) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region having the amino acid sequence of SEQ ID NO: 148, a light chain CDR2 region having the amino acid sequence of SEQ ID NO : 159, and a region of light chain CDR3 that shows the amino acid sequence of SEQ ID NO: 160, i) u a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 151, and a heavy chain CDR3 region of SEQ ID NO: 161, a CDR1 region of light chain which displays the amino acid sequence of SEQ ID NO: 142, a region of light chain CDR2 which displays the amino acid sequence of SEQ ID NO: 143, and a region of light chain CDR3 which displays the amino acid sequence of SEQ ID NO: 144, j) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 151, and a heavy chain CDR3 region of SEQ ID NO : 162 , a light chain CDR1 region which has the amino acid sequence of SEQ ID NO: 163, a light chain CDR2 region which has the amino acid sequence of SEQ ID NO: 164, and a light chain CDR3 region light which shows the amino acid sequence of SEQ ID NO: 165, k) a binding molecule comprising wherein a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region that displays the amino acid sequence of SEQ ID NO: 166, a region of light chain CDR2 which has the amino acid sequence of SEQ ID NO: 167, and a region of light chain CDR3 which has the amino acid sequence of SEQ ID NO: 168 , l) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a region of light chain CDR1 which has the amino acid sequence of SEQ ID NO: 169, a region of light chain CDR2 which has the amino acid sequence of SEQ ID NO: 149, and a region of light chain CDR3 which has the sequence of amino acids of SEQ ID NO: 150, m) a binding molecule comprising a heavy chain CDR1 region a of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 141, a light chain CDR1 region that shows the amino acid sequence of SEQ ID NO: 163, a region of light chain CDR2 which has the amino acid sequence of SEQ ID NO: 169, and a region of light chain CDR3 which has the amino acid sequence of SEQ ID NO: 170, n) a molecule link comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region which shows the amino acid sequence of SEQ ID NO: 171, a region of light chain CDR2 which shows the amino acid sequence of SEQ ID NO: 164, and a region of light chain CDR3 which shows the amino acid sequence of SEQ ID NO: 172, o) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a CD region Heavy chain R2 of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, a region of light chain CDR1 that shows the amino acid sequence of SEQ ID NO: 142, a region of CDR2 from light chain having the amino acid sequence of SEQ ID NO: 143, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 173, and p) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region showing the amino acid sequence of SEQ ID NO:142, a region of light chain CDR2 which has the amino acid sequence of SEQ ID NO:143, and a region of light chain CDR3 which has the amino acid sequence of SEQ ID NO:144.
[00075] In another embodiment, the human binding molecules according to the invention are selected from the group consisting of: a) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a CDR2 region chain CDR3 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, a light chain CDR1 region showing the amino acid sequence of SEQ ID NO: 146, a heavy chain CDR2 region light chain which has the amino acid sequence of SEQ ID NO: 174, and a light chain CDR3 region which has the amino acid sequence of SEQ ID NO: 147,b) a binding molecule which comprises a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region showing the amino acid sequence of SEQ ID NO : 171, a region of light chain CDR2 which shows the amino acid sequence of SEQ ID NO: 16 4, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 172, c) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, a light chain CDR1 region that displays the amino acid sequence of SEQ ID NO: 142, a light chain CDR2 region that displays the amino acid sequence of SEQ ID NO: 143, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 173, and d) a binding molecule comprising a heavy chain CDR1 region of SEQ ID NO : 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, a light chain CDR1 region showing the amino acid sequence of SEQ ID NO: 142, a light chain CDR2 region which shows the amino acid sequence of SEQ ID NO: 143, and a light chain CDR3 region the light which shows the amino acid sequence of SEQ ID NO: 144.
[00076] In another embodiment, the binding molecule according to the invention is selected from the group consisting of: a) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 2, b) a molecule A binding molecule comprising a heavy chain variable region of SEQ ID NO: 6, c) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 10, d) a binding molecule comprising a variable region of heavy chain of SEQ ID NO: 14, e) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 18, f) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 22 g) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 26, h) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 30, i) a binding molecule which comprises a heavy chain variable region of SEQ ID NO: 34,j) a molecule of linker comprising a heavy chain variable region of SEQ ID NO: 38,k) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 42,l) a linker molecule comprising a chain variable region heavy chain of SEQ ID NO: 46,m) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 50,n) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 54, o) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 58, and p) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 62.
[00077] In one embodiment, the binding molecule according to the invention is selected from the group consisting of a binding molecule comprising a heavy chain variable region of SEQ ID NO: 10, a binding molecule comprising a variable region of heavy chain of SEQ ID NO: 54, a binding molecule comprising a heavy chain variable region of SEQ ID NO: 58, and a binding molecule comprising a heavy chain variable region of SEQ ID NO: 62.
[00078] In a further embodiment, the binding molecules according to the invention comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO: 44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, and SEQ ID NO:64.
[00079] In yet another embodiment, the binding molecule is selected from the group consisting of: a) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 4,b) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 8, c) a binding molecule comprising a variable region chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 12,d) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 16, e) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 20, f) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 24, g) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 28,h) a binding molecule comprising a heavy chain variable region of SEQ ID NO :30 and a light chain variable region of SEQ ID NO: 32, i) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 36, j) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 38 and a light chain variable region of SEQ ID NO: 40, k) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 42 and a light chain variable region of SEQ ID NO: 44,1) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 48.m) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 50 and an r light chain variable region of SEQ ID NO: 52,n) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 54 and a light chain variable region of SEQ ID NO: 56,o) a molecule binding molecule comprising a heavy chain variable region of SEQ ID NO: 58 and a light chain variable region of SEQ ID NO: 60, and p) a binding molecule comprising a heavy chain variable region of SEQ ID NO: 62 and a light chain variable region of SEQ ID NO: 64.
[00080] In one embodiment, the human binding molecules according to the invention are selected from the group consisting of: a binding molecule comprising a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 12, a binding molecule comprising a heavy chain variable region of SEQ ID NO: 54 and a light chain variable region of SEQ ID NO: 56, a binding molecule comprising a chain variable region heavy chain variable region of SEQ ID NO: 58 and a light chain variable region of SEQ ID NO: 60, and a binding molecule comprising a heavy chain variable region of SEQ ID NO: 62 and a light chain variable region of SEQ ID NO: 64.
[00081] In a preferred embodiment, the binding molecules according to the invention are for use as a medicament, and preferably for use in the diagnostic, therapeutic and/or prophylactic treatment of influenza infection caused by influenza A and/or B viruses. .
[00082] Preferably, the Influenza virus which causes influenza infection and which can be treated using the binding molecules of the present invention is an influenza A virus of phylogenetic group 1 and/or 2, and/or an influenza B virus. The present invention also relates to a pharmaceutical composition comprising at least one binding molecule according to the invention, and a pharmaceutically acceptable excipient.
[00083] In yet another embodiment, the invention relates to the use of a binding molecule according to the invention in the preparation of a medicament for the diagnosis, prophylaxis, and/or treatment of an Influenza virus infection. Such infections can occur in small populations, but they can also spread around the world in seasonal epidemics or, worse, global pandemics where millions of individuals are at risk. The invention provides binding molecules that can neutralize infection from influenza strains that cause such seasonal epidemics as well as potential pandemics. Importantly, protection and treatment can now be visualized with the binding molecules of the present invention in relation to various influenza subtypes, since it has been described that the binding molecules of the present invention are capable of cross-neutralizing in various subtypes of influenza. influenza from both phylogenetic group 1, which includes subtypes H1, H2, H5, H6, H8, H9, and H11, and phylogenetic group 2, which includes subtypes H3, H4, H7, and H10, as well as influenza B subtypes .
[00084] Another aspect of the invention includes functional variants of the binding molecules as defined herein. But the molecules are considered functional variants of a binding molecule according to the invention, if the variants are able to compete with what binds specifically to an Influenza virus or a fragment thereof, with the "parental" or " of reference". In other words, molecules are considered to be functional variants of a binding molecule according to the invention when the functional variants are still capable of binding to the same epitope or overlapping epitope of the Influenza virus, or a fragment thereof. In connection with this application, "parent" and "reference" will be used synonymously, meaning that information from the reference or parent molecule, or the physical molecule itself, may form the basis for the variation. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, including those that show modifications in the Fc receptor or other regions involved with effector functions, and/or that contain, for example, in vitro or in vivo modifications. , chemicals and/or biochemicals that are not found in the parent binding molecule. Such modifications include inter alia acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation , and the like. Alternatively, functional variants may be binding molecules as defined in the present invention, which comprise an amino acid sequence containing substitutions, insertions, deletions, or combinations thereof of one or more amino acids, compared to the amino acid sequences of the parent binding molecules. Furthermore, functional variants may comprise truncations of the amino acid sequence at either one or both amino or carboxyl termini. Functional variants according to the invention may have the same or different binding affinities, both higher and lower, compared to the parent binding molecule, but are still capable of binding to the Influenza virus or a fragment thereof. For example, functional variants according to the invention may show greater or lesser binding affinities with an Influenza virus or a fragment thereof compared to parent binding molecules. Preferably, the amino acid sequences of the variable regions including, but not limited to, framework regions, hypervariable regions, in particular the CDR3 regions, are modified. In general, the light chain and heavy chain variable regions comprise three hypervariable regions, which comprise three CDRs, and more conserved regions, the so-called framework regions (FRs). Hypervariable regions comprise amino acid residues from CDRs and amino acid residues from hypervariable loops. Functional variants intended to be within the scope of the present invention have at least about 50% to about 99%, preferably at least about 60% to about 99%, more preferably at least about 70% to about 99% , even more preferably at least about 80% to about 99%, most preferably at least about 90% to about 99%, in particular at least about 95% to about 99%, and in particular at least at least about 97% to about 99% amino acid sequence identity and/or homology to the parent binding molecules as defined herein. Computer algorithms such as inter alia Gap or Bestfit, known to those skilled in the art, can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by altering the parent binding molecules or parts thereof by general molecular biology methods known in the art including, but not limited to, error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and heavy chain shuffling, and /or light. In one embodiment, functional variants of the invention exhibit neutralizing activity against group 1 and group 2 influenza A viruses, and/or influenza B viruses. Neutralizing activity can be either identical or greater or lesser compared to binding molecules. parental. Henceforth, when the term (human) binding molecule is used, it also includes functional variants of the (human) binding molecule. Assays to verify whether a variant binding molecule displays neutralizing activity are well known in the art (see WHO Manual on Animal Influenza Diagnosis and Surveillance, Geneva: World Health Organization, 2005 version 2002.5).
[00085] In a still further aspect, the invention includes immunoconjugates, i.e., molecules which comprise at least one binding molecule as defined herein, and which further comprise at least one label, such as inter alia a detectable moiety/agent. Also contemplated in the present invention are mixtures of immunoconjugates according to the invention, or mixtures of at least one immunoconjugate according to the invention and another molecule, such as a therapeutic agent or another binding molecule or immunoconjugate. In a further embodiment, the immunoconjugates of the invention may comprise more than one label. These labels may be the same or different from each other, and may be non-covalently attached/conjugated to the binding molecules. The tag(s) can also be joined/conjugated directly to human binding molecules via covalent bonding. Alternatively, the tag(s) may be joined/conjugated to the binding molecules by means of one or more binding compounds. Techniques for attaching tags to binding molecules are well known to those skilled in the art.
[00086] The labels of the immunoconjugates of the present invention can be therapeutic agents, but they can also be detectable fractions/agents. Suitable tags in therapy and/or prevention can be toxins or functional parts thereof, antibiotics, enzymes, other binding molecules that enhance phagocytosis or immune stimulation. Immunoconjugates comprising a detectable agent can be used diagnostically, for example to ensure that a subject has been infected with an Influenza virus or to monitor the development or progression of an Influenza virus infection as part of a clinical testing procedure, for example , to determine the efficiency of a given treatment regimen. However, they may also be used for other detection, and/or analytical, and/or diagnostic purposes. Detectable fractions/agents include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. The labels used to label the binding molecules for detection, and/or analytical, and/or diagnostic purposes depend on the specific detection/analysis/diagnostic techniques, and/or methods used, such as inter alia immunohistochemical staining of (tissue) samples, flow cytometry detection, laser scanning cytometry detection, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), bioassays (eg, phagocytosis assay), Western applications blotting, etc. Labels suitable for detection/analysis/diagnostic techniques and/or methods known in the art are well within the reach of those skilled in the art.
[00087] Furthermore, the human binding molecules or immunoconjugates of the invention can also be attached to solid supports, which are particularly used for in vitro immunoassays or purification of influenza viruses or fragments thereof. Such solid supports may be porous or non-porous, planar or non-planar. The binding molecules of the present invention can be fused to tag sequences, such as a peptide, to facilitate purification. Examples include, but are not limited to, hexahistidine tag, hemagglutinin (HA) tag, myc tag, or flag tag. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate. In another aspect, the binding molecules of the invention may be conjugated/attached to one or more antigens. Preferably, these antigens are antigens that are recognized by the immune system of a subject to which the binding molecule-antigen conjugate is administered. Antigens can be identical, but they can also differ from one another. Conjugation methods for attaching antigens and binding molecules are well known in the art and include, but are not limited to, the use of cross-linking agents. The binding molecules of the invention will bind to the Influenza HA virus and the antigens attached to the binding molecules will initiate a potent T cell attack on the conjugate, which eventually leads to the destruction of the Influenza virus.
[00088] After chemically producing immunoconjugates by conjugation, directly or indirectly, for example, via a linker, the immunoconjugates can be produced as fusion proteins comprising the binding molecules of the invention and a suitable label. Fusion proteins can be produced by methods known in the art such as, for example, recombinantly constructing nucleic acid molecules comprising nucleotide sequences which encode the binding molecules in alignment with the nucleotide sequence encoding the tag(s). appropriate(s) and then expressing the nucleic acid molecules.
