TW200837079A - Recombinant polyclonal antibody for treatment of respiratory syncytial virus infections - Google Patents

Abstract

Disclosed are novel polyclonal antibodies, which target respiratory syncyticilal virus (RSV), and novel high affinity antibody molecules reactive with RSV. The polyclonal antibodies may comprise antibody molecules which are reactive with both RSV protein F and RSV protein G, and preferably the polyclonal antibodies target a variety of epitopes on these proteins. The single antibody molecules of the invention are shown to exhibit affinities which provide for dissocation constants as low as in the picomolar range. Also disclosed are methods of producing the antibodies of the invention as well as methods of their use in treatment for RSV infection.

Description

200837079 IX. INSTRUCTIONS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to a recombinant multi-body antibody for preventing, treating or ameliorating one or more symptoms associated with viral infection of a respiratory fused cell. The present invention also relates to a multi-strain cell line producing an anti-rsv recombinant multi-strain antibody (anti-RSV rpAb). Furthermore, this application describes the use of diagnostic and pharmacological compositions comprising an anti-RSV rpAb and for preventing, treating or ameliorating one or more symptoms associated with Rsv infection. [Prior Art] Respiratory system fusion cell virus (RSV) is the main cause of respiratory diseases in infants and young children. Premature babies and children with potential health problems, such as chronic lung disease or congenital heart disease, are at great risk of developing serious diseases such as bronchiolitis and pneumonia after RSV infection. Recently, Rsv has also been regarded as an important pathogen for certain dangerous adults, such as adults with immune dysfunction, especially bone marrow transplant recipients, the elderly and chronic lung disease patients. The human RSV is a member of the subfamily of the paramyxovirus () subfamily, and is present in the eight and six subtypes. Rsv is a kind of envelope, non-segmented, negative ^ RNA, virus. The viral genome is encoded into; 11 proteins' three of which are envelope-associated proteins, F (fusion listener proteins), G (receptor-binding glycoproteins), and small hydrophobic proteins. Envelope proteins are present on the surface of the virus and, to some extent, on the surface of infected cells. ...promotes fusion of the virus with the cell membrane, thereby allowing the viral RNA to penetrate into the cytoplasm. The qF protein line consists of two subunits Fi and F2 linked by a disulfide bond 5 200837079, which has 574 amine groups. Proteolytic cleavage of acid inactive, N-glycosylation precursors. The G protein is a π-type transmembrane protein having 289-299 amino acids (depending on the virus strain). The precursor form is 32 kDa, which matures to a protein of 80-90 kDa after the addition of both Ν_ and 〇_ __. The rsv G protein is responsible for attaching virions to target cells. In addition to the membrane-bound form of prion protein, a truncated, soluble form is also produced. It has been suggested that this function redirects the immune response away from viruses and infected cells. In addition, it has been shown that G proteins are associated with many pro-inflammatory (pr〇-inflammat〇ry) effects, such as chemokine modification and cytokine expression, as well as white blood cell recruitment. The SH protein is a protein having 64-65 amino acids, which is present in a very small amount on the surface of the purified RSV particles, but is abundantly expressed on the surface of the RSV-infected cells. The function of the sH protein has not been determined, but it is possible to help the viral protein to be transported via a high-growth complex (Golgi c〇mplex) (Rixon, 2004, J. Gen. Virol 85:1 153-1 165). The function of blocking the G and F proteins is related to the prevention of RSV infection. In the past few decades, prevention and treatment of RSV infection has received widespread attention, including vaccine development, antiviral compounds (Ribavirin approved for treatment), antisense drugs, RNA interference (RNAi) Techniques and antibody products such as immunoglobulins and monoclonal antibodies (reviewed in Magg〇n and Barik, 2004, Rev. med. Virol 14: 149-168) ° In these methods, intravenous immunization Globulin RSV-IVIG and the monoclonal antibody palivizumab () have been approved for RSV prevention in high-risk children. 6 200837079 However, immunoglobulin products such as RSV-IVIG (RespiGam) are known to have several disadvantages, such as low specific activity, resulting in the need for large injections, which is difficult for children with limited venous access due to previous intensive treatment. In addition, there is a risk of transmitting viral diseases from serum-derived immunoglobulin products, as well as differences between batches. Finally, because only about 8% of normal donors have sufficiently high RSV neutralizing antibody valence, it is difficult to obtain sufficient donors to meet the need to produce highly immunological RSV immunoglobulin products. Individual antibodies against F or G proteins have been shown to have in vitro neutralizing and in vivo prophylaxis (eg, Beeler and Coelingh 1989.

J. Virol 63: 2941-50; Garcia-Barreno et al. 1989. J. Virol 63: 925-32; Taylor et al. 1984. Immunology 52: 137-142; Walsh et al. 1984, Infection and Immunity 43:756 -758; US 5,842,307 and US 6,818,216). Today, the monoclonal antibody palivizumab has almost completely replaced the use of RSV-IVIG. Neutralization experiments showed that palivizumab and RSV-IVIG performed identically for RSV subtype B, but palivizumab performed better against subtype A (Johnson et al. 1997. J. Infect. Dis. 176) :1215-24.). However, although monoclonal antibodies have good neutralization and prophylaxis as indicated by products like palivizumab and Numax, this may also be associated with certain disadvantages caused by the nature of the RSV virus. There are two different antigenic groups or subtypes of RSV, a and b. Most RSV proteins are highly conserved between the two subpopulations, with the ρ protein showing 91% amino acid similarity. However, the G protein shows a wide range of sequence variability with only 53% amino acid similarity between 7 200837079 A and the B subpopulation (Sullender 2(8)〇. Clin.MiCr〇bi〇l. Rev 13:b 15) . Most proteins also show some limited intra-subgroup variation, except for the G protein, which is up to 20% in the subgroup A and up to 9% in the subgroup B at the amino acid level for the G protein. A and B virus subtypes are commonly transmitted in most RSV epidemics, where relative frequencies vary from year to year. Therefore, individual antibodies must be carefully selected so that they can neutralize both subtypes and intra-subtype variations.

In addition to the problems of diversity within the two RSV subtypes and subtypes, human RSV, like most Rsv viruses, has the ability to undergo rapid mutations under selection pressure. It has been documented that RSV escaped dog variants were selected using mAb tubes (e.g., Garcia-Barreno et al. 1989 J Virol 63:925-32). Importantly, palivizumab has recently been found to select escape mutants in vitro and in vivo, and some isolated mutants are completely resistant to palivizumab in cotton rats (ζι^〇 and

Sullender 2005· J. Virol. 79:3962-8 and Zhao et al. 2004 JL Infect. Dis. 190:1941-6·). In addition, as demonstrated by the failure of murine antibodies of the origin of palivizumab to neutralize a clinical isolate (Βαία and