[00089] It is another aspect of the present invention to provide a nucleic acid molecule that encodes at least one binding molecule, functional variant or immunoconjugate according to the invention. Such nucleic acid molecules can be used as intermediates for cloning purposes, for example, in the affinity maturation process in the manner described above. In a preferred embodiment, the nucleic acid molecules are isolated or purified.
[00090] Those skilled in the art will understand that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are the nucleic acid sequence, which can be directly translated using standard genetic code, to provide an amino acid sequence identical to that translated from parent nucleic acid molecules.
[00091] Preferably, the nucleic acid molecules encode binding molecules comprising the CDR regions in the manner described above. In a further embodiment, the nucleic acid molecules encode binding molecules comprising two, three, four, five or all six CDR regions of the binding molecules of the invention.
[00092] In another embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising the heavy chain variable sequences, in the manner described above. In another embodiment, the nucleic acid molecules encode binding molecules that comprise a light chain comprising the light chain variable sequences, in the manner described above. The nucleotide sequence and amino acid sequences of the heavy and light chain variable regions of the binding molecules of the invention are provided below.
[00093] It is another aspect of the invention to provide vectors, i.e., nucleic acid constructs that comprise one or more nucleic acid molecules in accordance with the present invention.
[00094] Vectors can be derived from plasmids such as, inter alia, F, R1, RP1, Col, pBR322, TOL, Ti, etc.; cosmids; phages such as lambda, lambdoid, M13, Mu, PI, P22, Q, T-even, T-odd, T2, T4, T7, etc.; plant viruses. The vectors can be used to clone and/or express the binding molecules of the invention and can further be used for gene therapy purposes. Vectors that comprise one or more nucleic acid molecules according to the invention, operably linked to one or more nucleic acid molecules that regulate expression, are also included by the present invention. The choice of vector is dependent on the recombinant procedures to be followed and the host used. The introduction of vectors into host cells can be affected, inter alia, by calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofectamine transfection or electroporation. Vectors can be replicated autonomously or they can be replicated together with the chromosome on which they are integrated. Preferably, the vectors contain one or more selection markers. The choice of markers may depend on the host cells of choice, although this is not important to the invention, it is well known to those skilled in the art. They include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, Herpes simplex virus thymidine kinase (HSV-TK) genes, mouse dihydro folate reductase (dhfr) gene. Vectors comprising one or more nucleic acid molecules encoding the human binding molecules in the manner described above, operably linked to one or more nucleic acid molecules encoding proteins or peptides which can be used to isolate the human binding molecules, are also included in the invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose-binding protein, metal-binding polyistidine, green fluorescent protein, luciferase, and beta-galactosidase.
[00095] Hosts that contain one or more copies of the aforementioned vectors are an additional subject of the present invention. Preferably, the hosts are host cells. Host cells include, but are not limited to, mammalian, plant, insect, fungal or bacterial cells. Bacterial cells include, but are not limited to, cells of Gram-positive bacteria or Gram-negative bacteria, such as various species of the Escherichia genera, such as E. coli, and Pseudomonas. In the fungal cell group, preferably yeast cells are used. Expression in yeast can be achieved using yeast strains such as, inter alia, Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha. Furthermore, insect cells such as Drosophila and Sf9 cells can be used as host cells. Furthermore, the host cells can be plant cells such as, inter alia, crop plant cells such as forest plants, or plant cells that provide food or raw materials such as cereal plants, or medicinal plants, or plant cells. ornamental culture, or flower tuber cells. Transformed (transgenic) plants or plant cells are produced by known methods, for example, Agrobacterium-mediated gene transfer, leaf disc transformation, protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication, microinjection or holistic gene transfer. Additionally, a suitable expression system may be a baculovirus system.
[00096] Expression systems using mammalian cells such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells, NSO cells or Bowes melanoma cells are preferred in the present invention. Mammalian cells provide expressed proteins with post-translational modifications that are very similar to natural molecules of mammalian origin. Since the present invention relates to molecules that can be administered to humans, a fully human expression system may be particularly preferred. Therefore, even more preferably, the host cells are human cells. Examples of human cells, inter alia, are HeLa, 911, AT1080, A549, 293 and HEK293T cells. In preferred embodiments, the human producer cells comprise at least a functional part of a nucleic acid sequence that encodes an adenovirus E1 region in expressible format. In even more preferred embodiments, said host cells are derived from a human retina and immortalized with nucleic acids comprising adenoviral E1 sequences, such as 911 cells or the cell line deposited with the European Collection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 29 February 1996, under the number 96022940 and marketed under the trade name PER.C6® (PER.C6 is a registered trademark of Crucell Holland BV). For the purposes of this application "PER.C6 cells" refers to cells deposited with the number 96022940 or earlier, upstream or downstream passages, as well as descendants of these ancestors of the deposited cells, as well as derivatives of any of the foregoing. Production of recombinant proteins in host cells can be carried out according to methods well known in the art. The use of cells marketed under the trademark PER.C6® as a production platform for the proteins of interest has been described in WO 00/63403, the disclosure of which is incorporated herein by reference in its entirety.
[00097] In yet another embodiment, the binding molecules of the present invention may also be produced in transgenic and non-human mammals such as, inter alia, rabbits, goats or cows, and secreted, for example, into their milk.
[00098] In yet another alternative embodiment, the binding molecules according to the present invention can be generated by non-human transgenic mammals, for example, such as transgenic mice or rabbits that express human immunoglobulin genes. Preferably, the non-human transgenic mammals have a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of the human binding molecules, in the manner described above. Non-human transgenic mammals can be immunized with a purified or enriched preparation of Influenza virus or a fragment thereof. Protocols for immunizing non-human mammals are well established in the art. See Using Antibodies: A Laboratory Manual, Edited by: E. Harlow, D. Lane (1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York and Current Protocols in Immunology, Edited by: JE Coligan, AM Kruisbeek, DH Margulies , EM Shevach, W. Strober (2001), John Wiley & Sons Inc., New York, the disclosures of which are incorporated herein by reference. Immunization protocols often include multiple immunizations, both with and without adjuvants such as complete Freund's adjuvant, but may also include naked DNA immunizations. In another embodiment, the human binding molecules are produced by B cells, plasma and/or memory cells derived from the transgenic animals. In yet another embodiment, the human binding molecules are produced by hybridomas, which are prepared by fusion of B cells obtained from the previously described non-human transgenic mammals into immortalized cells. The B-cells, plasma cells and hybridomas obtainable from the non-human transgenic mammals described above, and human binding molecules obtained from the non-human transgenic mammals, B-cells, plasma and/or memory cells and hybridomas described above are also a part of the present invention.
[00099] In still a further aspect, the invention provides compositions comprising at least one binding molecule, preferably a human monoclonal antibody, according to the invention, at least one functional variant thereof, at least one immunoconjugate according to the invention , and/or a combination thereof. In addition to these, the compositions may comprise, inter alia, stabilizing molecules such as albumin or polyethylene glycol or salts. Preferably, the salts used are salts which maintain the desired biological activity of the binding molecules and do not impart any undesired toxicological effects. If necessary, the human binding molecules of the invention can be coated on these or a material to protect them from the action of acids or other natural or unnatural conditions that can inactivate the binding molecules.
[000100] In a still further aspect, the invention provides compositions comprising at least one nucleic acid molecule defined in the present invention. The compositions may comprise aqueous solutions, such as aqueous solutions containing salts (e.g. NaCl or salts as described above), detergents (e.g. SDS) and/or other suitable components.
[000101] Furthermore, the present invention pertains to pharmaceutical compositions comprising at least one binding molecule, such as a human monoclonal antibody of the invention (or fragment or functional variant thereof), at least one immunoconjugate according to the invention, at least one composition according to the invention, or combinations thereof. The pharmaceutical composition of the invention additionally comprises at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known to those skilled in the art. The pharmaceutical composition according to the invention may additionally comprise at least one other therapeutic agent. Suitable agents are also well known to those skilled in the art.
[000102] In a preferred embodiment, the pharmaceutical composition according to the invention comprises at least one additional binding molecule, i.e. the pharmaceutical composition may be a cocktail or mixture of binding molecules. The pharmaceutical composition may comprise at least two binding molecules according to the invention, or at least one binding molecule according to the invention and at least one additional Influenza virus that binds and/or neutralizes the molecule, such as another antibody. directed against the HA protein or against other antigenic structures present in influenza viruses, such as M2. In another embodiment, the additional binding molecule can be formulated for simultaneous, separate or sequential administration.
[000103] In one embodiment the pharmaceutical compositions may comprise two or more binding molecules which exhibit neutralizing activity against influenza A viruses and/or influenza B viruses. In one embodiment, the binding molecules exhibit synergistic neutralizing activity when used in combination. As used herein, the term "synergistic" means that the combined effect of the binding molecules, when used in combination, is greater than their additive effects when used individually. Binding molecules that act synergistically can bind to different structures in the same or different Influenza virus fragments. One way to calculate synergy is through the combination index. The concept of the combination index (CI) was described by Chou and Talalay (1984). The compositions, for example, may comprise a binding molecule with neutralizing activity and a binding molecule without neutralizing. Non-neutralizing and neutralizing binding molecules can also act synergistically in neutralizing the Influenza virus.
[000104] In one embodiment, the pharmaceutical composition may comprise at least one binding molecule according to the invention, and at least one additional neutralizing Influenza virus binding molecule. The binding molecules in the pharmaceutical composition are preferably capable of reacting with influenza viruses of different subtypes. Binding molecules may be of high affinity and may exhibit broad specificity. Preferably, both binding molecules are cross-neutralizing molecules, each of which neutralizes different influenza virus subtypes. Furthermore, they preferably neutralize the many strains of each different subtype of the Influenza virus as much as possible.
[000105] A pharmaceutical composition according to the invention may additionally comprise at least one other therapeutic, prophylactic and/or diagnostic agent. Preferably, the pharmaceutical composition comprises at least one other prophylactic and/or therapeutic agent. Preferably, said additional therapeutic and/or prophylactic agents are agents capable of preventing and/or treating an Influenza virus infection, and/or a condition resulting from such an infection. Therapeutic and/or prophylactic agents include, but are not limited to, antiviral agents. Such agents can be binding molecules, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, antiviral peptides, etc. Other agents that are currently used to treat patients infected with influenza viruses are M2 inhibitors (eg, amantidine, rimantadine) and/or neuraminidase inhibitors (eg, zanamivir, oseltamivir). These can be used in combination with the binding molecules of the invention. "In combination" herein means simultaneously, as separate formulations, or as a single combined formulation, or according to a sequential administration regimen as separate formulations, in any order. Agents capable of preventing and/or treating an Influenza virus infection and/or a condition resulting from such an infection, which are not in the experimental phase, can also be used as other therapeutic and/or prophylactic agents used in the present invention. .
[000106] The binding molecules or pharmaceutical compositions of the invention may be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, mouse, ferret, and monkey.
[000107] Typically, pharmaceutical compositions must be sterile and stable under the conditions of manufacture and storage. The binding molecules, immunoconjugates, nucleic acid molecules or compositions of the present invention may be in powder form for reconstitution in the appropriate pharmaceutically acceptable excipient, prior to or at the time of delivery. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient, and any additional desired ingredient from a previously sterilized solution thereof. by filtration.
[000108] Alternatively, the binding molecules, immunoconjugates, nucleic acid molecules or compositions of the present invention may be in solution, and the appropriate pharmaceutically acceptable excipient may be added and/or mixed before or during the delivery period to provide a form single-dose injectable. Preferably, the pharmaceutically acceptable excipient used in the present invention is suitable in high concentration of the drug, can maintain adequate fluidity and, if necessary, can delay absorption.
[000109] The choice of the optimal route of administration of pharmaceutical compositions will be influenced by several factors, including the physicochemical properties of the active molecules in the compositions, the urgency of the clinical situation, and the relationship of plasma concentrations of active molecules to the desired therapeutic effect. . For example, if necessary, the binding molecules of the invention can be prepared with carriers that will protect them against rapid release, such as a controlled release of the formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, inter alia, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Furthermore, it may be necessary to coat the binding molecules, or co-administer the binding molecules, with a material or compound that prevents inactivation of the human binding molecules. For example, binding molecules can be administered to a subject in an appropriate carrier, for example, liposomes or a diluent.
[000110] Routes of administration can be divided into two main categories, oral and parenteral administration. The preferred route of administration is intravenous or inhalation.
[000111] Oral dosage forms may be formulated, inter alia, as tablets, lozenges, tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules, soft gelatine capsules, syrups or elixirs, pills, dragees, liquids, gels or slurries. These formulations may contain pharmaceutical excipients including, but not limited to, inert diluents, granulating and disintegrating agents, binding agents, lubricating agents, preservatives, coloring, flavoring or sweetening agents, vegetable or mineral oils, wetting agents and thickening agents. .
[000112] The pharmaceutical compositions of the present invention may also be formulated for parenteral administration. Formulations for parenteral administration may be, inter alia, in the form of aqueous or non-aqueous, isotonic, sterile and non-toxic solutions or suspensions for injection or infusion. Solutions or suspensions may comprise agents that are non-toxic to recipients at the dosages and concentrations employed, such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oils, fatty acids, anesthetic agents. preservatives, buffers, viscosity or solubility enhancing agents, water soluble antioxidants, oil soluble antioxidants and metal chelating agents.
[000113] In a further aspect, binding molecules such as human monoclonal antibodies (fragments and functional variants thereof), immunoconjugates, compositions, or pharmaceutical compositions of the invention can be used as a medicament. Thus, a method of diagnosing, treating and/or preventing an Influenza virus infection using the binding molecules, immunoconjugates, compositions, or pharmaceutical compositions of the invention is another part of the present invention. The aforementioned molecules, inter alia, can be used in the diagnosis, prophylaxis, treatment, or combination thereof, of an Influenza virus infection, caused by the influenza virus, which comprises HA of subtype H1, H2, H3, H4, H5, H6 , H7, H8, H9, H10 and/or H11. In one embodiment, the aforementioned molecules can also be used in the diagnosis, prophylaxis, treatment or combination thereof of an Influenza virus infection caused by an influenza B virus. They are suitable for the treatment of untreated patients suffering from an influenza B virus. Influenza virus infection, and patients who have been or are being treated for an Influenza virus infection.