Coelingh 1989· J. Virol 63: 2941_50), there may also be wild-type RSV strains that are resistant to palivizumab. In addition, a significantly resistant virus has been identified following the prevention of palivizumab in immunocompetent cotton rats (j〇hnson et al. i997·J Infect Dis 6:12i5_24). Thus, under certain conditions, the use of a single, monospecific antibody may not be suitable or sufficient to treat RSV disease because of the presence of escape mutants or the generation of escape mutants over time due to treatment. 8 200837079 Another consideration related to the use of RSV-IVIG and palivizumab is the dose of the placebo/oral therapy product. Serum concentrations greater than 3 已 have been shown to be necessary to reduce i. 00-fold reduction in lung RSV replication in infected cotton and mouse models. For RSV-IVIG, intravenous administration of a monthly dose of 750 mg total protein/kg effectively reduced the incidence of RSV hospitalization in high-risk children, whereas for palivizumab, a monthly dose of 15 mg/kg proved effective. However, multiple intravenous or intramuscular high-dose administrations are inconvenient for patients and prevent these products from being widely used to prevent and treat a large number of adults at risk of RSV infection. Thus 'there is a need for an antibody product that is highly dependent on donor availability and that immunospecifically binds to one or more of the RSV antigens that cover subtypes A and b and any escape mutants caused by viral mutations' It has improved pharmacokinetic characteristics, and thus has overall improved therapeutic characteristics' and therefore requires less dosing frequency and/or lower dose administration. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a highly potent alternative anti-RSV immunoglobulin product which is recombinantly produced and exhibits reactivity to subtypes A and B of respiratory fused cellular viruses and is The activity of multiple epitopes on at least one of the major surface antigens limits the likelihood of evading mutations. It is also an object of the present invention to provide novel human anti-rSV antibody molecules and their bamboo organisms which exhibit improved characteristics over existing monoclonal antibodies against R S V antibodies and antibody derivatives. It is expected that multiple antibody sets targeting multiple epitopes on RSV 9 200837079 will minimize the development of escape mutants and may also provide protection against diverse, naturally transmitted viruses. In contrast to serum-derived RSV-IVIG, the polyclonal antibodies of the invention do not contain antibody molecules that bind to non-RSV antigens. The present invention provides a plurality of strains of anti-RSV antibodies. Preferably, the plurality of anti-RSV anti-systems are obtained from cells which do not naturally produce antibodies. This antibody is referred to as a recombinant multi-body antibody (rpAb). The anti-rSV rpAb of the invention is directed against multiple epitopes on the ρ or G protein. Particularly preferred are anti-V rpAbs directed against a plurality of epitopes on both G and F proteins. Preferably, the anti-rsV rpAb encompasses G protein epitopes belonging to a conserved population and possibly to a subtype-specific population and a strain-specific population. Furthermore, an antibody having reactivity against a third envelope protein (i.e., a small hydrophobic (SH) protein) is a desired component of the anti-RSV rpAb of the present invention. Furthermore, the present invention provides pharmaceutical compositions in which the active ingredient is an anti-RSV multi-strain antibody, and the use of such compositions in preventing, ameliorating or treating RSV infection. The invention further provides a procedure for isolating the original VH and VL gene pairs from the chimeric individuals and producing an antibody that retains the original pair to reflect a humoral immune response elicited by RSV infection.

It can also induce the induction of immune effect mechanisms. The antibody molecule is capable of binding to or reacting with a specific antigen base. Individual antibody molecules are generally considered to be monospecific, and the composition of the antibody molecule can be either single (ie, composed of the same antibody molecule) or multiple, ie, identical antigen or unique,

5 different antibody molecule composition of the same or different epitopes in the original reaction). Each antibody molecule has a unique structure that allows it to specifically bind to its corresponding antigen, and all natural antibody molecules have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known as immunoglobulins. The term anti-system as used herein is used in the broadest sense and encompasses antibodies, chimeric antibodies, humanized antibodies, fully human antibodies and single-chain antibodies, as well as binding fragments of antibodies, such as Fab, Fv# segments or Heart fragments, as well as multimeric forms, such as dimeric IgA molecules or five gM. In some instances, the present application uses the term "synthetic or semi-synthetic antibody analogue", which specifically refers to the non-natural production that exhibits antibody characteristics that exhibit specific binding to RSV antigens. Molecules and including CDRs derived from naturally occurring antibodies, such as represented by scFv fragments, diabo〇dies, and the like, but may, for example, be engineered to include anti-RS V as described herein. A naturally occurring antibody that is a CDR of an antibody molecule (e.g., by a grafting technique known in the art), for example, such an antibody analog can comprise an antibody molecule disclosed herein that is incorporated into another animal species. Or incorporation of CDRs in different antibody isotypes or species of the same species. The term "anti-RSV recombinant polyclonal antibody" or "anti-RS V rpAb" describes a composition of recombinantly produced antibody molecules in which individual members are able to fuse with the respiratory system. At least one epitope on the cellular virus 11 200837079, and wherein the plurality of compositions are generally capable of neutralizing RSV. Preferably, the anti-RSVrpAb composition neutralizes both the Rsv subtype human and B. Even more preferably, the anti-RSV rpAb additionally comprises binding reactivity to G and ρ proteins. Preferably, the composition is produced from a single multi-plant cell line. <cognate VH and VL coding pair" describes a pair of Vh and VIII original coding sequences contained in or derived from the same cell. Thus, homologous, VH and VL pairs represent % and Vl pairs originally present in the donor from which the cell is derived. The term "an antib〇dy expressed ^ VL cochng pair" means that the antibody or antibody fragment is produced by a vector, a plastid or an analog containing the vH& vL coding sequence. When the homologous Vh and the eight-coupling pair are expressed, both retain the binding affinity and specificity of the antibody originally expressed by the cell from which it is derived in the form of an intact antibody or a stable fragment thereof. Libraries of homologous pairs are also known as repen〇ires or collections and can be stored separately or collectively. The term "a distinct member 〇fa recombinant p〇lyclonai antib〇dy" means an individual antibody molecule of a recombinant multi-drug antibody group comprising one or more stretches in the variable region (stretch ) 'characterized by the fact that compared to other individual members of the multi-plant protein, the monthly female-based sequence is different. These stretches are especially located in the cDR1, cdR2 and CDR3 regions. The "epitope" is generally used to describe a part of a caravan macromolecule (e.g., an antigen or antigenic site) having antigen or immunogenic activity in an animal (a ritual animal) and an optimal human. It has the activity of immunogen 12 200837079, the original determinant, which is the larger molecule of the antibody that causes the antibody reaction in animals. An antigenic epitope having a portion of a larger molecule to which an antibody immunospecifically binds, such as by any method well known in the art, for example, by immunoassay as described herein. The 疋 antigenic epitope is not necessarily immunogenic. The antigen is a substance or a substance in which the body fragment is immunospecifically bound, such as a toxin, a disease, a bacterium, a protein, or a DNA. An antigen or antigenic site often has a fi1 epitope, unless it is very small, and it is often capable of stimulating the location of the immunoreactive dioptic epitope, and is resistant to binding to different antigens on the same antigen. It has a different effect on the activity of the antigen to which it binds. An antibody that binds to an epitope at the active site of the antigen may completely block the function of the antigen, while another antibody that binds to a different epitope may have no or only a minor effect on the antigen. However, such antibodies may still activate complement and thereby cause elimination of the antigen, and may (inducing synergy when combined with one or more antibodies that bind to different epitopes on the same antigen. In the present invention, epitopes The macromolecule which is a part thereof is preferably a part of the RSV polypeptide. The antigen of the present invention is preferably an RSV-related protein, a polypeptide or a fragment thereof, and the antibody or antibody fragment is specifically immunologically bound thereto. The RSV-related antigen may also be an RSV. An analog or derivative of a polypeptide or fragment thereof, and an antibody or antibody fragment thereof immunospecifically binds thereto. For example, the term "fully human" is used in connection with DNA, RNA or protein sequences and is described as 98 to ι. 〇〇〇/〇 human sequence. The term "imiminogiobulin" is generally used as the collective name for a mixture of antibodies found in blood 13 200837079 fluid or serum, but may also be used to indicate antibodies from other sources. Mixture. When the term "mirrors the humoral immune response" is used in association with multiple antibodies A nucleic acid sequence encoding an individual antibody member is derived from an antibody composition that produces an increased frequency of plasma cells against an RSV-specific antibody. The donor may be recovering from RSV infection and has been in intimate contact with the infected individual. , or have been vaccinated with RSV (for examples of RSV vaccines, see, for example, Magg0n and Barik, 2004, Rev. med. Virol. 14: 149_168). To reflect the affinity of antibodies produced in donors after infection or work. And specificity, the sequence encoding the variable heavy chain (VH) and the variable light chain (vL) should be retained in the pair or combination (homologous pair) of the gene originally present in the donor when it is isolated. The diversity of humoral immune responses in the body, based on the screening program to select all sequences encoding the antibodies that bind to RSV. The variable regions, especially the diversity of the cdr regions, are also isolated for the VH and VL families. The sequence is analyzed. Based on these analyses, a population of homologous pairs representing the overall diversity of the RSV binding antibody is selected. The multi-strain antibody typically has at least 5, 1 〇, 2 〇, 3 〇, 40, 50, 1 〇〇, 1〇〇〇 or ι〇4 The same member. If the administration of the composition can be tolerated by the recipient patient, it is said to be "pharmacologically acceptable", and of course also applies to the excipients and vehicles as part of the composition ( Vehicle, carrier and diluent. The term "polyclonal antib〇dy" describes a composition of different (diverse) antibody molecules that are capable of reacting with different antigens on the same or different antigens. The epitope/epitope binds or reacts with each of the antibodies in the composition to react with a particular epitope. Typically, the variability of multiple antibodies is in the so-called multi-strain antibody variable region, particularly in the CDR1, CDR2 and CDR3 regions. In the present invention, a plurality of antibodies may be produced together from a plurality of cell lines, or may be a mixture of different polyclonal antibodies. Mixtures of monoclonal antibodies are not considered as multiple antibodies as such, as they are produced in individual batches and are not necessarily produced by the same cell line, which will cause, for example, post-translational modification differences. However, if a mixture of monoclonal antibodies provides the same antigen/antigenic coverage as the polyclonal antibodies of the present invention, it is considered to be the equivalent of multiple antibodies. When it is stated that a member of a plurality of antibodies specifically binds to an antigen/antigen site/antigenic epitope or has specific reactivity against the latter, it means that the binding constant is less than 1 〇〇 nM, preferably lower than i. 〇nM, even better than 1 nM. The term "recombinant antibody" is used to describe an antibody molecule or molecules expressed by a cell or cell line transfected with an expression vector comprising a coding sequence for an antibody that is not naturally associated with the cell. If the antibody molecules in the recombinant antibody composition are diverse or different, the term "recombinant polyclonal antibody (rpAb)" is applied according to the definition of a plurality of antibodies. The term "recombinant polyclonal ceu line" or "polyclonal cell line" refers to a protein-expressing cell transfected with a spectrum of a variant nucleic acid sequence (eg, a nucleic acid sequence encoding an antibody). The mixture/population 'these sequences are not naturally associated with the transfected cells. Preferably, the transfection is performed such that the individual cells of the recombinant strain 15 200837079 each have a transcriptional active copy of a single different nucleic acid sequence of interest encoding a member of the recombinant polyclonal antibody of interest. Even more preferably, a single copy of a different nucleic acid sequence is integrated only at a specific site within the genome. The cell lines that make up the recombinant multi-cell line are selected for their ability to retain an integrated copy of the different nucleic acid sequences of interest, e.g., by antibiotic selection. The cells which can constitute the plurality of cell lines can be, for example, fine sputum, true scorpion, eukaryotic cells, such as yeast, insect cells, plant cells, mammalian cells, especially immortalized mammalian cells, such as CH 〇 fine (cell, COS) Cells, BHK cells, myeloma cells (eg, Sp2/〇 cells, NSO), NIH3T3, YB2/0, and immortalized human cells, such as cells, HEK 293 cells, or PER.C6. The term "codes VH and VL pairs The sequence encoding VH and VL pairs or VH and & encoding (seqUence pair S) means that each molecule contains a sequence encoding the expression of a variable heavy chain and a variable light chain. a nucleic acid molecule such that if a human (suitable promoter and/or IRES region is present and operably linked to the sequence, the linkages can be expressed in pairs by the nucleic acid molecule. The nucleic acid molecule can also encode a heavy bond and/or a light chain a portion of the constant region or a complete constant region such that if a human promoter and/or IRES region is present and operably linked to the sequence, the Fab fragment, full length antibody or other antibody fragment is present. When the amount of the antibody administered is physiologically sufficient, for example, to prevent or attenuate RSV infection in an animal or human, it is said to be administered in a "therapeutically effective amount". 16 200837079 [Embodiment] Multi-Functional Antibody Compositions The multi-strain anti-system of the present invention consists of a number of different antibody molecules in the same composition. Each molecule is selected based on its ability to bind to RSV-associated antigens. The binding activity of the combined binding activity of different antibody molecules of the antibody composition. The anti-RSV multi-strain antibody of the present invention preferably comprises a comprehensive binding activity against both G and F proteins and even better against multiple epitopes to be developed The risk of escaping mutants is minimized and the highest possible neutralizing capacity is achieved. At least five major antigenic sites recognized by neutralizing antibodies have been identified on the F protein (Lopez et al. 1998. J. Virol. 72:6922- 8) All antigenic sites have been mapped to the F! chain and include sites I, π, iv, V and VI, where positions j and η can also be referred to as b and a, respectively. Located in the anti-protease region of the N-terminal fragment, and positions iv, v and vi are located at the C-terminus of the cysteine-rich region of the protein. Site I is located in the middle of the cysteine cluster. The other antigenic site is site C, and the epitope F2 including amino acid positions 241 and 242 is located in this site C. In addition, a single antibody binds to an antigenic site called F1, and the antigenic site The spots contain epitopes called Fla, Flb and Flc. Currently, this antigenic site has not been mapped to a specific site on the F protein. Most of these sites/antigen thiol groups cause extensive neutralizing antibodies, but some antibodies specific for antigenic site 1 have been shown to have subtype A specificity. The antibodies with site I also have a marginal effect on virus neutralization (marginai). As judged by the escape mutation of the selected amino acid position 272 (Zhao et al. 17 200837079 2004. J. Infect. Dis. 19〇: 1941_6), the epitope recognized by palivizumab is located at the antigenic site. „中. In addition, three types of epitopes have been identified on the G protein: 丨j % ^ 1 J-protected epitope, which exists in