[000114] The aforementioned molecules or compositions can be employed together with other molecules used in diagnosis, prophylaxis and/or treatment. These can be used in vitro, ex vivo or in vivo. For example, binding molecules such as human monoclonal antibodies (or functional variants thereof), immunoconjugates, compositions or pharmaceutical compositions of the invention can be co-administered with an Influenza virus vaccine (if available). Alternatively, the vaccine can also be administered before or after administration of the molecules of the invention. Instead of a vaccine, antiviral agents can also be used together with the binding molecules of the present invention. Suitable antiviral agents are mentioned above.
[000115] The molecules are typically formulated into the compositions and pharmaceutical compositions of the invention in a therapeutically or diagnostically effective amount. Alternatively, they may be formulated and administered separately. For example, other molecules such as antiviral agents can be applied systemically, while the binding molecules of the invention can be applied intravenously.
[000116] Treatment can be targeted at groups of patients who are susceptible to influenza infection. Such patient groups include, but are not limited to, e.g. older patient (e.g. >50 years, >60 years, and preferably >65 years), young (e.g. <5 years, <1 year) , hospitalized patients and already infected patients who were treated with an antiviral compound but showed an inadequate antiviral response.
[000117] Dosage regimens can be adjusted to provide optimal desired responses (eg, a therapeutic response). A suitable dosage range, for example, may be 0.01-100 mg/kg body weight, preferably 0.1-50 mg/kg body weight, preferably 0.01-15 mg/kg body weight. Furthermore, for example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionately reduced or increased, as indicated by the requirements of the therapeutic situation. Molecules and compositions according to the present invention are preferably sterile. Methods for rendering these molecules and compositions sterile are known in the art. The other molecules used in diagnosis, prophylaxis and/or treatment can be administered in a similar dosage regimen, in the manner proposed for the binding molecules of the invention. If the other molecules are given separately, they can be given to a patient earlier (e.g. 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks before), at the same time, or after (e.g. 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks after) to the administration of one or more of the human binding molecules or pharmaceutical compositions of the invention. The exact dosing regimen is generally chosen during clinical trials in human patients.
[000118] Human binding molecules and pharmaceutical compositions comprising the human binding molecules are particularly used, and often preferred, when they are administered to humans as therapeutic agents in vivo, since the immune response of the recipient to the administered antibody will be often and substantially less than that occasioned by the administration of a chimeric or humanized murine monoclonal binding molecule.
[000119] In another aspect, the invention relates to the use of binding molecules, such as neutralizing human monoclonal antibodies (fragments and functional variants thereof), immunoconjugates, nucleic acid molecules, compositions or pharmaceutical compositions according to the invention in the preparation of a medicament for the diagnosis, prophylaxis, treatment, or combination thereof, of an Influenza virus infection, in particular an infection caused by the Influenza virus comprising HA of subtypes H1, H2, H3, H4, H5, H6, H7 , H8, H9, H10 and/or H11 and/or influenza B viruses.
[000120] Hereinafter, kits comprising at least one binding molecule such as a neutralizing human monoclonal antibody (fragments and functional variants thereof), at least one immunoconjugate, at least one nucleic acid molecule, at least one composition, at least a pharmaceutical composition, at least one vector, at least one host according to the invention or a combination thereof are also a part of the present invention. Optionally, the above-described components of the kits of the invention are packaged in suitable containers and labeled for the diagnosis, prophylaxis and/or treatment of the indicated conditions. The aforementioned components may be stored in single-dose or multi-dose containers as an aqueous solution, preferably sterile, or as a lyophilized formulation, preferably sterile, for reconstitution. The containers may be formed from a variety of materials such as glass or plastic, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by an injection needle). hypodermic). The kit may further comprise further containers comprising a pharmaceutically acceptable buffer. Other materials desirable from a commercial and user point of view may additionally be included, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts, and possibly even at least one other therapeutic, prophylactic or diagnostic agent. Routine instructions included in commercial packages of therapeutic, prophylactic or diagnostic products may be associated with the kits, which contain information on, for example, the indications, use, dosage, manufacture, administration, contraindications and/or precautions regarding the use of such therapeutic, prophylactic or diagnostic products.
[000121] The binding molecules according to the present invention can also be advantageously used as a diagnostic agent in an in vitro method for the detection of Influenza viruses. The invention thus relates to a method of detecting Influenza virus phylogenetic group 1 or group 2, or Influenza virus subtype influenza B in a sample, wherein the method comprises the steps of: (a) contacting a sample with a diagnostically of a binding molecule (fragments and functional variants thereof) or an immunoconjugate according to the invention, and (b) determining whether the binding molecule or immunoconjugate specifically binds to a molecule in the sample. The sample may be a biological sample including, but not limited to, blood, serum, feces, sputum, nasopharyngeal aspirates, bronchial washes, urine, tissue, or other biological material from (potentially) infected subjects, or a non-biological sample such as water, drink, etc. Infected subjects (potentially) can be human subjects, but also animals that are suspected to be carriers of Influenza virus can be tested for the presence of the virus using the human binding molecules or immunoconjugates of the invention. the sample can first be manipulated to better prepare it for the detection method. Handling means, inter alia, treating the sample suspected of containing and/or containing the virus in such a way that the virus is disintegrated into antigenic components such as proteins, polypeptides or other antigenic fragments. Preferably, the human binding molecules or immunoconjugates of the invention are contacted with the sample under conditions which allow the formation of an immune complex between the human binding molecules and the virus, or antigenic components thereof which may be present in the sample. The formation of an immune complex, if any, indicating the presence of the virus in the sample, is then detected and measured by appropriate means. Such methods include, inter alia, homogeneous and heterogeneous binding immunoassays, such as radioimmunoassay (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analysis.
[000122] The preferred assay techniques, especially for large-scale clinical selection of patient sera and blood, and blood products are ELISA and Western blot techniques. ELISA tests are particularly preferred. For use as reagents in these assays, the binding molecules or immunoconjugates of the invention are conveniently attached to the inner surface of microtiter plates. The binding molecules or immunoconjugates of the invention can be linked directly into the microtiter well. However, maximal binding of the binding molecules or immunoconjugates of the invention to the wells can be accomplished by pretreating the wells with polylysine prior to the addition of the binding molecules or immunoconjugates of the invention. Furthermore, the binding molecules or immunoconjugates of the invention can be covalently attached by known means to the wells. In general, binding molecules or immunoconjugates are used in a concentration between 0.01 to 100 μg/mL for coating, although higher and lower amounts can also be used. Samples are then added to wells coated with the binding molecules or immunoconjugates of the invention.
[000123] Furthermore, the binding molecules of the invention can be used to identify Influenza virus specific binding structures. The binding structures can be epitopes on proteins and/or polypeptides. It can be linear, but also structural and/or conformational. In one embodiment, linkage structures can be analyzed by means of PEPSCAN analysis (see, inter alia, WO 84/03564, WO 93/09872, Slootstra et al, 1996). Alternatively, a random peptide library, which comprises peptides from an Influenza virus protein, can be selected for peptides capable of binding the binding molecules of the invention.
[000124] The invention is further illustrated in the following examples and figures. The examples are not intended to limit the scope of the invention in any way. EXAMPLESExample 1
[000125] Construction of Fvsc phage display libraries using RNA extracted from peripheral blood mononuclear cells
[000126] Peripheral blood was collected from normal healthy donors by venipuncture into EDTA anticoagulant sample tubes. Fvsc phage display libraries were obtained in the manner described in WO 2008/028946, which is incorporated herein by reference. RNA was isolated from peripheral blood mononuclear cells and cDNA was prepared. A two-cycle PCR amplification approach was applied using the oligonucleotide primer sets shown in Tables 1 and 2 to isolate the immunoglobulin VH and VL regions from the respective donor repertoire.
[000127] The first round of amplification on the respective cDNAs using the oligonucleotide primer sets mentioned in Table 1 yielded 7, 6 and 9 products of about 650 base pairs respectively for the VH, Vkappa and Vlambda regions. For amplification of the VH region of IgM, the OCM constant primer oligonucleotide was used in combination with OH1 to OH7. The thermocycler program for the first amplification cycle was: 2 minutes at 96 °C (denaturing step), 30 cycles of 30 seconds at 96 °C/ 30 seconds at 60 °C/ 60 seconds at 72 °C, 10 min at 72 °C with final elongation and 6 °C for cooling. The products were placed and isolated on a 1% agarose gel using gel extraction columns (Macherey Nagel), and eluted in 50 μL of 5 mM Tris-HCl pH 8.0. Ten percent of the products from the first round (3 to 5 μL) were subjected to the second round of amplification using the oligonucleotide primers mentioned in Table 2. These oligonucleotide primers were extended with restriction sites that allow directional cloning of the respective VL and VH regions in the phage display vector PDV-C06. The PCR program for the second amplification cycles was as follows: 2 minutes at 96 °C (denaturing step), 30 cycles of 30 seconds at 96 °C / 30 seconds at 60 °C / 60 seconds at 72 ° C, 10 minutes at 72 °C for final elongation and 6 °C for refrigeration. The second cycle products (~350 base pairs) were first grouped according to naturally occurring J segments found in immunoglobulin gene products, resulting in 7, 6 and 9 groupings for the VH, Vkappa and Vlambda regions, respectively (see tables 3 and 4). To obtain a normalized distribution of immunoglobulin sequences in the immune library, light chain pools 6 Vkappa and 9 Vlambda were mixed according to the percentages mentioned in table 3. This final single VL pool (3 μg) was digested overnight. with the restriction enzymes Sail and Notl, loaded and isolated from a 1% agarose gel (~350 base pairs), using Macherey Nagel gel extraction columns, and ligated into the PDV-C06 vector with cut of Sall-Notl (~5,000 base pairs) as follows: 10 μL of PDV-C06 vector (50 ng/μL), 7 μL of VL insert (10 ng/μL), 5 μL of binding buffer 10 X (NEB), 2.5 T4 DNA Ligase (400 U/μL) (NEB), 25.5 μL ultrapure water (vector to insertion ratio was 1:2). Binding was carried out overnight in a 16 °C water bath. Then, the volume was doubled with water, extracted with an equal volume of phenol-chloroform-isoamyl alcohol (75:24:1) (Invitrogen), followed by extraction with chloroform (Merck) and precipitated with 1 μL of Pellet Paint ( Novogen), 10 μL of sodium acetate (3 M pH 5.0) and 100 μL of isopropanol for 2 hours at -20 °C. The sample obtained was subsequently centrifuged at 20,000 x g for 30 minutes at 4 °C. The precipitate obtained was washed with 70% ethanol and centrifuged for 10 minutes at 20,000 x g at room temperature. Ethanol was removed by vacuum aspiration and the precipitate was air-dried for several minutes, then dissolved in 50 μL of buffer containing 10 mM Tris-HCl, pH 8.0. 2 μL of ligation mixture was used for the transformation of 40 μL of electrocompetent TG-1 cells (Agilent) in a cooled 0.1 cm electroporation cuvette (Biorad) using a Genepulser II apparatus (Biorad) set at 1.7 kV, 200 Ohm, 25 μP (time constant ~4.5 msec). Immediately after the pulse, the bacteria were rinsed in the cuvette with 1000 μL of SOC medium (Invitrogen) containing 5% (w/v) glucose (Sigma) at 37 °C and transferred to a 15 mL round bottom culture tube. . An additional 500 μL of SOC/glucose was used to wash residual bacteria in the cuvette and was added to the culture tube. Bacteria were recovered by culturing for exactly one hour at 37 °C in a Hasker incubator at 220 rpm. The transformed bacteria were placed in large 240 mm petri dishes (NUNC) containing 150 mL of 2TY agar (16 g/L of bactotryptone, 10 g/L of bacto-yeast extract, 5 g/L of NaCl, 15 g/L agar, pH 7.0) supplemented with 50 μg/ml ampicillin and 5% (w/v) glucose (Sigma). A 1 in 1000 dilution was plated for counting purposes in 15 cm Petri dishes containing the same medium. This transformation procedure was repeated ten times sequentially and each complete transformation was plated in a separate square Petri dish and grown overnight in a culture oven at 37 °C. Typically around 1 x 10 cfu (1 x 10 6 per Petri dish) were obtained using the above protocol. The intermediate VL light chain library was collected from the plates by gently streaking the bacteria in 10 ml of 2TY medium per plate. Cell mass was determined by measuring OD600 and twice 500 OD of bacteria were used for the preparation of maxi plasmid DNA using two P500 maxiprep columns (Macherey Nagel) according to the manufacturer's instructions.
[000128] Analogous to the VL variable regions, the products of the second VH-JH cycle were first mixed together to obtain the normal J segment usage distribution (see table 4), resulting in 7 VH subgroups named PH1 to PH7. The pools were mixed to acquire a normalized sequence distribution using the percentages shown in Table 4, obtaining the VH fraction that was digested with Sfil and Xhol restriction enzymes and ligated into the Sfil-Xhol cut PDV-VL intermediate library. , obtained in the manner described above. The ligation setup, purification method, subsequent TGI transformation and bacterial collection was essentially as described for the VL intermediate library (see above), with the exception that 20 transformations and 20 Petri dishes were used. The final library (approximately 1 x 10 cfu) was checked for insertion frequency with a colony PCR, using an oligonucleotide primer set flanking the inserted VH-VL regions. 90% of colonies showed correct insertion size. Colony PCR products were used for subsequent DNA sequence analysis to verify sequence variation and to assess the percentage of colonies that exhibit a complete ORF. This resulted in 76%. Finally, the library was recovered and amplified using CT helper phage (see WO 02/103012), and used for phage antibody selection by choosing methods in the manner described below. Example 2
[000129] Selection of phages carrying single-chain Fv fragments against Influenza A and Influenza B hemagglutinin
[000130] Antibody fragments were selected using phage display libraries for antibody, constructed essentially in the manner described above, and general phage display technology and MABSTRACT® technology, essentially in the manner described in US patent 6,265,150 and in WO 98/15833 (both of which are incorporated herein by reference). Furthermore, methods and helper phages in the manner described in WO 02/103012 (which is incorporated herein by reference) were used in the present invention.
[000131] Selection was performed against recombinant hemagglutinin (HA) of influenza A subtype H1 (A/New Caledonia/20/99), H3 (A/Wisconsin/67/2005), H4 (A/Duck/Hong Kong/24 /1976), H5 (A/Chicken/Vietnam/28/2003), H7 (A/Netherlands/219/2003) and H9 (A/HongKong/1073/99). HA antigens were diluted in PBS (5.0 μL), added to MaxiSorp™ Nunc-Immuno tubes (Nunc) and incubated overnight at 4°C on a shaker. Immunotubes were emptied and washed three times in block buffer (2% skimmed milk powder (ELK) in PBS). Subsequently, the immunotubes were filled completely with buffer block and incubated for 1-2 hours at room temperature. Aliquots of phage display library (500-1000 µL, 0.5 x 10 13 - 1 x 10 13 cfu, amplified using CT helper phage (see WO 02/103012)) were blocked in blocking buffer supplemented with 10% bovine serum non-heat inactivated fetal tissue and 2% mouse serum for 1-2 hours at room temperature. The blocked phage library was added to the immunotubes, incubated for 2 hours at room temperature, and washed with wash buffer (0.05% (v/v) Tween-20 in PBS) to remove unbound phage. Bound phages were eluted from the respective antigen by incubation with 1 ml of 100 mM triethylamine (TEA) for 10 minutes at room temperature. Subsequently, the eluted phage were mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 to neutralize the pH. This mixture was used to infect 5 ml of an E. coli XL 1-blue culture that was grown at 37 °C, at an OD of 600 nm of approximately 0.3. The phages naturally infected the bacteria in XL1-blue for 30 minutes at 37 °C. Next, the mixture was centrifuged for 10 minutes at 3,000 x g at room temperature, and the bacterial precipitate was resuspended in 0.5 mL of 2-tryptone yeast extract (2TY) medium. The bacterial suspension obtained was divided into two 2TY agar plates supplemented with tetracycline, ampicillin and glucose. After overnight incubation of the plates at 37°C, colonies were removed from the plates and used to prepare an enriched phage library, essentially in the manner described by De Kruif et al. (1995) and WO 02/103012. Briefly, striated bacteria were used to inoculate 2TY medium containing ampicillin, tetracycline and glucose and were grown at a temperature of 37 °C, at an OD600 nm of ~0.3. CT helper phages were added and naturally infect the bacteria, after which the medium was changed to 2TY containing ampicillin, tetracycline and kanamycin. Incubation continued overnight at 30°C. The next day, bacteria were removed from the 2TY medium by centrifugation, after which the phages in the medium were precipitated using polyethylene glycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml of PBS with 1% bovine serum albumin (BSA), sterile filtered and used in the second round of selection. The second round of selection is performed both on the same HA subtype and on HA of a different subtype.