a virus-specific group of epitopes, which are present in all viruses of the same-subtype; called strain-specific or variable epitopes, which exist only in the same-subtype In the subgroup of virus strains. Conservative epitopes and group-specific epitopes have been mapped to the central portion of the G protein's four semi-deaminating acids (amino acid residues 173, 176, 182 and 186 containing the same sequence in all human RSV isolates) Clusters and short amino acid fragments (residues 164-176). The cysteine cluster is maintained by a disulfide bond between position 173 "83 and 176-182 and constitutes the G protein cysteine rich region (GCRR) in the range of amino acid residues 171_丨87. The central portion, whereby GCRR overlaps with the conserved regions of the 13 amino acids. G glycoproteins appear to play an important role in inducing protective immunity and pathogenicity of the disease. For example, a 'study on mice has shown G glycoprotein The Th2 CD4+ T cell response is initiated, which is characterized by the production of IL_4, IL_5, IL-13 and pulmonary eosinophilia. The eosinophil recruitment and activation system is promoted by several factors, such as IL-4 and IL-5. The expression of RS v prion protein during acute infection in mice has been associated with improved innate immune responses characterized by decreased expression of Th 1 cytokines (eg, IL 2 and gamma interferon) and changes in chemokine mRNA expression. (eg, ΜΙΡ-1 α, ΜΙΡ-1 β, ΜΙΡ-2, IP-10, MCP-1) and reduced sputum cells transported to infected lungs. In particular, GCRR has been shown to play an important role in regulating congenital inflammatory responses, Which may delay RSV clearing (p〇iack Et al. 2005. PNAS 102:8996- 18 200837079 9001). GCRR contains a CX3C motif at amino acid positions 182 to 186. It has been shown that a decrease in respiratory rate in RSV-infected mice is associated with this CX3C motif 'because Antibodies against this motif prevent a decrease in respiration rate (Tripp et al. 2003 J. Virol. 77: 6580-6584 and US 2004/0009177 (Shenzhen Brothers 1 0/420, 3 87)). The virus-specific epitope has been localized to the N-terminus of the variable region of the cysteine cluster in the extracellular domain of the G protein, but the virus-specific epitope is preferentially located in the G-peptide, and the C-terminus Within one subsection (Martinez et al. 1997. J. Gen. Virol. 78:2419-29). Figure 1 shows G from L〇ng strain (subtype A) and 18537 strain (subtype B) Protein alignment, indicating the regions of the g protein. Typically, a single anti-G protein antibody has a marginal effect on Rsv neutralization 'however' has been reported to enhance the in vitro and in vivo RSV neutralization of a mixture of individual anti-G protein antibodies. (Walsh et al. 989 [Μ νϋ 70:2953-61 and Martinez and trace (7) ΐ998 79:2215-20). Although iron, forgotten ,, no small part of one of the diseases and still resist, but a single strain the different anti-G antibodies bind antigen ^ + ^ j when decision Cloth group, apparently to reach a significant role recorded to find the antibody composition.卜μ冰' has been,,,, and has been shown to have a combination of two different anti-F-chain antibodies with different epitope specificities and an anti-F antibody combined with an anti-G antibody. And the effect of Q Anderson et al. iqSq τ ” 合 单 抗体 抗体 此 ^ ^ ^ ^ ^ 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 似乎 似乎 似乎 似乎 似乎 似乎 似乎 似乎The individual special $ from, for example, hunting hinders the active site. The reason for this is that the mechanism of RSV neutralization is not yet understood to be more complex and not fully understood. Many different epitopes identified on the protein alone and 19 200837079 G (including Conservative, subtype-specific, and strain-specific epitopes, as well as the potential for escape mutants, suggesting that a wide range of antibody specificities are required to meet all neutralization mechanisms that play a role in preventing RSV infection. It is extremely difficult (in a reasonable manner) to select new virus strains that are capable of preventing RSV strains of subtypes A and B, as well as escape mutants and from RSV strains known today. RSV-infected monoclonal antibody mixture. One aspect of the present invention provides a multi-strain anti-RSv antibody having abundant diversity and extensive anti-RSV specificity. The plurality of anti-RSv antibodies of the present invention are independent of the donor at the moment of production. Availability and inter-assay variation is much lower than that observed for donor-derived anti-RSV immunoglobulin products (eg, RS v iviG ). In multiple anti-RSV antibodies of the invention, all individual antibody members are able to bind Rsv-related proteins and multiple antibodies can neutralize Rsv subtypes A and B. The different antibodies of multiple antibodies preferably bind to epitopes that are not bound by any other member of the anti-moon bean. Antibodies will bind to RSV antigens in a multivalent manner, which often leads to synergistic neutralization, improved phagocytosis of infected cells by macrophages, improved antibody-dependent cellular cytotoxicity (ADCC) against infected cells, and increased complementation. In addition, unlike the case of RS V IVIG (wherein a dose of 750 mg total protein/kg is required), the polyclonal antibodies of the present invention are not diluted by non-binding proteins.The percentage of RSV-specific antibodies in 750 mg of total protein is unknown, but it is unlikely to be greater than about 1%, and it is likely that the in vitro efficacy of the sigma seleparizumab is better than that of the RSV IVIG. b 冋25-30 times (j〇hnson et al. 1997.JJnfect.Dis. 20 200837079 176·1=15·24) when making a complement to k by reducing the specific activity of reading mG □ this ' # RSV_IVIG The immunoglobulin molecule contained is only specific for the loop RSV, and the active dose of the rsv_ivig multi-strain antibody is only g kg which is lower than the active dose of the monoclonal antibody palivizumab. For these reasons, it is expected that the recombinant multi-strain Rsv-specific antibody of the present invention will be significantly more potent than the monoclonal antibody, and thus A will be more likely to be administered with an effective dose of palivizumab and an effective dose of RSV IVIG. A small amount of multiple antibodies of the invention. Therefore, it is considered that the plurality of anti-RSV antibodies of the present invention are suitable for prevention and/or high-risk adults, especially bone marrow transplant recipients, elderly people and chronic lung disease patients. Another advantage of the multi-strain anti-RSV antibodies of the present invention is that the concentration of individual antibody members is significantly lower than the concentration of monoclonal antibodies (even if the same is used), so individual antibodies will be in the immune system of the individual being treated. The likelihood of being identified as a foreign object is reduced, even if the individual antibodies are eliminated by the patient's immune system 'however, because the remaining antibody members remain unaffected', this may not affect the neutralizing ability or clearance rate of multiple anti-RSV antibodies. A specific embodiment of the present invention is a multi-strain anti-RSV antibody capable of neutralizing RSV subtypes A and B, and wherein the multi-strain antibody comprises different antibody members, and the members specifically bind at least one Rsv envelope protein together At least two different epitopes on the substrate. Preferably, the F protein line is specifically bound by at least three different antibody members, and the epitopes are preferably located at different antigenic sites. Another embodiment of the invention is a recombinant multi-strain anti-RSV antibody capable of neutralizing RSV subtypes A and B, and wherein the multi-strain antibody comprises a different antibody 21 200837079 member, the members together providing for at least two RSV envelopes Protein specific reactivity. The two envelope proteins may be selected from the group consisting of Rsv G protein, rsv F protein, and RSV SH protein. Preferably, the plurality of anti-Rsv antibodies of the invention comprise anti-G and anti-F reactivity. The anti-G and anti-resistance of the multi-drug antibody? Preferably, the reactivity is comprised of at least two different anti-G antibodies and at least one different anti-F antibody. Preferably, at least three different antibodies bind to different epitopes to cover at least three different epitopes, and the antibodies together are capable of neutralizing RSV subtype A and subtype B strains equally well. Even more preferably, the anti-G and anti-F reactivity of the plurality of anti-RSV antibodies of the present invention consists of any combination of anti-G and anti-F reactivity described below. Most preferably, the plurality of anti-RSV anti-systems of the invention consist of anti-G and anti-F reactivity against all antigenic sites/antigenic groups mentioned below. In order to obtain the broadest possible specificity of the plurality of anti-RSV antibodies of the invention, more than one antibody is required to cover one or more, preferably all, antigenic sites. Thus, several epitopes on the same antigen or antigenic site are preferably bound by different members of the polyclonal antibodies of the invention. For the anti-G activity of a plurality of anti-RSV antibodies of the present invention, this activity is preferably directed to a conserved epitope. Even more preferably, the anti-G activity is comprised of a first anti-G antibody capable of specifically binding to a conserved epitope on the G protein and a second capable of specifically binding to a G protein cysteine rich region (GCRR). Anti-G antibody composition. The plurality of anti-Rsv antibodies preferably comprise at least two different anti-G proteins, wherein at least one of the first antibodies is capable of specifically binding to a conserved epitope on the G protein, and at least one of the second antibodies is capable of specifically binding to a different conserved antigen A determinant-based group-specific epitope recognized by subtype A or 22 200837079 subtype B. Preferably, the plurality of antibodies comprise at least two different anti-G proteins, wherein the first antibody is capable of specifically binding to a conserved epitope on the G protein, and the second antibody is capable of specifically binding to the G protein of subtype A and The third antibody is capable of specifically binding to the G protein of subtype B. The G-protein cysteine-rich region (GCRR) partially overlaps with the region of the 丨3 amino acids upstream of the epitope and the region where the group-specific epitope is located. Therefore, if an antibody capable of specifically binding a conserved epitope and an antigenic determinant recognized by a group-specific antibody are located in the GCRR, the antibodies can bind to the GCRR. Preferably, at least one of the different antibodies that are specifically bound to a conserved epitope or a virus-specific epitope also recognizes the GCRR. From the standpoint of virus neutralization, antibodies that bind to the CX3C motif of GCRR are particularly preferred. However, antibodies that bind to CX3C motifs may also bind to many other unrelated human antigens, such as Frautalkin(R) and other human a% chemotactics, and thus produce undesirable side effects, which means that a reasonable method should be tested. Cross-activity of such antibodies (eg, as evidenced by certain antibodies in the Examples and subsequent testing of the same antibodies in a suitable model system. In any event, it will always be necessary to test a particular drug in a clinical trial, such as an antibody of the invention Then, it can be established that the side effects are not present, less or at least two days. In addition to the conservative and group-specific anti-G activity, additional anti-G activity against the terry epitope of the virus strain can also be included in In the present invention, (d) is directed to a virus strain which has been caused to have a (four) sense in the last five years: sex 2: strain of the genus: the virus-specific specific epitope of the strain. In the present invention, Viral strain-specific epitope 23 200837079 is understood to be an epitope present only on a small number of RSV strains. Add group-specific and/or strain-specific anti-G resistance Additional diversity may be provided for the anti-RSV antibodies of the invention, and synergistic effects may be induced when combined with antibodies active against the conserved regions of the G protein. Preferably, the anti-G antibodies of the invention directly neutralize RSV, preventing viral entry. Cells, preventing cell migration, inhibiting inflammatory responses and/or preventing formation of fused cells. In terms of anti-F reactivity of a plurality of anti-RSV antibodies of the present invention, the reactivity is preferably directed against antigenic sites I, II, IV, At least one epitope on one or more of V, VI, C or F 1. In other embodiments of the invention, different antibodies of the plurality of anti-RSV antibodies of the invention cover at least two, three, Four, five, six or all of these antigenic sites/antigenic determinants. Preferably, the anti-F antibodies of the invention directly neutralize RSV and/or prevent viral entry into cells and/or prevent formation of fused cells. In the multi-strain anti-RSV antibody composition of the present invention, when the composition does not contain binding reactivity against all antigenic sites on the F protein, it is preferred to have at least one antigen that specifically binds to the antigenic site. base Different anti-F antibodies. Even more preferably, the site II-specific anti-F antibody binds to the same epitope or antigenic site as the antibody palivizumab. In addition to the site II-specific antibody, one or A variety of different site IV specific anti-F antibodies, which preferably bind to the same epitope as rSVF2-5. Subtype-specific anti-F antibodies are also known in the art. However, since F protein is The two subgroups a and b show 91% amino acid similarity' subtype-specific anti-F antibodies are less than anti-G antibodies. However, these viruses 24 200837079 strain-specific anti-F antibodies will help to obtain The broadest possible specificity, and therefore also the desired components of multiple strains of anti-RSV antibodies of the invention.