[000132] Two consecutive rounds of selection were performed prior to the isolation of individual single-chain phage antibodies. After the second round of selection, individual E. coli colonies were used to prepare phage monoclonal antibodies. Essentially, individual colonies were grown to log phase in a 96-well plate format and infected with VCS-M13 helper phage, after which phage antibody production naturally continued overnight. Phagemids were sequence analyzed and all unique phagemids were used for further analysis. Supernatants containing phage antibodies were used directly in ELISA to bind HA antigens. Alternatively, phage antibodies were PEG/NaCl precipitated and filter sterilized by either Elisa or flow cytometry analysis. Example 3
[000133] Validation of HA-specific single-chain phage antibodies
[000134] The selected supernatants containing single-chain phage antibodies, which were obtained in the selections described above, were validated in ELISA for specificity, ie binding on different HA antigens. For this purpose, baculoviruses that expressed recombinant HAs of H1 (A/New Caledonia/20/99), H3 (A/Wisconsin/67/2005), H5 (A/Vietnam/1203/04) H7 (A/Netherlands/ 219/2003), and B (B/Ohio/01/2005) (Protein Sciences, CT, USA) were coated onto Maxisorp™ ELISA plates. After coating, plates were washed three times with PBS containing 0.1% Tween-20 v/v and blocked in PBS containing 3% BSA or 2% ELK for 1 hour at room temperature. Selected single chain phage antibodies were incubated for 1 hour in an equal volume of PBS containing 4% ELK to obtain blocked phage antibodies. The plates were emptied, washed three times with PBS/0.1% Tween-20 and blocked single-chain phage antibodies were added to the wells. The incubation was naturally continued for one hour, the plates were washed with PBS/0.1% Tween-20 and bound phage antibodies were detected (using OD 492nm measurement) using a peroxidase-conjugated anti-M13 antibody conjugate. As a control, the procedure was performed simultaneously without single-chain phage antibody and with an unrelated negative control single-chain phage antibody. From the selections of the different HA antigens with the phage libraries, 13 unique single-chain phage antibodies that specifically bind to recombinant HA from influenza A H1, H3, H5, H7 and influenza B were obtained (SC09-003, SC09-004, SC09-005, SC09-006, SC09-007, SC09-008, SC09-009, SC09-010, SC09-011, SC09-030, SC09-112, SC09-113 and SC09-114). See table 5.
[000135] Alternatively, phage antibodies precipitated with PEG/NaCl and sterile-filtered were used to validate binding and specificity by FACS analysis. For this purpose, full-length recombinant influenza A HAs, subtypes H1 (A/New Caledonia/20/1999), H3 (A Wisonsin/67/2005) and H7 (A/Netherlands/219/2003), were expressed in PER.C6 cell surface. Cells were incubated with single-chain phage antibodies for 1 hour, followed by three washing steps with PBS+0.1% BSA. Bound phage were detected using M13-antibody conjugated FITC. From the selections on the different HA antigens with the phage libraries, 14 single-chain phage antibodies that specifically bind to influenza A subtypes H1, H3 and H7 HA were found (SC09-003, SC09-004, SC09- 005, SC09-006, SC09-007, SC09-008, SC09-009, SC09-010, SC09-011, SC09-012, SC09-030, SC09-112, SC09-113 and SC09-114). See table 6.
[000136] All 16 phage antibodies, SC09-003, SC09-004, SC09-005, SC09-006, SC09-007, SC09-008, SC09-009. SC09-010, SC09-011, SC09-012, SC09-029, SC09-030, SC09-031, SC09-112, SC09-113 and SC09-114, were used for the construction of fully human immunoglobulins. Example 4
[000137] Construction of fully human immunoglobulin molecules (human monoclonal antibodies) from selected single-chain Fvs
[000138] From the selected specific single-chain phage (Fvsc) antibodies, plasmid DNA clones were obtained and the nucleotide and amino acid sequences were determined according to standard techniques. The heavy and light chain variable regions of the scFvs were cloned directly by restriction digestion for expression into the pIg-C911-HCgamma1 (see SEQ ID NO: 175), pIG-C909-Ckappa (see SEQ ID NO) : 176), or plg-C910-Clambda (see SEQ ID NO: 177). The VH and VL gene identity (see Tomlinson IM et al. V-BASE Sequence Directory. Cambridge, UK: MRC Center for Protein Engineering (1997)) of the Fvscs was determined (see Table 7).
[000139] The nucleotide sequence for all constructs was verified according to standard techniques known to those skilled in the art. The resulting expression constructs expressing human IgG1 heavy and light chains were transiently expressed in combination with 293T cells, and supernatants containing human IgG1 antibodies were obtained and produced using standard purification procedures.
[000140] The amino acid sequence of the CDRs of the heavy and light chains of selected immunoglobulin molecules is given in Table 7.
[000141] The number of amino acid differences and % identity of all heavy and light chain variable domains are given in Table 8. Example 5
[000142] Cross-linking reactivity of IgGs
[000143] A panel of five of the previously described IgG antibodies, CR9005, CR9030, CR9112, CR9113 and CR9114, was validated in ELISA for binding specificity, ie, that it binds to different HA antigens. For this purpose, baculoviruses that expressed recombinant HAs H1 (A/New Caledonia/20/1999), H3 (A/Wisconsin/67/2005), H5 (A/Vietnam/1203/04), H7(A/Netherlands/219 /2003) and H9 (A/HongKong/1073/99) (Protein Sciences, CT, United States) were coated onto Maxisorp™ ELISA plates. After coating, the plates were washed three times with PBS containing 0.1% v/v Tween-20 and blocked in PBS containing 3% BSA or 2% ELK for 1 hour at room temperature. The plates were emptied, washed three times with PBS/0.1% Tween-20 and IgG antibodies were added to the wells. The incubation continued naturally for one hour, the plates were washed with PBS/0.1% Tween-20 and bound antibodies were detected (using OD 492nm measurement) using a peroxidase-conjugated anti-human IgG antibody. As a control, an unrelated IgG CR4098 was used.
[000144] CR9005, CR9030, CR9112, CR9113 and CR91 14 were shown to show heterosubtypic cross-linking activity in all recombinant HAs tested. See table 9.
[000145] Additionally, selected antibodies were used to test for heterosubtypic binding by FACS analysis. For this purpose, the full-length recombinant influenza A HAs subtypes H1 (A/New Caledonia/20/1999), H3 (A/Wisonsin/67/2005) and H7 (A/Netherlands/219/2003) were expressed in surface of PER.C6 cells. Cells were incubated with IgG antibodies for 1 hour, followed by three washing steps with PBS+0.1% BSA. Bound antibodies were detected using PE-conjugated anti-human antibody. As a control, untransfected PER.C6 cells were used. CR9005, CR9030, CR9112, CR9113, and CR9114 exhibited cross-linking activity with HA subtypes of influenza A H1, H3, and H7, but not with wild-type PER.C6 cells. See table 9. Example 6
[000146] Cross-activity of neutralizing IgGs
[000147] In order to determine whether the selected IgGs were able to block multiple strains of influenza A, further in vitro virus neutralization (VNA) assays were performed. VNAs were performed in MDCK cells (ATCC CCL-34). MDCK cells were cultured in MDCK cell culture medium (MEM medium supplemented with antibiotics, 20 mM Hepes and 0.15% (w/v) sodium bicarbonate (complete MEM medium), supplemented with 10% (v/v) of fetal bovine serum). The H1 strains (A/WSN/33, A/NewCaledonia/20/1999, A/Solomon Islands/IVR-145 (high multiplication recombination from A/Solomon Islands/3/2006), A/Brisbane/59/2007, A/NYMC/X-181 (high multiplication recombinant from A/California/07/2009), H2 (A/Env/MPU3156/05), H3 (A/Hong Kong/1/68, A/Johannesburg/33/ 94, A/Panama/2000/1999, A/Hiroshima/52/2005, A/Wisconsin/67/2005 and A/Brisbane/10/2007), H4 (A/WF/HK/MPA892/06), H5 ( PR8-H5N1-HK97 (6:2 recombinant A/Hong Kong/156/97 and A/PR/8/34) and A/Eurasian Wigeon/MPF461/07), H6 (A/Eurasian Wigeon/MPD41 1/07 ), H7 (NIBRG-60 (6:2 recombinant A/Mallard/Netherlands/12/2000) and PR8-H7N7-NY (7:1 recombinant A/New York/107/2003 (H7N7) and A/PR /8/34)), H8 (A/Eurasian Wigeon/MPH571/08) H9 (A/Hong Kong/1073/99 and A/Chick/HK/SSP 176/09), H10 (A/Chick/Germany/N /49) and H14 (PR8-H14N5 (6:2 recombinant A/mallard/Astrakhan/263/1982 (H14N5) and A/PR/8/34)) that were used in the assay were all diluted to a titer of 5 .7 x 103 TCID50/mL (50% dose i tissue culture per mL), with the titration calculated according to the method of Spearman and Karber. IgG preparations (200 µg/mL) were serially diluted 2-fold (1:2 - 1:512) in complete MEM medium in quadruplicate wells. 25 μL of the respective IgG dilution was mixed with 25 μL of virus suspension (100 TCID50/25 μL) and incubated for one hour at 37°C. The suspension was then transferred in quadruplicate to 96-well plates containing confluent MDCK cultures, in 50 µL of complete MEM medium. Prior to use, MDCK cells were seeded at 3 x 10 4 cells per well in MDCK cell culture medium, grown until cells reached confluence, washed with 300-350 μL of PBS, pH 7.4, and finally 50 μL of complete MEM medium was added to each well. Inoculated cells were cultured for 3-4 days at 37 °C and observed daily for the development of cytopathogenic effect (CPE). The CPE was compared with the positive control.
[000148] CR9005, CR9112, CR9113 and CR9114 show neutralizing heterosubtypic cross-activity with representative strains of all tested influenza A virus subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9 and H10. See table 10. Example 7
[000149] Pan-influenza antibodies bind to the pre-fusion conformation of HA
[000150] In order to determine whether the selected IgGs were able to bind to the pre- or post-fusion conformation of the HA molecule, an in vitro pH shift experiment was performed. For this purpose, the full-length recombinant influenza A HA subtypes H1 (A/New Caledonia/20/99), H3 (A/Wisonsin/67/2005), H5 (A/Vietnam/1203/04), H7 ( A/Netherlands/219/03) and H9 (A/Hong Kong/1073/99) were expressed on the surface of PER.C6 cells. To ensure binding of mAbs in different HA structural conformations, cells were detached from the plastic support using PBS-EDTA, and were subsequently trypsinized (TrypLE™Select, Gibco) for 5 minutes at RT, washed (1% BSA in PBS) and incubated for 15 minutes in citric acid-sodium phosphate buffer (pH 4.9). Cell samples were set aside after each processing step (untrypsinized/HA0; trypsinized/HA1-HA2; pH 4.9/HA fusion) and fractions from each treatment were incubated with mAb CR9114 for 1 hour. Cells were then incubated for 30 minutes with phycoerythrin-conjugated anti-human IgG (Southern Biotech) in 1% BSA. Stained cells were analyzed using a FACS Canto with FACS Diva software (Becton Dickinson). FACS binding of IgG1s to surface expressed HA occurred after sequential treatment with trypsin and pH 4.9 buffered medium, and was expressed as percent binding on untreated HA (A). See figure 1A.
[000151] Antibody CR9114 shows a marked decrease in binding after pH change, indicating specificity for an epitope present just before low pH induced conformational change of the HA molecule.
[000152] Alternatively, to test whether IgGs can block the conformational change of HA induced by low pH, antibody CR9114 was added before the low pH step. Samples from consecutive treatments were split and stained with each phycoerythrin-conjugated anti-human IgG (Southern Biotech). Stained cells were analyzed using a FACS Canto with FACS Diva software (Becton Dickinson). See figure 1B.
[000153] Antibody CR9114 shows a high level of residual binding with various HAs after pH shift, indicating that when these antibodies are bound to the HA molecule, the conformational change induced by low pH does not occur. Example 8
[000154] Affinity measurements of Fabs in various influenza A and B HAs.
[000155] Recombinant soluble HA from A/New Caledonia/20/1999 (HI), A/Brisbane/59/2007 (HI), A/Wisconsin/67/2005 (H3), A/Brisbane/10/2007 (H3) , B/Florida/4/2006 (B), B/Brisbane/60/2008 (B) and B/Malaysia/2506/2004 (B) produced using baculovirus vectors in insect cells were obtained from Protein Sciences Corp (CT , USA) and biotinylated at room temperature (RT) for 40 minutes using EZ-linked sulfo-NHS-LC-LC-LC-biotin (Pierce).The buffer exchange step for PBS was performed using Amicon Ultra 0. 5 mL (Millipore). Biotinylated HA was bound to the streptavidin sensors at 37 °C for 1200 seconds. The association of Fab fragment of CR9005, CR9112, CR9113 and CR9114 in HA was measured in Octet QK (ForteBio) for 700 seconds at 37 °C by exposing the sensors to 100 nM antibody in 1x conetic buffer (ForteBio) Dissociation of Fab fragments was evaluated by exposing the sensors to 1x kinetic buffer for 9000 seconds at 37°C. Fab fragments of CR9005, CR9112 , CR91 13 and CR9114 all bound with micro to pico-molar affinities to HA H1, H3 of influenza B. Example 9
[000156] Competition for binding with other central binding antibodies
[000157] Recombinant soluble HA from A/New Caledonia/20/1999 (H1N1) and A/Wisconsin/67/2005 (H3N2) produced using baculovirus vectors in insect cells was obtained from Protein Sciences Corp (CT, USA) and biotinylated at room temperature (RT) for 40 minutes using EZ-linked sulfo-NHS-LC-LC-biotin (Pierce). The buffer exchange step for PBS was performed using Amicon Ultra 0.5 mL centrifuge filters (Millipore). Biotinylated HA was bound to streptavidin sensors at 37 °C for 1200 seconds. The association of CR9114 and CR6261 antibodies with HA H1 was measured in Octet QK (ForteBio) for 700 seconds at 37 °C, exposing the sensors to 100 nM of antibody in 1 x kinetic buffer (ForteBio), after which the degree of binding Additional testing was evaluated by exposing the sensors to a second antibody (100 nM in 1x kinetic buffer) in the presence of the first antibody (100 nM) for 700 seconds at 37 °C. As a control, mAb CR9020, which binds to the globular main part of H1, was used. The association of CR9114 and CR8020 antibodies in HA H3 was measured in Octet QK (ForteBio) for 900 seconds at 37 °C, exposing the sensors to 100 nM of antibody in 1 x kinetic buffer (ForteBio), after which the degree of binding Additional was evaluated by exposing the sensors to a second antibody (100 nM in 1x kinetic buffer) in the presence of the first antibody (100 nM) for 900 seconds at 37°C. As a control, mAb CR8057, which binds to the globular backbone of H3, was used.
[000158] CR9114 competes to bind to HA H1 with CR6261 and aHA H3 with CR8020. Therefore, CR9114 likely binds to an epitope that overlaps with both CR6261 and CR8020 epitopes in the central region of HA. (See figure 2) Example 10
[000159] Prophylactic activity of human IgG monoclonal antibody CR9114 against lethal challenge of influenza B in vivo
[000160] A study was performed to test the prophylactic effect of monoclonal antibody CR9114 against a lethal challenge with influenza B virus in vivo. MAb CR9114 was tested for prophylactic efficiency in a mouse model with lethal challenge, with mouse-adapted B/Florida/04/2006 influenza virus (Central Veterinary Institute (CVI), Lelystad, Netherlands). The B/Florida/04/2006 virus was adapted to mice after 5 lung-to-lung passages. The mouse-adapted 5-pass influenza B virus was propagated in embryonated chicken eggs in a CVI laboratory. All mice (Balb/c, female, 6-8 weeks, n=10 per group) were acclimatized and maintained for a period of at least 4 days prior to the start of the experiment. MAb CR9114 was dosed at 15 mg/kg intravenously into the tail vein (coccygeal vein) on day 1 before challenge, assuming an average weight of 18 g per mouse and a fixed dose volume of 0.2 mL. A control group was obtained and dosed with vehicle control. Mice were then challenged on day 0 with 25 LD50 of influenza B virus B/Florida/04/2006 by intranasal inoculation. Clinical signs and body weights were determined daily from day 1 pre-challenge to day 8. Clinical signs were scored using a scoring system (0=no clinical signs; l=rough coat; 2=rough coat, less reactive during handling; 3=rough coating, coiled, forced breathing, less reactive during handling; 4=rough coating, coiled, forced breathing, inactive response to handling/handling). At a rating of 4, the animal was euthanized.
[000161] All mice were active and appeared healthy without showing signs of illness during the acclimatization period. Figure 3A shows survival rates of mice after mAb administration. Mice dosed with 15 mg/kg of mAb CR9114 showed a 100% survival rate, whereas no control mAb group survived 50%.
[000162] In figure 3B, the change in the average body weight of mice during the 8-day study period after mAb administration is shown. In the mAb CR9114 group mice did not lose weight during the 8 day period in the study, whereas in the vehicle control group weight loss was observed. The median clinical scores of mice are shown in Figure 3C. Of the mice treated with 15 mg/kg of mAb CR9114 on day 1 pre-challenge, all survived and none of the animals showed any clinical signs during the observation period (from day 0 to day 8, post-infection). These results showed that the human anti-influenza antibody CR9114, identified and developed in the manner described herein, is capable of providing protection against a lethal dose of influenza B virus in vivo. When administered one day before infection, at a dose of 15 mg/kg or more, mAb CR9114 was able to competitively prevent the clinical manifestation of influenza B infection in mice.Table 1. First cycle of Vkappa amplifications, Vlambda and VH








Table 5: Cross-linking activity of PEG/NACl-precipitated and HA-filter-sterilized single-chain phage antibodies of different subtypes as measured by ELISA. +=link (background >4x); +/-= little binding (2-4 x background); -= undetectable binding; H1= HA of influenza A subtype H1; H3= HA of influenza A subtype H3; H5= HA of influenza A subtype H5; H7= HA of influenza A subtype H7; B= HA of influenza B virus; Rabies= Rabies virus glycoproteins

Table 6: FACS analysis of PEG/NACI-precipitated and filter-sterilized phage antibodies. +=link (background >4x); +/-= little binding (2-4 x background); -= undetectable binding; PER.C6=non-transfected PER.C6 cells (control); mH1, mH3, mH7= membrane bound HA of subtypes H1, H3 and H7, respectively.




Table 9: Cross-linking reactivity of IgGs as measured by ELISA and FACS. H1= soluble recombinant A/NewCaledonia/20/1990 H1 HA; H3= soluble recombinant A/Wisconsin/67/2005 H3 HA; H5= soluble recombinant A/Vietnam/1203/04 H5 HA, H7= soluble recombinant A/Netherlands/219/2003 H7 HA, H9= soluble recombinant A/Hong Kong/1073/99 H9 HA, B= soluble recombinant B/Ohioq01/05 influenza B HA, Rabies= Rabies glycoprotein, PER.C6= untransfected PER.C6 cells (control); mH1= PER.C6 expressed in A/New Caledonia/20/1990 H1 HA; mH3= PER.C6 expressed in A/Wisconsin/67/2005 H3 HA; mH7= PER.C6 expressed in A/Netherlands/219/2003 H7 HA, ND= not performed; += binding (background > 10x) +/-= little binding (background 2-10 times); -= undetectable link.
Table 10. Cross-neutralizing activity of IgGs; titrations (indicated in μg/mL) are geometric means of IC50 values, determined according to the Spearman-Karber method of experiments at least in duplicate; > 100= non-neutralizing at the concentration tested (100 μg/mL)