In addition to the RS V G and F protein antigens mentioned above, the RS virus exhibits a second envelope protein, a small hydrophobic (SH) protein. It has been shown that high immune sera generated against peptides derived from SH protein are not capable of neutralizing RSV in vitro (Akerlind-Stopner et al. 1993 J. Med. Vir〇l. 40:1 12-120). However, since this protein is mainly expressed on infected cells, our antibody to SH protein will act on fusion inhibition and may be involved in combating RS V infection in vivo. This view is supported by the fact that the replication capacity of the rS v strain lacking the sh gene in the upper respiratory tract is 10 times less (Bukreyev et al. 1997 J. Virol 71:8973-82).

Another specific example of this disclosure is the ability to neutralize RS V subtypes a and B and to contain anti-SH activity and anti-G or anti-! ^Reactive multi-strain anti-RSV antibodies. The SH protein is exposed to the cell surface at the c-terminus (subtype A/B) in the range of 41 to 64/65. Therefore, there is a need for anti-SH reactivity against antigenic epitopes located in this region. The c-terminus of the SH protein varies between subtypes VIII and B, and therefore it is necessary to include anti-SH reactivity against both subtypes A and B in the polyclonal antibodies of the present invention. This SH reactivity can be provided by at least two different anti-SH antibodies, wherein the first antibody is capable of specifically binding to sh subtype A and the purine antibody is capable of specifically binding to SH subtype B. In one embodiment of the invention, the plurality of anti-Rsv antibodies comprise specific reactivity against SH subtype A and/or B and specific reactivity against prion protein. The reactivity against the G protein may consist of any of the reactivity mentioned above. 25 200837079 In an alternative embodiment, the specific reactivity against SH subtype A and/or B can be combined with any of the anti-F reactivity described above to form a plurality of anti-RSV antibodies. In a preferred embodiment of the invention, the plurality of anti-RSV antibodies comprise reactivity against all three envelope proteins F, G and SH. The reactivity contained in the plurality of anti-RSV antibodies of the invention may constitute any possible combination of different antibodies having specific binding reactivity to the antigen/antigen site and/or epitope as outlined in Table 1, as long as The combination can neutralize the RSV subtypes A and B. Preferably, the combination contains activity against at least two RSV envelope proteins. Preferably, the individual different antibody members of the polyclonal antibodies of the invention themselves have neutralizing and/or anti-inflammatory properties. However, antibodies without such specific properties can also play a role in RSV clearance, for example, via complement activation. Table 1: Overview of RSV-associated antigens, antigenic sites and epitopes

Antigen antigen site / epitope F protein epitope I antigen site II antigen site IV antigen site V antigen site VI antigenic site C F1 epitope G protein conserved region (amino acid 164-176) Subtype A specific subtype B specific GCRR (amino acid 171-187) (conservative and strain specific) CX3C motif (amino acid 182-186) virus strain specific SH protein subtype A subtype B 26 200837079 Preferably, the multi-strain resistance system of the present invention is produced in a single batch or batches from a plurality of cell lines that do not naturally exhibit antibody molecules (also referred to as recombinant multi-body antibodies). In contrast to mixed monoclonal antibodies, one advantage of producing recombinant polyclonal antibodies is the ability to simultaneously produce an unlimited number of different antibody molecules (costs are similar to the cost of f-single-monoclonal antibodies). Therefore, it is possible to include antibodies reactive with a large number of RSV-associated antigens without significantly increasing the cost of the final product. Especially for the complex goal of RSV, when the biology is not fully understood, individual antibodies that have not been independently neutralized or defended against RSV may induce synergy when combined with other antibodies. It may be beneficial to include different antibodies in addition to the above antibodies in multiple antibody groups, and the only criterion is that individual antibodies bind to RSV-related proteins (e.g., by binding to cells infected with RSV). ). Preferably, all of the plurality of anti-RSV antibody compositions described above are recombinant multi-strain anti-Rsv antibody (anti-Rsy print) compositions. One method of obtaining a potentially relevant antibody that binds to an RSV target antigen that has not been verified as a related antigen (but possibly a related antigen) is to produce a multi-drug antibody composition that is immunoreactive with a donor that has been infected with Rsv (full The individual antibodies produced by the immune response). In addition to obtaining antibodies that display a full immune response to RSV, positive selection of antibodies that bind to antigens that may have a specific relevance in preventing, neutralizing, and/or eliminating RSV infection may be performed. In addition, if an antibody against a specific antigen, antigenic site or epitope that is considered to be relevant in preventing, neutralizing, and/or eliminating RSV is not identified in the donor's total immune response, then the antibody may be The 27 200837079 donor is immunized/vaccinated with this particular antigen (selected immune response). It is generally used to evaluate the neutralization effect (eg, Johnson #人1997了城(五) 176:1215 by the test tube t and the test, such as plaque reduction, micro-neutralization, and 融合10 丄 talent test or fusion inhibition test. -twenty four ). Thus, an antibody or antibody composition that has a significant effect in such assays is considered to be neutralized when compared to a negative control. Usually by, for example, a cotton rat model (eg J〇hns〇n et al.) JJnfect.Dis. ^642^-24) or a murine model (eg Tayi〇r et al. 1984. Immunology 52, 137_142 and 乂Heart waiter

Antimicrob·Agents Chemother· 49: 4700-4707 ) In vivo in vivo toxicity tests to assess protection. In vivo viability tests can be performed in a prophylactic manner, in which antibodies are administered prior to viral dysfunction, or can be administered in a therapeutic manner, wherein antibodies are administered after viral dysfunction, or a combination of the two. The multi-drug antibody composition of the present invention may be composed of an antibody capable of binding to an Rsv antigen, which antigen is not necessarily known or necessarily an envelope protein (the antibody binds to the infected cell but does not bind to the selected antigen or antigenic site), but Wherein the anti-system is obtained from a total immune response following RSV infection, for example by obtaining a nucleic acid sequence encoding one or more different antibodies from a donor having rSV infection or recovering from rSV infection. Secondly, antibodies selected from the same total immune response, based on their ability to bind to specific antigens, antigenic sites and/or epitopes, can be included in the polyclonal antibodies of the present invention. Further, 'different antibodies encoded by νΗ and VL pairs derived from one or more donors can be included in the polyclonal antibody compositions of the invention, wherein the donors have been immunized/vaccinated with a particular RSV-associated antigen Inoculation, resulting in a "selected" immune response in these donors. Thus, antibodies obtained by any of the techniques mentioned in the present invention 28 200837079 can be incorporated into a single multi-strain antibody. Preferably, the nucleic acid encoding the antibody of the invention is obtained from a human donor and the antibody produced is a fully human antibody. The motivation for producing the polyclonal antibody compositions of the present invention is that if the donor is infected with RSV and produces a humoral immune response against the antigen, such antibodies may contribute to viral clearance (at least to a certain extent) and are thus suitable Included in multiple antibody products. The other malignant strains of the present invention produce anti-RSV rpAbs in which compositions of different antibody members reflect humoral immune responses in terms of diversity, affinity and specificity of the RSV envelope antigen. Preferably, the response of the humoral response is established by ensuring that one or more of the following 35 are satisfied: i) the nucleic acid sequence encoding the individual antibody members of the anti-RSV rpAb and the V1 region is derived from the a donor for a humoral immune response to RS V (eg, after RS v infection); ii) separating the VH and vL coding sequences from the donor to maintain the original pairing of the VH and VL coding sequences present in the donor; iii) selection The individual members encoding the anti-RSV rpAb are such that the CDR regions are as diverse as possible; or lv) the specificity of the individual members of the anti-Rsv rpAb are selected such that the antibody composition collectively binds to an antigen that elicits a significant antibody response in the mammal. Preferably, the antibody composition collectively binds to an antigen, antigenic site and/or epitope at which a significant antibody titer is produced in a serum sample of the donor. As noted above, such antigens, antigenic sites and/or epitopes are outlined in Table 1, but may also specify unknown antigens, antigenic sites and/or epitopes, and non-enveloped antigens. Preferably, the donor is a human, and the plurality of strains are completely human antibodies. & 29 200837079 The present invention has identified a series of Vh& ^ pairs which can be expressed as full length antibodies, fragments or other antibody fragments having binding specificity to RSV-associated antigens. The specificity, and the pair are identified by the pure number in Table $ of Example 2. The antibody containing the Vh&^ pair identified in Table 5 is preferably a scorpion human antibody. However, chimeric antibodies can also be produced if desired. The preferred anti-RSV recombinant multi-strain system of the invention consists of different members comprising the heavy and light chain cDR2 and CDR3 regions selected from the group consisting of the VH and VL pairs listed in Table 5, for example. Preferably, the pairings shown in 纟5 retain the CDR regions and insert them into the desired framework. Alternatively, the CDR @ (CDRH) of the first pure heavy chain is combined with the CDR region (CDRL) of the second pure line of light bonds (% and Vl are mixed (Cafu ^)). The CDR regions can also be hybridized within the light or heavy chain', e.g., by combining the CDL1 region of the first pure line with the CDRL2 and cdrl3 regions of the second pure line. Preferably, the hybrid is performed between pure lines that bind the same antigen. The CDR regions of the invention may also undergo an affinity of 35, e.g., by point mutation. The method of isolating and selecting a variable heavy chain and variable light chain encoding to produce an anti-RSV recombinant multi-strain antibody composition comprises isolating a variable heavy chain (Vh) and a variable light chain (Vl) encoding from a suitable source. The sequence, which produces a spectrum of vH and vj code pairs. Usually 'a suitable source of % and coding sequence is a cell fraction containing lymphocytes, such as blood, spleen or bone marrow samples'. The samples are from an animal or human being recovered from Rsv infection or recovering from rsv sense = or from A sham RSV strain or an animal or human derived from a protein or DNA immunization/vaccination of the strain. Preferably, the lymphocyte-containing fraction is obtained from a human or a transgenic animal having the human immunoglobulin gene 30 200837079. The collected lymphocyte-containing cell fraction can be further enriched to obtain a specific lymphocyte population, such as cells from the B lymphocyte lineage. Preferably, magnetic bead cell sorting (MACS) and/or fluorescence activated cell sorting (FACS) is used,

This enrichment is performed, for example, by lineage-specific cell surface marker proteins of B cells, plasma mother cells, and/or plasma cells. Preferably, the cell fraction containing lymphocytes is enriched in B cells, plasma mother cells and/or plasma cells. Even more preferably, cells having high CD38 performance and moderate CDi9 and/or CD45 expression are isolated from blood. Such cells are sometimes referred to as circulating plasma cells, early plasma cells, and/or plasma mother cells. For the sake of simplicity, only the plasma cells are referred to in the present invention, but other terms are used interchangeably. Isolation of the VH and VL coding sequences can be performed in a standard manner, wherein the Vh and VL coding sequences are randomly combined in a vector to generate a combinatorial library of v-sequence pairs. However, in the present invention, it is preferred to reflect (10) the diversity, affinity and specificity of antibodies produced in the humoral immune response after infection. This includes maintaining a pair that is originally present in the donor, from: generating a sequence pair sequence, wherein each pair encodes a VhA V1 pair corresponding to an antibody originally present in the antibody produced by the donor from its isolated sequence. Variable weight two (VH) and variable light chain (Vl). This is also known as homologous, and :: and: the body is called a homologous antibody. Preferably, the set of the invention: or homologous VL and VL 'flat code pairs are obtained from a human donor, and thus a sequence of classes. 7 a Feng Yuanquan method There are several different methods for generating homologous vH and vL coding sequences, including amplification and isolation of VH and VL coding sequences from a single cell from 31. The vH and vL coding sequences can be amplified separately and paired in the second step or they can be paired during amplification (C〇roneila et al. 2000. Nucleic Acids Res 28: E85; Babcook et al. 1996. PNAS 93: 7843_7848 with W〇05/042774). The second method involves intracellular amplification and pairing of the % and & coding sequences (Embleton et al. 1992. Nucleic Acids Res 2: 383 1-3837, Chapal et al. 1997. Chest (10) is called (10) 23 5i8_ 524) . The second method is the selected lymphocyte antibody method which incorporates a hemolytic plaque assay with the selection of Vh& Vl cDNA (Secret (3)^ et al. 1996. PNAS 93:7843_7848). In order to obtain a spectrum of Vh& coding sequences similar to the diversity of vH& vL sequence pairs in vivo, it is preferred to use a high yield method with as few as possible and Vl versus hybrid (random combination), such as WO 05. /042774 (incorporated herein by reference). In a preferred embodiment of the invention, the S vH and VL coding pairs are generated according to a method comprising the steps of: a pair of genes reflecting a humoral immune response caused by a lining infection: i) a sense of RSV A donor that recovers from RSV infection provides a fraction of cells containing lymphocytes; ii) enriches cardiac cells or plasma cells in the fraction of the cells as appropriate; iii) obtains a single cell population that is isolated' The cell part: the cells are individually distributed into a plurality of containers; iv) using a template derived from the single cells of the sisters, amplifying and realizing νΗ and VL coding pairs in a multiple overlap extension RT_PCR protocol Linking; and ν) performing nested PCR (nested PCR) of the linked vH and VL coding pairs as appropriate. (iv) 32 200837079 The 'isolated homologous V Η and VL encoding pairs are subjected to the screening procedure as described below. Once the VH and VL sequence pairs are generated, a screening procedure is performed to identify the VH and v pairs of coding sequences that have binding reactivity to the RSV-associated antigen. Preferably, the RSV-associated antigen is an RSV envelope protein, particularly RSv G protein, RSV F protein and RSV SH protein. If the Vh and Vl sequence pairs are combined, the VH and VL pairs encoding the antibody fragments that bind to RSV can be enriched prior to screening using a phage display program. To reflect the diversity, affinity, and specificity of antibodies produced by humoral immune responses following RSV infection, the present invention has developed screening procedures for homologous pairs to achieve the widest possible diversity. For screening purposes, a bacterial or mammalian selection vector that has been transfected into a suitable host cell is used, and the homologous VH and VL coding pairs are individually expressed as antibody fragments (e.g., scFv or Fab) or full length antibody. The Fab/antibody profile was screened for reactivity against viral particles of one or more RSV strains. Preferably, at least two strains of virus, i.e., one of subtype A and one of subtype b, are used. The subtype A virus strain is, for example, Long (ATCC VR-26), A2 (ATCC VR-1540) or a renewed Long (L〇ng_Uke) subtype A isolate. The subtype B virus strain is, for example, 1 8537 (ATCC VR-1580), B1 (ATCC VR-1400), 9320 (ATCC VR-955) or a renewed 1 8537 isolate. At the same time, the Fab/antibody profile is screened against selected antigens, such as recombinant prion protein, recombinant F protein, and peptide derived from the Rsv antigen. The antigenic peptide may, for example, be selected from the conserved region of the G protein (amino acid 164_176) and the cysteine core region of the G protein (subtype a and subtype b virus strain 33 200837079 amino acid 171-187) and SH protein The extracellular domain (amino acid 42-64 of subtype A and amino acid 42-65 of subtype b). Preferably, the peptides are biotinylated to facilitate immobilization on the beads or plates during screening. Alternative fixing methods can also be used. Antibodies can be provided that bind to such antigens based on knowledge of the RSV biology and expected and/or protective effects of the antigen. This screening procedure is equally applicable to combinatorial phage display libraries. The recombinant G and/or F protein used for screening can be expressed in bacteria, cercaria cells, mammalian cells or other suitable expression systems. 〇 and / or? The protein can be expressed as a soluble protein form (no transmembrane region) or it can be fused to a third protein to enhance: stability. If the G and/or F protein is expressed as a fusion marker, the fusion partner can be broken down prior to screening. Preferably, the expression represents both (} and/or F protein of subtype A and subtype B and is used for screening. In addition, virus strain-specific prion protein can be expressed for screening. In addition to the above primary screening. , Executable: secondary screening to ensure that none of the selected sequences encode a false positive. In the second (four), 're-integrated RSV/ for all strains and selected antigens in the first screening. The antigens are screened. Typically, immunoglobulin assays are suitable for screening performed in the present invention. The assays are well known in the art and include, for example, SPOTS' eusa, FLISA, membrane assays ( For example, Western blotting method, manufacturing method = Ways on fU and FA~ such tests can be performed using the peptides produced by the self-extraction sequence without any prior:: set steps. In V Η and VL In the case where the coding pair is π 曰 as the 对 source pair, when the screening I, , and the error is represented by, for example, phage display, the immunoassay is better... and, in the selection group, Oral sigma set method execution or after enrichment method 34 200837079, the rich Set methods such as phage display, ribosome presentation, bacterial surface presentation, yeast presentation, eukaryotic virus presentation, RNA presentation, or co-existence (described in FitzGerald, K., 2000. Drug Discoν· Today 5, 253 -258). The selected VH and VL sequences in the screening are typically sequenced and analyzed for the diversity of the variable regions. Particular attention is paid to the diversity of the CDR regions, but also to the VH and VL family expressions. For such analysis, select the Vh and VL pair coding sequences that represent the overall diversity of RSV binding antibodies isolated from one or more donors. Preferably, select all of the cdr regions (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and Sequences having differences in CDRL3). If there are sequences having one or more identical or extremely similar CDR regions belonging to different Vh or Vi families, such sequences are also selected. Preferably, at least the variable heavy chain CDR3 region ( CDRH3) differs between pairs of selected sequences. It is possible to select Vh & ^ sequence pairs based only on the variability of the CDRH3 region. During the initiation and amplification of the sequence, in the framework region of the variable region (especially the first Frame area Mutations may occur within the region. Preferably, errors occurring in the first framework region are corrected to ensure that the sequence corresponds exactly to the donor sequence or at least corresponds to 98%, for example such that the sequence is a fully human sequence. The overall diversity of the set of sequences encoding the VH&Vl pair represents a high degree of antibody expected to be expressed from the selected Vh& Vl coding set when the diversity is seen at the genetic level in the humoral response to RSV infection. Specificity is also representative of the specificity of antibodies produced by Rsv-infected donors. Whether the specificity of the antibody expressed by the selected VH& VL coding pair represents the specificity of the antibody produced by the infected donor 35 200837079 can also be expressed by the combination of the selected Vh and 'coding pairs The comparison of the opposite sex is obtained for the virus strain and the antibody titer of the selected antigen for the donor blood. In addition, the #八J progressive knife analyzes the terry nature of the antibody expressed from the selected and ντ coding pairs. The degree of king specificity is related to the number of different antigens that can detect binding reactivity. In another embodiment of the present invention, the selected pair and the ^ coding pair are analyzed by antigen-based localization. The specificity of the individual antibodies that are expressed.