SEQUENCES> SC09-003 VH DNA (SEQ ID NO: 1) GAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTCAAGAAGGCTG GGTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCA ACAACTTTGGTATCAGCTGGGTACGACAGGCCCCTGGCCAAGGC CTTGAGTGGATGGGCGGGATCAGCCCAATCTTTGGTTCGACAGTC TACGCACAGAAATTTCAGGGCAGAGTCACTATTTCCGCGGACATA TTTTCACACACTGCCTACATGGAGATGAACAGCCTGACATCTGAG GACACGGCCGTCTATTTCTGTGCGAGGCACGGAAATTATTATTTCT ACTCCGGTATGGACCTCTGGGGCCAAGGGACCACGGTCACC> SC09-003 VH PROTEIN (SEQ ID NO: 2) EVQLVESGAEVKKAGSSVKVSCKSSGGTSNNFGISWVRQAPGQGLE WMGGISPIFGSTVYAQKFQGRVTISADIFSHTAYMEMNSLTSEDTAVY FCARHGNYYFYSGMDLWGQGTTVT> SC09-003 VL DNA (SEQ ID NO: 3 ) TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGG ACAGACGGCCACGATTTCCTGTGGGGGAGACAACGTTGGAAGTA ACAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTG CTGGTCGTCTATGATGATCGCGACCGACCCTCAGGGATCCCTGA GCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCAT CAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGG TGTGGGATAGTAGTAGTGATCATCGAGTCTTCGGAACTGGGACCA AGGTCACCGTCCTAG> VL SC09-003 PROTEIN (SEQ ID NO: 4) SYVLTQPPSVSVAPGQTATISCGGDNVGSNSVHWYQQKPGQAPV LV VYDDRDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDS SSDHRVFGTGTKVTVL> SC09-004 VH DNA (SEQ ID NO: 5) CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGCGGCACCTCCAA TAACTATGCCATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGGTCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGTCTGACATCTGAGG ACACGGCCGTCTATTATTGTGCGAGACACGGGAATTATTATTACAA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACC> SC09-004 VH PROTEIN (SEQ ID NO: 6) QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGVSPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTA VYYCARHGNYYYNSGMDVWGQGTTVT> SC09-004 VL DNA (SEQ ID NO : 7) CAGTCTGTGCTGACGCAGCCGCCCGCAGTGTCTGGGACCCCCGG GCAGAGGGTCACCATCTCGTGTTCTGGAAGTGATTCCAACATCGG GAGAAGAAGTGTAAACTGGTACCAGCAGTTCCCAGGAACGGCCC CCAAACTCCTCATCTATAGTAACGATCAGCGGCCCTCAGTGGTCC CTGACCGATTCTCTGGCTCCAAGTCCGGCACCTCAGCCTCCCTGG CCATCAGTGGGCTCCAGTCTGAAGATGAGGCCGAATATTACTGTG CAGCATGGGATGACAGCCTGAAGGGGGCTGTGTTCGGAGGAGGC ACCCAGCTGACCGTCCTCG> SC09-004 VL PROT EIN (SEQ ID NO: 8) QSVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPK LLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWD DSLKGAVFGGGTQLTVL> SC09-005 VH DNA (SEQ ID NO: 9) CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTCAAGAGGCCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATTAGTTGGGTGCGACAGGCCCCTGGACAAGGCCT TGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGTCTA CGCACAGAAATTCCAGGGCAGAGTCACTATTTCCGCGGACATATT TTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGGA CACGGCCGTATATTTCTGTGCGAGGCACGGGAACTATTATTACTA CTCCGGTATGGACCTCTGGGGCCAAGGGACCACGGTCACC> SC09-005 VH PROTEIN (SEQ ID NO: 10) QVQLVQSGAEVKRPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTVYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YFCARHGNYYYYSGMDLWGQGTTVT> SC09 -005 VL DNA (SEQ ID NO: 11) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGA CAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTCGGT GGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCC CCCAAACTCCTGATTTTTGATGTCAGTGATCGGCCCTCAGGGGTT TCTGATCGCTTCTCTGGCTCCAAGTCTGCGGACACGGCCTCCCTG ACCATCTCTGGACTCCAGGCTCAGGACGAGGCTGATTATTACTGC TGCTCATATGCAG GTAGTGCCAAGGGCGTCTTCGGAACTGGGAC CAAGGTCACCGTCCTAG> VL SC09-005 PROTEIN (SEQ ID NO: 12) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAP KLLIFDVSDRPSGVSDRFSGSKSADTASLTISGLQAQDEADYYCCSY AGSAKGVFGTGTKVTVL> SC09-006 VH DNA (SEQ ID NO: 13) GAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTCAAGAGGCCTG GGTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCA ATAACTATGCTATTAGTTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGTCT ACGCACAGAAATTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTATATTTCTGTGCGAGGCACGGGAACTATTATTACT ACTCCGGTATGGACCTCTGGGGCCAAGGGACCACGGTCACC> SC09-006 VH PROTEIN (SEQ ID NO: 14) EVQLVESGAEVKRPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTVYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YFCARHGNYYYYSGMDLWGQGTTVT> SC09-006 VL DNA (SEQ ID NO: 15) TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGG ACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAA AACTGTGCATTGGTACCAGCAGAACTCAGGCCAGGCCCCTGTGCT GGTCGTCTATGGTGATAGCGACCGGCCCTCAGGGATCCCTGAGC GATTCTCTGGCTCCAACTCTGGGACCACGGCCACCCTGA Ccatca District GCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTG TGGGATAGTAGTAGTGATCATCCCGGTGCTGTGTTCGGAGGAGG CACCCAGCTGACCGTCCTCG> VL SC09-006 PROTEIN (SEQ ID NO: 16) SYVLTQPPSVSVAPGQTARITCGGNNIGSKTVHWYQQNSGQAPVLV VYGDSDRPSGIPERFSGSNSGTTATLTISRVEAGDEADYYCQVWDS SSDHPGAVFGGGTQLTVL> SC09-007 VH DNA (SEQ ID NO: 17) CAGGTGCAGCTGGTGCAATCTGGAGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAGGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGAGGGATCAGCCCTATCTTTGGTTCAGCAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTACCGCGGACATAT TTTCGAACACAGTGTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTGTATTACTGTGCGAGACACGGGAATTATTATTACT ACTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCGAGC> SC09-007 VH PROTEIN (SEQ ID NO: 18) QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSAAYAQKFQGRVTITADIFSNTVYMELNSLTSEDTAV YYCARHGNYYYYSGMDVWGQGTTVTVSS> SC09-007 VL DNA (SEQ ID NO: 19) CAA TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGG GCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGG AAGTAATACTGTAAACTGGTACCAGCAGGTCCCCGGAACGGCCCC ACTCCTCATCTATGGTGATGATCAGCGGCCCTCAGGGGTCCC TGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGC CATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGC AACATGGGATGACAGCCTGAATGGTCATGTGTTCGGAGGAGGCA CCCAGCTGACCGTCCTCG> VL SC09-007 PROTEIN (SEQ ID NO: 20) SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPKL LIYGDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWD DSLNGHVFGGGTQLTVL> SC09-008 VH DNA (SEQ ID NO: 21) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAGAGTCTCCTGTAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTATATTTCTGTGCGAGGCACGGGAATTATTATTACTA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CGAGC> SC09-008 VH PROTEIN ( SEQ ID NO: 22)EVQLVQSGAEVKKPGSSVRVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YFCARHGNYYYSGMDVWGQGTTVTVSS >SC09-008 VL DNA (SEQ ID NO: 23)TCCTATGTGCTGACTCAGCCACCCTCGGCCTGAGGACA TTACCTGTGGGGGAAACAACATTGGAAGTAA AACTGTGCATTGGTACCAGCAGAACTCAGGCCAGGCCCCTGTGCT GGTCGTCTATGGTGATAGCGACCGGCCCTCAGGGATCCCTGAGC GATTCTCTGGCTCCAACTCTGGGACCACGGCCACCCTGACCATCA GCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTG TGGGATAGTAGTAGTGATCATCCCGGTGCTGTGTTCGGAGGAGG CACCCAGCTGACCGTCCTCG> VL SC09-008 PROTEIN (SEQ ID NO: 24) SYVLTQPPSVSVAPGQTARITCGGNNIGSKTVHWYQQNSGQAPVLV VYGDSDRPSGIPERFSGSNSGTTATLTISRVEAGDEADYYCQVWDS SSDHPGAVFGGGTQLTVL> SC09-009 VH DNA (SEQ ID NO: 25) CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAATTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGCCTGGCATCTGAGG ACACGGCCGTATATTTCTGTGCGAGGCACGGGAATTATTATTACTA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CGAGC> VH SC09-009 PROTEIN (SEQ ID NO: 26)QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLASEDTAV YFCARHGNYYYSGMDVWGQGTTVTVSS>SC09-009 V U DNA (SEQ ID NO: 27) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTA GGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCATATTAGC AGTTGGTTAGCCTGGTATCAGCAGAAGCCAGGGAAAGGCCCTCA GCTCCTGATCTATTCTGCATCCCGTTTGCAAAGTGGGGTCCCATC AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT CAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACA GGCTAACAGTTTCCCCCTCACTTTCGGCCCTGGGACCAAAGTGGA TATCAAAC> VL SC09-009 PROTEIN (SEQ ID NO: 28) DIQMTQSPSSVSASVGDRVTITCRASQHISSWLAWYQQKPGKGPQL LIYSASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSF PLTFGPGTKVDIK> SC09-010 VH DNA (SEQ ID NO: 29) GAGGTGCAGCTGGTGGAGTCCGGGGCTGAGGTCAAGAAGCCTG GGTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCA ATAATTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCCAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTATATTACTGTGCGAGGCACGGGAATTATTATTACT ACTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCGAGC> SC09-010 VH PROTEIN (SEQ ID NO: 30) EVQLVESGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLD WMGGISPIFGSTAYAQKF QGRVTISADIFSNTAYMELNSLTSEDTAVY YCARHGNYYYYSGMDVWGQGTTVTVSS> SC09-010 VL DNA (SEQ ID NO: 31) TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGG ACAGACGGCCAGGATTACCTGTGGGGGAAACAACATYGGAAGTA AAACTGTGCATTGGTACCAGCAGAACTCAGGCCAGGCCCCTGTG CTGGTCGTCTTTGTTGATAGCGACCGTCCCTCAGGGATCCATGAG CGATTCTGTGGCTCCAACTCTGGGTCCACGGCCACCCTGACCATC AGCAGCGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGT GTGGGATAGTAATAGCGATCATCCCGGTGCTGTGTTCGGAGGAG GCACCCAGCTGACCGTCCTCG> VL SC09-010 PROTEIN (SEQ ID NO: 32) SYVLTQPPSVSVAPGQTARITCGGNNIGSKTVHWYQQNSGQAPVLV VFVDSDRPSGIHERFCGSNSGSTATLTISSVEAGDEADYYCQVWDS NSDHPGAVFGGGTQLTVL> SC09-011 VH DNA (SEQ ID NO: 33) GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAGGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGGCAGGCCCCTGGACAAGGCC TTGACTGGATGGGAGGGATCAGCCCTATCTTTGGTTCAGCAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTACCGCGGACATAT TTTCGAACACAGTGTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTGTATTACTGTGCGAGACACGGGAATTATTATTACT ACTCCGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCGAGC> SC09-011 VH PROTEIN (SEQ ID NO: 34) EVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSAAYAQKFQGRVTITADIFSNTVYMELNSLTSEDTAV YYCARHGNYYYYSGTDVWGQGTTVTVSS> SC09-011 VL DNA (SEQ ID NO: 35) TCCTATGTGCTGACTCAGCCACCCGCAGTGTCTGGGACCCCCGG GCAGAGGGTCACCATCTCGTGTTCTGGAAGTGATTCCAACATCGG GAGAAGAAGTGTAAACTGGTACCAGCAGTTCCCAGGAACGGCCC CCAAACTCCTCATCTATAGTAACGATCAGCGGCCCTCAGTGGTCC CTGACCGATTCTCTGGCTCCAAGTCCGGCACCTCAGCCTCCCTGG CCATCAGTGGGCTCCAGTCTGAAGATGAGGCCGAATATTACTGTG CAGCATGGGATGACAGCCTGAAGGGGGCTGTGTTCGGAGGAGGC ACCCAGCTGACCGTCCTCG> VL SC09-011 PROTEIN (SEQ ID NO: 36) SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPK LLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWD DSLKGAVFGGGTQLTVL> SC09- 012 VH DNA (SEQ ID NO: 37) GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAGGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAATTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCT TGACTGGATGGGAGGGATCAGCCCTATTTTTGGTTCAGCAGTCTA CGCACAGAAGTTCCAGGGCAGAGTCACTATTACCGCGGACATATT TTCGAACACAGTGTACATGGAGCTGAACAGCCTGACATCTGAGGA CACGGCCGTGTATTACTGTGCGAGACACGGGACTTA TTATTACTA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CGAGC> SC09-012 VH PROTEIN (SEQ ID NO: 38) EVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSAVYAQKFQGRVTITADIFSNTVYMELNSLTSEDTAV YYCARHGTYYYYSGMDVWGQGTTVTVSS> SC09-012 VL DNA (SEQ ID NO: 39) CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGG GCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCG GGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGGACA GCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCCTCAGGG GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCC CTGGCCATCACTGGGCTCCAGGTTGAGGATGAGGCTGATTATTAC TGCCAGTCCTATGACCAGAACCTGAGTGAGGGGGTCTTCGGCGG AGGGACCAAGCTGACCGTCCTAG> VL SC09-012 PROTEIN (SEQ ID NO : 40) QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAP KLLIYGNNNRPSGVPDRFSGSKSGTSASLAITGLQVEDEADYYCQSY DQNLSEGVFGGGTKLTVL> SC09-029 VH DNA (SEQ ID NO: 41) GAGGTGCAGCTGGTGGAGTCCGGGGCTGAGGTCAAGAAGCCTG GGTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCA ATAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGC CTTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCC TACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATA TTTTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAG GACACGGCCGTATATTACTGTGCGAGGCACGGGAATTATTATTAC TACTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGT CTCGAGC> SC09-029 VH PROTEIN (SEQ ID NO: 42) EVQLVESGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLD WMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAVY YCARHGNYYYYSGMDVWGQGTTVTVSS> SC09-029 VL DNA (SEQ ID NO: 43) GAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCT GGGGAAAGAGGCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAG CAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCCCAGA CAGGTTCACTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCA GTATGGGAGCTCACCATTCGCTTTCGGCCCTGGGACCAAGGTGG AGATCAAA> VL SC09-029 PROTEIN (SEQ ID NO: 44) EIVMTQSPGTLSLSPGERGTLSCRASQSVSSYLAWYQQKPGQAPRL LIYGASTRATGIPDRFTGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS PFAFGPGTKVEIK> SC09-030 VH DNA (SEQ ID NO: 45) CAGATGCAGCTGGTGCAGTCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGC CCTATCTTTGGTTCGACAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTATATTTCTGTGCGAGGCACGGGAATTATTATTACTA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CGAGC> SC09-030 VH PROTEIN (SEQ ID NO: 46) QMQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YFCARHGNYYYYSGMDVWGQGTTVTVSS> SC09-030 VL DNA (SEQ ID NO: 47) TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGG ACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAA AAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGC TGGTCGTCTATGGTGATAGCGACCGGCCCTCAGGGATCCCTGAG CGATTCTCTGGCTCCAACTCTGGGACCACGGCCACCCTGACCATC AGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGT GTGGGATAGTAGTAGTGATCATCCCGGTGCTGTGTTCGGAGGAG GCACCCAGCTGACCGTCCTCG> VL SC09-030 PROTEIN ( SEQ ID NO: 48)SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLV VYGDSDRPSGIPERFSGSNSGTTATLTISRVEAGDEADYYCQVWDS SSDHPGAVFGGGTQLTVL>SC09-031 VH DNA (SEQ ID NO: 49)CAGGTCTCCAGCTGGTACAGTCTGGGGCTGAGGTCGAGAGGCCTAGGTCGCAG GGCACCTCCAA TAACTATGCCATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGTCTGACATCTGAGG ACACGGCCGTCTATTATTGTGCGAGACACGGGAATTATTATTACAA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CGAGC> SC09-031 VH PROTEIN (SEQ ID NO: 50) QVQLVQSGAEVERPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YYCARHGNYYYNSGMDVWGQGTTVTVSS> SC09-031 VL DNA (SEQ ID NO: 51) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGG GCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCG GGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGAAACAG CCCCCAAACTCCTCATTTATGATAACAACAATCGTCCCTCAGGGGT TTCTGACCGATTCTCTGGCTCCAAGTCTGGCACTTCAGCCTCCCT GGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTG CCAGTCCTATGACAGCGGCCTGAGTGCTTCGCCTTATGTCTTCGG AGCTGGGACCAAGGTCACCGTCCTAG> VL SC09-031 PROTEIN (SEQ ID NO: 52)QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPETAP KLLIYDNNNRPSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCQSY DSGLSASPYVFGAGTKVTVL>SC09-112 VH DNA (S EQ ID NO: 53) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAGTTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTATATTACTGTGCGAGGCACGGGAATTATTATTACT ACTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCGAGC> SC09-112 VH PROTEIN (SEQ ID NO: 54) QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YYCARHGNYYYYSGMDVWGQGTTVTVSS> SC09-112 VL DNA (SEQ ID NO: 55) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGG GCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCGCCAACATCG GGGCAGGTTATGATGTCCACTGGTACCAGCAGTTTCCAGGAACAG CCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCCTCAGGGG TCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCC TGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACT GCCAGTCCTATGACAGCAGCCTGAGTGGTGCGTTATTCGGCGGA GGGACCAAGCTGACCGTCCTAG>SC09-112 VL PROTEIN (SEQ ID NO: 56)QSVLTQPPSVSGAPGQRVTISCTGSSANIGAGYDVHWYQQFPGTAP K LLIYGNNNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSY DSSLSGALFGGGTKLTVL> SC09-113 VH DNA (SEQ ID NO: 57) CAGATGCAGCTGGTGCAGTCTGGGGCTGAGGTCAAGAAGGCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGAGTGGATGGGCGGGATCAGTCCAATCTTTGGTTCGACAGTCT ACGCACAGAAATTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCACACACTGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGCATATTTCTGTGCGAGGCACGGAAACTATTATTACT ACTCCGGTATGGACCTCTGGGGCCAAGGGACCACGGTCACCGTC TCGAGC> SC09-113 VH PROTEIN (SEQ ID NO: 58) QMQLVQSGAEVKKAGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL EWMGGISPIFGSTVYAQKFQGRVTISADIFSHTAYMELNSLTSEDTAA YFCARHGNYYYYSGMDLWGQGTTVTVSS> SC09-113 VL DNA (SEQ ID NO : 59) CAGTCTGTGCTGACTCAGCCACCCGCAGTGTCTGGGACCCCCGG GCAGAGGGTCACCATCTCGTGTTCTGGAAGTGATTCCAACATCGG GAGAAGAAGTGTAAACTGGTACCAGCAGTTCCCAGGAACGGCCC CCAAACTCCTCATCTATAGTAACGATCAGCGGCCCTCAGTGGTCC CTGACCGATTCTCTGGCTCCAAGTCCGGCACCTCAGCCTCCCTGG CCATCAGTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGTG CAGCATGGGATGCCAGCCTGAGTGGTCCTGTGTTCGGAGGAGGC ACCCAGCTGACCGTCCTCG> SC 09-113 VL PROTEIN (SEQ ID NO: 60) QSVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPK LLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQAEDEADYYCAAWD ASLSGPVFGGGTQLTVL> SC09-114 VH DNA (SEQ ID NO: 61) CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTCAAGAAGCCTGG GTCCTCGGTGAAAGTCTCCTGCAAGTCTTCTGGAGGCACCTCCAA TAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCC TTGACTGGATGGGCGGGATCAGCCCTATCTTTGGTTCGACAGCCT ACGCACAGAAATTCCAGGGCAGAGTCACTATTTCCGCGGACATAT TTTCGAACACAGCCTACATGGAGCTGAACAGCCTGACATCTGAGG ACACGGCCGTATATTTCTGTGCGAGGCACGGGAATTATTATTACTA CTCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CGAGC> SC09-114 VH PROTEIN (SEQ ID NO: 62) QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGL DWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAV YFCARHGNYYYYSGMDVWGQGTTVTVSS> SC09-114 VL DNA (SEQ ID NO: 63) TCCTATGTGCTGACTCAGCCACCCGCAGTGTCTGGGACCCCCGG GCAGAGGGTCACCATCTCGTGTTCTGGAAGTGATTCCAACATCGG GAGAAGAAGTGTAAACTGGTACCAGCAGTTCCCAGGAACGGCCC CCAAACTCCTCATCTATAGTAACGATCAGCGGCCCTCAGTGGTCC CTGACCGATTCTCTGGCTCCAAGTCCGGCACCTCAGCCTCCCTGG CCATCAGTGGGCTCCAGTCTGAAGATGAGGCCG AATATTACTGTG CAGCATGGGATGACAGCCTGAAGGGGGCTGTGTTCGGAGGAGGC ACCCAGCTGACCGTCCTCG> VL SC09-114 PROTEIN (SEQ ID NO: 64) Pig-SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPK LLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWD DSLKGAVFGGGTQLTVLVector HCgamma1-C911 (SEQ ID NO: 175) tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 60 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 120 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 180gcttagggtt aggcgttttg cgctgcttcg ctaggtggtc aatattggcc attagccata 240 ttattcattg gttatatagc ataaatcaat attggctatt ggccattgca tacgttgtat 300 ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat 360 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 420 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 480ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 540 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 600 tatcatatgc caagtacgcc ccctattgac gtcaa tgacg gtaaatggcc cgcctggcat 660 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 720 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 780 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 840 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 900ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 960 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc 1020ctccgcggcc gggaacggtg cattggaagc tggcctggat atcctgactc tcttaggtag 1080ccttgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac aagacaggtt 1140taaggagatc aatagaaact gggcttgtcg agacagagaa gactcttgcg tttctgatag 1200gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt ccactcccag 1260 ttcaattaca gctcgccacc atgggatgga gctgtatcat cctcttcttg gtactgctgc 1320 tggcccagcc ggccagtgac cttgaccggt gcaccacttt tgatgatgtt caagctccta 1380 attacactca acatacttca tctatgaggg gggtttacta tcctgatgaa atttttagat 1440 cggacactct ttatttaact caggatttat ttcttccatt ttattctaat gttacagggt 1500 ttcatactat taatcatacg tttggcaacc ctgtcatacc ttttaaggat ggtatttatt 1560 ttgctgccac agagaaatca aatgttgtcc gtggttgggt ttttggttct accatgaaca 1620 acaagtcaca gtcggtgatt attattaaca attctactaa tgttgttata cgagcatgta 1680 actttgaatt gtgtgacaac cctttctttg ctgtttctaa acccatgggt acacagacac 1740 atactatgat attcgataat gcatttaatt gcactttcga gtacatatct gatgcctttt 1800cgcttgatgt ttcagaaaag tcaggtaatt ttaaacactt acgagagttt gtgtttaaaa 1860 ataaagatgg gtttctctat gtttataagg gctatcaacc tatagatgta gttcgtgatc 1920 taccttctgg ttttaacact ttgaaaccta tttttaagtt gcctcttggt attaacatta 1980 caaattttag agccattctt acagcctttt cacctgctca agacatttgg ggcacgtcag 2040 ctgcagccta ttttgttggc tatttaaagc caactacatt tatgctcaag tatgatgaaa 2100 atggtacaat cacagatgct gttgattgtt ctcaaaatcc acttgctgaa ctcaaatgct 2160 ctgttaagag ctttgagatt gacaaaggaa tttaccagac ctctaatttc agggttgttc 2220 cctcaggaga tgttgtgaga ttccctaata ttacaaactt gtgtcctttt ggagaggttt 2280 ttaatgctac taaattccct tctgtctatg catgggagag aaaaaaaatt tctaatt gtg 2340 ttgctgatta ctctgtgctc tacaactcaa catttttttc aacctttaag tgctatggcg 2400 tttctgccac taagttgaat gatctttgct tctccaatgt ctatgcagat tcttttgtag 2460 tcaagggaga tgatgtaaga caaatagcgc caggacaaac tggtgttatt gctgattata 2520attataaatt gccagatgat ttcatgggtt gtgtccttgc ttggaatact aggaacattg 2580 atgctacttc aactggtaat tataattata aatataggta tcttagacat ggcaagctta 2640 ggccctttga gagagacata tctaatgtgc ctttctcccc tgatggcaaa ccttgcaccc 2700 cacctgctct taattgttat tggccattaa atgattatgg tttttacacc actactggca 2760 ttggctacca accttacaga gttgtagtac tttcttttga acttttaaat gcaccggcca 2820 cggtttgtgg accaaaatta tccactgacc ttattaagaa ccagtgtgtc aattttaatt 2880 ttaatggact cactggtact ggtgtgttaa ctccttcttc aaagagattt caaccatttc 2940 aacaatttgg ccgtgatgtt tctgatttca ctgattccgt tcgagatcct aaaacatctg 3000 aaatattaga catttcacct tgctcttttg ggggtgtaag tgtaattaca cctggaacaa 3060 atgcttcatc tgaagttgct gttctatatc aagatgttaa ctgcactgat gtttctacag 3120 caattcatgc agatcaactc acaccagctt ggcgcatata ttctactgga 318 aacaatgtat 0 tccagactca ggcaggctgt cttataggag ctgagcatgt cgacacttct tatgagtgcg 3240 acattcctat tggagctggc atttgtgcta gttaccatac agtttcttta ttacgtagta 3300 ctagccaaaa atctattgtg gcttatacta tgtctttagg tgctgatagt tcaattgctt 3360 actctaataa caccattgct atacctacta acttttcaat tagcattact acagaagtaa 3420 tgcctgtttc tatggctaaa acctccgtag attgtaatat gtacatctgc ggagattcta 3480 ctgaatgtgc taatttgctt ctccaatatg gtagcttttg cacacaacta aatcgtgcac 3540 tctcaggtat tgctgctgaa caggatcgca acacacgtga agtgttcgct caagtcaaac 3600aaatgtacaa aaccccaact ttgaaatatt ttggtggttt taatttttca caaatattac 3660 ctgaccctct aaagccaact aagaggtctt ttattgagga cttgctcttt aataaggtga 3720 cactcgctga tgctggcttc atgaagcaat atggcgaatg cctaggtgat attaatgcta 3780 gagatctcat ttgtgcgcag aagttcaatg gacttacagt gttgccacct ctgctcactg 3840 atgatatgat tgctgcctac actgctgctc tagttagtgg tactgccact gctggatgga 3900 catttggtgc tggcgctgct cttcaaatac cttttgctat gcaaatggca tataggttca 3960 atggcattgg agttacccaa aatgttctct atgagaacca aaaacaaatc gccaaccaat4020ttaacaa GGC gattagtcaa attcaagaat cacttacaac aacatcaact gcattgggca4080agctgcaaga cgttgttaac cagaatgctc aagcattaaa cacacttgtt aaacaactta 4140 gctctaattt tggtgcaatt tcaagtgtgc taaatgatat cctttcgcga cttgataaag 4200 tcgaggcgga ggtacaaatt gacaggttaa ttacaggcag acttcaaagc cttcaaacct 4260atgtaacaca acaactaatc agggctgctg aaatcagggc ttctgctaat cttgctgcta 4320 ctaaaatgtc tgagtgtgtt cttggacaat caaaaagagt tgacttttgt ggaaagggct 4380 accaccttat gtccttccca caagcagccc cgcatggtgt tgtcttccta catgtcacgt 4440 atgtgccatc ccaggagagg aacttcacca cagcgccagc aatttgtcat gaaggcaaag 4500catacttccc tcgtgaaggt gtttttgtgt ttaatggcac ttcttggttt attacacaga 4560 ggaacttctt ttctccacaa ataattacta cagacaatac atttgtctca ggaaattgtg 4620 atgtcgttat tggcatcatt aacaacacag tttatgatcc tctgcaacct gagcttgact 4680 cattcaaaga agagctggac aagtacttca aaaatcatac atcaccagat gttgattttg 4740 gcgacatttc aggcattaac gcttctgtcg tcaacattca aaaagaaatt gaccgcctca 4800 atgaggtcgc taaaaattta aatgaatcac tcattgacct tcaagaactg ggaaaatatg 4860 agcaatatat taaatg GCCT ctcgacgaac aaaaactcat ctcagaagag gatctgaatg 4920ctgtgggcca ggacacgcag gaggtcatcg tggtgccaca ctccttgccc tttaaggtgg 4980tggtgatctc agccatcctg gccctggtgg tgctcaccat catctccctt atcatcctca 5040 tcatgctttg gcagaagaag ccacgttagg cggccgctcg agtgctagca ccaagggccc 5100cagcgtgttc cccctggccc ccagcagcaa gagcaccagc ggcggcacag ccgccctggg 5160ctgcctggtg aaggactact tccccgagcc cgtgaccgtg agctggaaca gcggcgcctt 5220gaccagcggc gtgcacacct tccccgccgt gctgcagagc agcggcctgt acagcctgag 5280cagcgtggtg accgtgccca gcagcagcct gggcacccag acctacatct gcaacgtgaa 5340ccacaagccc agcaacacca aggtggacaa acgcgtggag cccaagagct gcgacaagac 5400ccacacctgc cccccctgcc ctgcccccga gctgctgggc ggaccctccg tgttcctgtt 5460 cccccccaag cccaaggaca ccctcatgat cagccggacc cccgaggtga cctgcgtggt 5520ggtggacgtg agccacgagg accccgaggt gaagttcaac tggtacgtgg acggcgtgga 5580ggtgcacaac gccaagacca agccccggga ggagcagtac aacagcacct accgggtggt 5640gagcgtgctc accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt 5700gagcaacaag gccctgcctg g cccccatcga agaccatc agcaaggcca agggccagcc 5760 ccgggagccc caggtgtaca ccctgccccc cagccgggag gagatgacca agaaccaggt 5820gtccctcacc tgtctggtga agggcttcta ccccagcgac atcgccgtgg agtgggagag 5880caacggccag cccgagaaca actacaagac caccccccct gtgctggaca gcgacggcag 5940cttcttcctg tacagcaagc tcaccgtgga caagagccgg tggcagcagg gcaacgtgtt 6000cagctgcagc gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct 6060gagccccggc aagtgataat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt 6120gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 6180 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 6240 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 6300agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 6360cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg 6420tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 6480 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 6540 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgac CCCA aaaaacttga 6600 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 6660 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 6720 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 6780 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta 6840 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 6900tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 6960atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta 7020 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 7080 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga 7140 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa 7200 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 7260gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 7320gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 7380 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctgg ccacg 7440acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 7500ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 7560 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 7620 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 7680gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 7740aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 7800ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 7860 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 7920 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 7980 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 8040 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 8100atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 8160 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 8220 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 8280 g ttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 8340 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8400 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8460tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8520 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 8580gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8640 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 8700agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8760cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8820ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 8880tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 8940 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 9000 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9060gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 9120ctggtaacag GATT agcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 9180ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 9240ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9300 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 9360 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 9420 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 9480 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 9540 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 9600 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 9660gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 9720agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 9780 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 9840 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 9900 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 9960 ttg cgatcgttgt cagaagtaag gccgcag tgttatcact catggttatg gcagcactgc 10020 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 10080 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 10140gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt10200cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc10260gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 10320 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 10380tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 10440 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 10500aagtgccacc tgacg 10515Vector pig-C909-Ckappa (SEQ ID NO: 176) tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 60 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 120 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgtt aattaacatg 180aagaatctgc ttagggttag gcgttttgcg ctgcttcgct aggtggtcaa tattggccat 240tagccatatt attcattggt tatatagcat aaatcaatat tg gctattgg ccattgcata 300 cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat 360 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 420 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 540ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 600 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 660 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 720 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 780 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 840 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 900 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc 960gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc 1020gatccagcct ccgcggccgg gaacggtgca ttggaatcga tgactctctt aggtagcctt 1080 gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag 1 140gagatcaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac1200ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac tcccagttca 1260attacagctc gccaccatgc ggctgcccgc ccagctgctg ggccttctca tgctgtgggt 1320 gcccgcctcg agatctatcg atgcatgcca tggtaccaag cttgccacca tgagcagcag 1380 ctcttggctg ctgctgagcc tggtggccgt gacagccgcc cagagcacca tcgaggagca 1440ggccaagacc ttcctggaca agttcaacca cgaggccgag gacctgttct accagagcag 1500cctggccagc tggaactaca acaccaacat caccgaggag aacgtgcaga acatgaacaa 1560cgccggcgac aagtggagcg ccttcctgaa ggagcagagc acactggccc agatgtaccc 1620cctgcaggag atccagaacc tgaccgtgaa gctgcagctg caggccctgc agcagaacgg 1680cagcagcgtg ctgagcgagg acaagagcaa gcggctgaac accatcctga acaccatgtc 1740caccatctac agcaccggca aagtgtgcaa ccccgacaac ccccaggagt gcctgctgct 1800ggagcccggc ctgaacgaga tcatggccaa cagcctggac tacaacgagc ggctgtgggc 1860ctgggagagc tggcggagcg aagtgggcaa gcagctgcgg cccctgtacg aggagtacgt 1920ggtgctgaag aacgagatgg ccagggccaa ccactacgag gactacggcg actactggag 1980aggcgactac gaagt gaacg gcgtggacgg ctacgactac agcagaggcc agctgatcga 2040ggacgtggag cacaccttcg aggagatcaa gcctctgtac gagcacctgc acgcctacgt 2100gcgggccaag ctgatgaacg cctaccccag ctacatcagc cccatcggct gcctgcccgc 2160ccacctgctg ggcgacatgt ggggccggtt ctggaccaac ctgtacagcc tgaccgtgcc 2220cttcggccag aagcccaaca tcgacgtgac cgacgccatg gtggaccagg cctgggacgc 2280 ccagcggatc ttcaaggagg ccgagaagtt cttcgtgagc gtgggcctgc ccaacatgac 2340ccagggcttt tgggagaaca gcatgctgac cgaccccggc aatgtgcaga aggccgtgtg 2400ccaccccacc gcctgggacc tgggcaaggg cgacttccgg atcctgatgt gcaccaaagt 2460gaccatggac gacttcctga ccgcccacca cgagatgggc cacatccagt acgacatggc 2520ctacgccgcc cagcccttcc tgctgcggaa cggcgccaac gagggctttc acgaggccgt 2580gggcgagatc atgagcctga gcgccgccac ccccaagcac ctgaagagca tcggcctgct 2640gagccccgac ttccaggagg acaacgagac cgagatcaac ttcctgctga agcaggccct 2700gaccatcgtg ggcaccctgc ccttcaccta catgctggag aagtggcggt ggatggtgtt 2760taagggcgag atccccaagg accagtggat gaagaagtgg tgggagatga agcgggagat 2820cgtgggcgtg gtggagcccg cg tgccccacga agacctac tgcgaccccg ccagcctgtt 2880ccacgtgagc aacgactact ccttcatccg gtactacacc cggaccctgt accagttcca 2940gttccaggag gccctgtgcc aggccgccaa gcacgagggc cccctgcaca agtgcgacat 3000cagcaacagc accgaggccg gacagaaact gttcaacatg ctgcggctgg gcaagagcga 3060gccctggacc ctggccctgg agaatgtggt gggcgccaag aacatgaatg tgcgccccct 3120gctgaactac ttcgagcccc tgttcacctg gctgaaggac cagaacaaga acagcttcgt 3180 gggctggagc accgactgga gcccctacgc cgaccagagc atcaaagtgc ggatcagcct 3240gaagagcgcc ctgggcgaca aggcctacga gtggaacgac aacgagatgt acctgttccg 3300gagcagcgtg gcctatgcca tgcggcagta cttcctgaaa gtgaagaacc agatgatcct 3360gttcggcgag gaggacgtga gagtggccaa cctgaagccc cggatcagct tcaacttctt 3420cgtgaccgcc cccaagaacg tgagcgacat catcccccgg accgaagtgg agaaggccat 3480ccggatgagc cggagccgga tcaacgacgc cttccggctg aacgacaact ccctggagtt 3540cctgggcatc cagcccaccc tgggccctcc caaccagccc cccgtgagca tctggctgat 3600cgtgtttggc gtggtgatgg gcgtgatcgt ggtgggaatc gtgatcctga tcttcaccgg 3660 catccgggac cggaagaaga agaacaaggc ccggagcggc gagaacccc t acgccagcat 3720cgatatcagc aagggcgaga acaaccccgg cttccagaac accgacgacg tgcagaccag 3780cttctgataa tctagaacga gctcgaattc gaagcttctg cagacgcgtc gacgtcatat 3840 ggatccgata tcgccgtggc ggccgcaccc agcgtgttca tcttcccccc ctccgacgag 3900cagctgaaga gcggcaccgc cagcgtggtg tgcctgctga acaacttcta cccccgggag 3960gccaaggtgc agtggaaggt ggacaacgcc ctgcagagcg gcaacagcca ggagagcgtg 4020accgagcagg acagcaagga ctccacctac agcctgagca gcaccctcac cctgagcaag 4080gccgactacg agaagcacaa ggtgtacgcc tgcgaggtga cccaccaggg cctgagcagc 4140 cccgtgacca agagcttcaa ccggggcgag tgttaataga cttaagttta aaccgctgat 4200cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 4260 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 4320 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 4380gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 4440aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 4500taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 456 agcgccctag 0cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 4620 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 4680 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 4740 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 4800 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg gccatttcgg 4860 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 4920 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 4980catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 5040aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 5100catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 5160 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 5220 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 5280 cggatctgat cagcacgtga tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt 5340tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc 5400tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc 5460 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat 5520 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg 5580 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc 5640ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg 5700cccattcgga ccacaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat 5760tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt 5820 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct 5880cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt 5940cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt 6000 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc 6060ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact 6120 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga 6180 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc 6240ggccgtctgg g accgatggct tgtagaagt actcgccgat agtggaaacc gacgccccag 6300cactcgtccg agggcaaagg aatagcacgt gctacgagat ttcgattcca ccgccgcctt 6360ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 6420 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 6480 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 6540 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 6600 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 6660 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 6720agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 6780 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6840gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6900 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6960aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 7020ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 7080gaggtggcga aacccgacag minutes gactataaag ccaggcg tttccccctg gaagctccct 7140cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 7200 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 7260 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 7320cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 7380cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 7440gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 7500 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 7560cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7620tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 7680 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 7740 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 7800 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 7860 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 7920gcgagaccca cgctcaccgg ctccagattt atcagcaata aacca gccag ccggaagggc 7980cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 8040 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 8100 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 8160 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 8220 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 8280 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 8340 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 8400 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 8460ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 8520 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 8580 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 8640catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 8700 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 8760 aaaagtgcca cctgacg 8777Vector pig-C910-Clambda ( SEQ ID NO: 177) tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 60 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 120 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgtt aattaacatg 180aagaatctgc ttagggttag gcgttttgcg ctgcttcgct aggtggtcaa tattggccat 240 tagccatatt attcattggt tatatagcat aaatcaatat tggctattgg ccattgcata 300 cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat 360 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 420 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 540ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 600 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 660 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 720 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 780 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 840tttggcacca aaatcaacg g gactttccaa aatgtcgtaa caactccgcc ccattgacgc 900 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc 960gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc 1020gatccagcct ccgcggccgg gaacggtgca ttggaatcga tgactctctt aggtagcctt 1080 gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag 1140gagatcaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac1200ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac tcccagttca 1260 attacagctc gccaccatgc ggttctccgc tcagctgctg ggccttctgg tgctgtggat 1320 tcccggcgtc tcgagatcta tcgatgcatg ccatggtacc aagcttgcca ccatgagcag 1380cagctcttgg ctgctgctga gcctggtggc cgtgacagcc gcccagagca ccatcgagga 1440gcaggccaag accttcctgg acaagttcaa ccacgaggcc gaggacctgt tctaccagag 1500cagcctggcc agctggaact acaacaccaa catcaccgag gagaacgtgc agaacatgaa 1560 caacgccggc gacaagtgga gcgccttcct gaaggagcag agcacactgg cccagatgta 1620ccccctgcag gagatccaga acctgaccgt gaagctgcag ctgcaggccc tgcagcagaa 1680cggcagcagc gtgctgagcg aggacaagag caagc ggctg aacaccatcc tgaacaccat 1740gtccaccatc tacagcaccg gcaaagtgtg caaccccgac aacccccagg agtgcctgct 1800gctggagccc ggcctgaacg agatcatggc caacagcctg gactacaacg agcggctgtg 1860ggcctgggag agctggcgga gcgaagtggg caagcagctg cggcccctgt acgaggagta 1920cgtggtgctg aagaacgaga tggccagggc caaccactac gaggactacg gcgactactg 1980gagaggcgac tacgaagtga acggcgtgga cggctacgac tacagcagag gccagctgat 2040cgaggacgtg gagcacacct tcgaggagat caagcctctg tacgagcacc tgcacgccta 2100cgtgcgggcc aagctgatga acgcctaccc cagctacatc agccccatcg gctgcctgcc 2160cgcccacctg ctgggcgaca tgtggggccg gttctggacc aacctgtaca gcctgaccgt 2220gcccttcggc cagaagccca acatcgacgt gaccgacgcc atggtggacc aggcctggga 2280cgcccagcgg atcttcaagg aggccgagaa gttcttcgtg agcgtgggcc tgcccaacat 2340gacccagggc ttttgggaga acagcatgct gaccgacccc ggcaatgtgc agaaggccgt 2400gtgccacccc accgcctggg acctgggcaa gggcgacttc cggatcctga tgtgcaccaa 2460 agtgaccatg gacgacttcc tgaccgccca ccacgagatg ggccacatcc agtacgacat 2520ggcctacgcc gcccagccct tcctgctgcg gaacggcgcc tt aacgagggct cacgaggc 2580cgtgggcgag atcatgagcc tgagcgccgc cacccccaag cacctgaaga gcatcggcct 2640gctgagcccc gacttccagg aggacaacga gaccgagatc aacttcctgc tgaagcaggc 2700cctgaccatc gtgggcaccc tgcccttcac ctacatgctg gagaagtggc ggtggatggt 2760gtttaagggc gagatcccca aggaccagtg gatgaagaag tggtgggaga tgaagcggga 2820gatcgtgggc gtggtggagc ccgtgcccca cgacgagacc tactgcgacc ccgccagcct 2880gttccacgtg agcaacgact actccttcat ccggtactac acccggaccc tgtaccagtt 2940 ccagttccag gaggccctgt gccaggccgc caagcacgag ggccccctgc acaagtgcga 3000catcagcaac agcaccgagg ccggacagaa actgttcaac atgctgcggc tgggcaagag 3060cgagccctgg accctggccc tggagaatgt ggtgggcgcc aagaacatga atgtgcgccc 3120cctgctgaac tacttcgagc ccctgttcac ctggctgaag gaccagaaca agaacagctt 3180cgtgggctgg agcaccgact ggagccccta cgccgaccag agcatcaaag tgcggatcag 3240cctgaagagc gccctgggcg acaaggccta cgagtggaac gacaacgaga tgtacctgtt 3300ccggagcagc gtggcctatg ccatgcggca gtacttcctg aaagtgaaga accagatgat 3360cctgttcggc gaggaggacg tgagagtggc caacctgaag ccccggatca gcttcaactt 3420cttcgt GACC gcccccaaga acgtgagcga catcatcccc cggaccgaag tggagaaggc 3480catccggatg agccggagcc ggatcaacga cgccttccgg ctgaacgaca actccctgga 3540gttcctgggc atccagccca ccctgggccc tcccaaccag ccccccgtga gcatctggct 3600gatcgtgttt ggcgtggtga tgggcgtgat cgtggtggga atcgtgatcc tgatcttcac 3660 cggcatccgg gaccggaaga agaagaacaa ggcccggagc ggcgagaacc cctacgccag 3720catcgatatc agcaagggcg agaacaaccc cggcttccag aacaccgacg acgtgcagac 3780cagcttctga taatctagaa cgagctcgaa ttcgaagctt ctgcagacgc gtcgacgtca 3840tatggatccg atatcgccgt ggcggccgca ggccagccca aggccgctcc cagcgtgacc 3900ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctcatc 3960agcgacttct accctggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 4020gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 4080tacctgagcc tcacccccga gcagtggaag agccaccgga gctacagctg ccaggtgacc 4140cacgagggca gcaccgtgga gaagaccgtg gcccccaccg agtgcagcta atagacttaa 4200gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 4260 cctcccccgt c gccttccttg cctggaag gtgccactcc cactgtcctt tcctaataaa 4320 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 4380 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 4440gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc 4500cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4560ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4620 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4680 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4740cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4800 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4860 ttttggccat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4920 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 4980 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 5040ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc5100gcccctaact ccgcccatcc cgcccctaac CBGT cccagt tccgcccatt ctccgcccca5160tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt 5220 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc 5280ttgtatatcc attttcggat ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac 5340 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 5400 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 5460ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 5520 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta 5580ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 5640cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca 5700gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 5760tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 5820 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 5880cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 5940ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattcc c aatacgaggt 6000cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 6060 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 6120tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 6180 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 6240cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 6300aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga 6360ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 6420 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 6480 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 6540 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 6600 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 6660 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 6720ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 6780 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 6840cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 6900 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 6960aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 7020aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 7080tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 7140ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 7200 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 7260 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 7320 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 7380 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 7440agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 7500 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 7560aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 7620atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 7680 acgttaag gg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 7740 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 7800 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 7860 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 7920 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 7980gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 8040tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 8100 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 8160 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 8220tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 8280 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 8340 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 8400 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 8460 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 8520 ttcgatgtaa cccact cgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 8580 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 8640gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 8700 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 8760 gcgcacattt ccccgaaaag tgccacctga cg 8792REFERÊNCIASAir MA (1981), Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus. Proc Natl Acad Sci USA 78(12):7639-7643 .De Kruif J et al. (1995), Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library. Proc Natl Acad Sci USA 92:3938.Ferguson et al., (2003), Nature 422:428-443.Fouchier AM et al. (2005), Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79(5):2814-2822.The World Health Organization Global Influenza Program Surveillance Network (2005), Evolution of H5N1 Avian Influenza Viruses in Asia. Emerg Infect Dis 11:1515-1521.