The epitope localization can be performed by a number of methods which are not necessarily mutually exclusive. A method of locating the epitope-specificity of a pure antibody to evaluate the binding of a peptide of a different length to the primary structure derived from the target antigen. The peptides can be linear and conformal and can be used in a variety of assay formats, including ELIS A, FLIS A, and surface plasma resonance (SPR, Biac〇re). Furthermore, the peptides can be reasonably selected to represent, for example, the extracellular region or conserved region of the target antigen using available sequence and structural data, or the peptide can be designed as a set of overlapping peptides representing the selected antigenic portion or the entire antigen (Meloen RH, Puijk WC). , Schaaper WMM. Epitope mapping by PEPSCAN. In: Immunology Methods Manual. Ed Iwan Lefkovits 1997, Academic Press, 苐 9 8 2 - 9 8 8 pages). The specific reactivity of an antibody to one or more such peptides will generally indicate epitope specificity. However, in many cases, the peptide is an adverse mimetic of the epitope recognized by the antibody raised against the protein antigen, due to a lack of conformation and due to antibody versus protein antigen compared to antibody and The generally larger buried surface area of the interaction between the peptides. A second epitope locating method that allows specificity to be directly defined on a protein antigen is achieved by selective masking using existing established anti-36 200837079 bodies. A decrease in binding of the second probe antibody to the antigen after blocking typically indicates sharing or overlapping epitopes. The epitope localization by selective masking can be performed by a number of immunoassays including, but not limited to, ELISA and Biacore, which are well known in the art (e.g., Ditzel et al. 1997. J. Mol Biol. 267: 684-695; Aldaz-Carroll et al. 2005. J. Virol. 79: 6260-6271). Another possible method for determining the specificity of the antigenic determinant of an antiviral antibody is to select a virus evading mutant in the presence of the antibody. The sequencing of the gene of interest from these escape mutants will typically reveal amino acids in the antigen that are important for recognition by the antibody and thus constitute part of the epitope. Preferably, the individual members of the anti-RSV rpAb of the invention are selected such that the specificity of the antibody composition collectively covers the rSV subtypes a and B, as well as the RSV associated proteins F and g, and preferably also SH. Generation of recombinant polyclonal antibodies from selected VH and V^ coding pairs Multiple strains of antibodies of the invention are produced from multiple display cell lines in one or several bioreactors or their equivalents. Following this method, the anti-RS V rpAb can be purified from the reactor into a single formulation without the need to separate individual components of the anti-RSV rpAb in the process. If multiple antibodies are produced in more than one bioreactor, the supernatant from each bioreactor can be confluent and subsequently purified, or the supernatant can be purified separately from each bioreactor. The obtained antibodies are confluent to obtain a purified anti-RSV rpAb 〇W02004/061104 and WO 2006/007850 (PCT/DK2005/000501) 37 200837079 (the disclosures of which are incorporated herein by reference) A method of multiple antibodies. The method described therein is based on the specific integration of the antibody coding sequence into the genome of an individual host cell, thereby ensuring that the original pairing of the VH and VL protein chains is maintained during production. In addition, site-specific integration minimizes site effects and therefore the growth and performance characteristics of individual cells in multiple cell lines are expected to be extremely similar. Typically, the method involves the following: i) a host cell having one or more recombinase recognition sites; η) a expression vector having at least one recombinase recognition site compatible with the recombinase recognition site of the host cell ;Hi) by generating a set of expression vectors by transferring the selected vH and VL coding pair to the expression vector such that the full length antibody or antibody fragment can be expressed by the vector (if the selection vector is identical to the expression vector, the transfer is not required) (iv) transfecting a host cell with a vector of expression vectors encoding a recombinase that binds a recombinase recognition site within the genome of the host cell with a recombinase recognition site in the vector; v) from the transfected host The cells acquire/produce multiple cell lines; and vi) display and collect multiple antibodies from multiple cell lines. Preferably, mammalian cells such as CHO cells, COS cells, BHK cells, myeloma cells (eg, Sp2/〇 cells or NSO cells), fibroblasts (such as NIH 3T3), and immortalized human cells can be used, such as HeLa cells, HEK 293 cells or PER.C6. However, non-mammalian eukaryotic or prokaryotic cells such as plant cells, insect cells, yeast cells, fungi, Escherichia coli and the like can also be used. Suitable host cells contain one or more suitable recombinase recognition sites within their genome. The host cell should also contain a selection pattern operably linked to the integration site to enable integration of the 38 200837079 integrant (i.e., an anti-Rsv Ab expression vector or expression vector fragment at the integration site). The cells are chosen. For example, US 5,677,177 describes the preparation of cells having FRT sites at predetermined positions within the genome. Preferably, the host cell has only a single integration site located at a site (so-called hot-spot) where the integrant is highly expressed. Suitable expression vectors comprise a recombinant recognition site that matches the recombinase recognition site of the host cell. Preferably, the recombinase recognition site is ligated to a suitable selection gene different from the selection gene used to construct the host cell. Selection genes are well known in the art and include the face amine indoleamine synthase gene (GS), the dihydrofolate reductase gene (DHFR), and neomycin, wherein GS or (10) is available for insertion. % and , sequence gene amplification. The vector may also contain two different recombinase recognition sites to achieve recombinase-mediated cassette exchange (rmce) of the antibody coding sequence rather than complete integration of the vector. RMCE is described in Langer et al. 2〇02· Nucleic Acids Res. 30, 3067_3077; ScMake& B〇de 1994