权利要求:
Claims (5)
[0001]
1. Isolated antibody capable of specifically binding an epitope in the major region of the hemagglutinin (HA) protein of influenza A virus subtypes of phylogenetic group 1, and influenza A virus subtypes of phylogenetic group 2, and capable of neutralizing at least one one or more influenza A virus group 1 subtypes selected from the group consisting of influenza A virus comprising HA of subtype H1, H2, H5, H6, H8, H9 and H11, and at least one or more subtypes of influenza A group 2, selected from the group consisting of influenza A viruses comprising HA of subtype H3, H4, H7 and H10, characterized in that the antibody is also capable of specifically binding to the hemagglutinin (HA) protein of influenza B virus subtypes , wherein the antibody is selected from the group consisting of: an antibody comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, and a the light chain CDR1 region of SEQ ID NO: 146, a light chain CDR2 region of SEQ ID NO: 174, and a light chain CDR3 region of SEQ ID NO: 147, a heavy chain CDR1 region of SEQ ID NO :139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 145, and a light chain CDR1 region of SEQ ID NO: 148, a light chain CDR2 region of SEQ ID NO: 149, and a light chain CDR3 region of SEQ ID NO: 150, a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a Heavy chain CDR3 of SEQ ID NO: 145, and a light chain CDR1 region of SEQ ID NO: 142, a light chain CDR2 region of SEQ ID NO: 143, and a light chain CDR3 region of SEQ ID NO: 173, an antibody comprising a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, and a CDR1 region light chain of SEQ ID NO: 148, a light chain CDR2 region of SEQ ID NO: 149, and a light chain CDR3 region of SEQ ID NO: 150, a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, and a light chain CDR1 region of SEQ ID NO: 156, a light chain CDR2 region of SEQ ID NO: 157, and a light chain CDR3 region of SEQ ID NO: 158, a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, and a light chain CDR1 region of SEQ ID NO: 171, a light chain CDR2 region of SEQ ID NO: 164, and a light chain CDR3 region of SEQ ID NO: 172, and a heavy chain CDR1 region of SEQ ID NO: 139, a heavy chain CDR2 region of SEQ ID NO: 134, and a heavy chain CDR3 region of SEQ ID NO: 152, and a light chain CDR1 region of SEQ ID NO: 142, a CDR2 region light chain of SEQ ID NO: 143, and a light chain CDR3 region of SEQ ID NO: 144.
[0002]
2. Antibody, according to claim 1, characterized in that the antibody does not have any hemagglutination inhibition activity.
[0003]
3. Antibody according to claim 1 or 2, characterized in that it is for use as a medicine.
[0004]
4. Antibody, according to claim 3, characterized in that it is for the diagnostic, therapeutic and/or prophylactic treatment of influenza infection.
[0005]
5. Pharmaceutical composition, characterized in that it comprises an antibody, as defined in claim 1 or 2, and a pharmaceutically acceptable excipient.

类似技术:

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US11175293B1|2021-01-04|2021-11-16|University Of Utah Research Foundation|Rapid assay for detection of SARS-CoV-2 antibodies|

法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-08-21| B25D| Requested change of name of applicant approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V (NL) |

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2019-12-17| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-03-31| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-29| B25G| Requested change of headquarter approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V. (NL) |

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2021-09-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 12/07/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161572417P| true| 2011-07-14|2011-07-14|

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EP11173953.8|2011-07-14|

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EP11173953|2011-07-14|

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US61/572,417|2011-07-14|

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PCT/EP2012/063637|WO2013007770A1|2011-07-14|2012-07-12|Human binding molecules capable of neutralizing influenza a viruses of phylogenetic group 1 and phylogenetic group 2 and influenza b viruses|

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