Biochemistry 33, ^746-1^ and Belteki et al. 2〇〇3· Nat biotech· 21,321-324. Suitable recombinase recognition sites are well known in the art and include FRT, lox and read/attB sites. Preferably, the integration vector is a homotypic coding vector, wherein the definitive region (preferably including an intron) is present in the vector (or if performed on the full length antibody) prior to the transfer of the vector and the coding pair. For screening, the constant region is already present in the screening vector). The constant region in the vector can be A throughout the heavy bond hatchery region (CH1 to CHS or to CH4) or the constant region encoding the Fc portion of the antibody (CH2 to 39 200837079)

CH3 or to ch4). The light chain may be present in the "stationary region" before the transfer. The number of strange regions (if any) present depends on the I selection and transfer system used. The heavy chain constant region may be selected from the same type of IgGi, igG2L. igG4, IgA1, IgA2, IgM, IgI^IgE〇__"igGi and/or IgG3. Furthermore, expression vectors for site-specific integration of nucleic acids encoding anti-RSV antibodies contain suitable promoters or equivalent sequences that direct high expression levels of each of the Vh and VL chains. Figure 3 illustrates a possible method of designing a performance vector, but many other designs are possible. The selected 乂 and vL coding pairs are transferred from the screening vector by conventional restriction enzyme cleavage and ligation such that each expression vector molecule contains a Vh and V coding pair. Preferably, the VH and vL coding pairs are separated. Transfer, however, L can also be transferred as a whole when needed. When all of the selected VH and ^ coding pairs are transferred to the expression vector, a collection or library of expression vectors is obtained. Alternative transfer methods can also be used if desired. The selection vector is the same as the expression vector, and the library of the expression vector consists of the Vh and vL sequence pairs selected during the screening, and the sequence pairs are located in the screening/expression vector. ▲ The method of transfecting the nucleic acid sequence into the host cell is here. It is well known in the art. In order to ensure site-specific integration, it is also necessary to provide a suitable recombinase to the host cell. This is preferably achieved by co-transfection of a plastid encoding a recombinase. Suitable recombinant enzymes are, for example, Flp. , Cre or sputum φ€3ΐ integrase, 宿主 a host cell/vector system with a corresponding recombinase recognition site. The host cell can be transfected in bulk, which means In a separate reaction, a library of expression vectors is transfected into a cell line to obtain a plurality of cell lines. Alternatively, a collection of expression vectors can be separately transfected into host cell 200837079 cells to produce a collection of individual cell lines ( Each cell line produces an antibody with specific specificity.) The cell line (individual or multi-strain) produced after transfection is then selected for the site-specific integrant, and if the cell line is not transfected, The characteristics of suspension growth in serum-free medium make it suitable for suspension growth in serum-free medium. If transfection is performed separately, individual cell lines are further analyzed for growth characteristics and antibody production. Preferably, the selection has similar proliferation. Rate and antibody expression of cell lines to produce multiple cell lines. Multiple cell lines are then produced by mixing individual cell lines at a predetermined ratio. Typically, multiple cell lines (PMCB), multiple plants are established from multiple cell lines. A cell bank (pRCB) and/or a plurality of working cell banks (pWCB) are studied. A plurality of cell lines are produced by mixing individual cell lines at a predetermined ratio. Multiple cell lines are distributed into ampoules, resulting in multiple research cell banks (pMCB) or master cell banks (pMCB), which can be generated by cell expansion from the cell bank or the master cell bank. pWCB). The research cell bank is mainly used to prove the conceptual study, in which multiple cell lines may not contain as many individual antibodies as many cell lines in the main cell bank. Usually, further Xiao pMCB is amplified to The aim is to establish a pWCB. Once the pWCB is depleted, the new ampoule from pMcB can be subjected to amplification to establish a new pWCB. One specific embodiment of the present invention is a multi-cell cell line capable of expressing the recombinant multi-strain anti-RSV antibody of the present invention. Another embodiment of the invention is a multi-strain cell line wherein each cell is capable of expressing a single vH and vL coding pair, and the plurality of cell lines are generally capable of representing a collection of vH and vL coding pairs, wherein each Vh and Vl pair encodes抗41 200837079 Like the antibody. Preferably, the set of coding pairs is a homologous pair produced according to the method of the invention. The recombinant polyclonal antibody of the present invention is expressed by culturing a pWCB in an appropriate medium for a period of time sufficient for the antibody to be expressed and the plurality of cell lines to be stable (with a time limit of between about 15 days and 50 days). A culture method such as fed-batch or perfusion can be used. Multiple strains of antibodies were obtained from the culture medium and purified by conventional purification techniques. Affinity chromatography is often used in conjunction with subsequent purification steps such as ion exchange chromatography, hydrophobic interactions, and gel filtration to purify IgG. After purification, the presence of all individual members in a plurality of antibody compositions is assessed, e.g., by ion exchange chromatography. The characteristics of a plurality of antibody compositions are described in detail in WO/2006/007853 (PCT/DK2005/000504), which is incorporated herein by reference. An alternative method of expressing an antibody mixture in a recombinant host is described in WO 04/009618. This method produces antibodies with different heavy chains associated with the same light chain from a single cell line. This method can be applied if an anti-RSV rpAb is produced from a recombinant library. Therapeutic composition Another aspect of the present invention is a pharmaceutical composition comprising as an active ingredient an anti-RSV rpAb or an anti-RSV recombinant multi-plant Fab or another anti-RSV recombinant multi-strain antibody fragment. Preferably, the active ingredient of the composition is an anti-RSV recombinant polyclonal antibody as described in the present invention. Such compositions are intended to be used to prevent and/or treat RSV infection. Preferably, the composition is administered to a human, livestock or pet. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient. 42 200837079 Anti-Rsv rpAb or a plurality of fragments thereof can be administered in units of pharmaceutically acceptable excipients, carriers or excipients. A suitable pharmaceutical formulation can be used to provide a suitable formulation or composition for administration to a patient, or a patient who may be at high risk if infected with RSV. In the specific case of the car father, the drug has a preventive effect from 曰 _ _ 々 在 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 另一 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在Any suitable route of administration may be employed, for example, parenteral, intravenous, r intraarterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository or oral administration. For example, the medicinal whistles may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a key, capsule, chewing gum or paste, and for intranasal administration, the formulation may be For powders, nasal drops or aerosols. The medical composition of the present invention is prepared by a method known per se, for example, by means of a conventional dissolution, tempering, mixing, granulating or morphing process. The pharmaceutical composition can be formulated according to conventional pharmaceutical specifications (see, for example, Remington: The Science _ 2〇 version), A.R. Edit, 2〇〇〇,% 出咖&洲(四)

Philadelphia, PA and Encycl〇pedia 〇f Pharmaceutica] Technology, eds. J. Swarbrick and J. C. B〇ylan, 1988.1999,

Marcel Dekker, New York, NY) 医药 The pharmaceutical compositions of the present invention are preferably prepared using a solution or suspension of the active ingredient, and especially an isotonic aqueous solution or suspension. In the case of a dry composition comprising only active ingredients or active ingredients and carriers (e.g., mannitol), (if possible) the solutions may be prepared prior to use. The pharmaceutical composition can be sterilized and sterilized, #丨匕3 excipients, such as cockroaches and humectants, humectants and/or emulsifiers, solubilizers, and agents known to be wounded by methods known per se. For example, by means of a solution or freeze-drying process. The solutions or suspensions m , ^ m τ ' contain a thickening agent from J; methylcellulose sodium, slow methylcellulose, dextrose, polyethylene ether, or different gelatin.制成 Preparation of the injectable compositions under sterile conditions by conventional methods "The compositions are also introduced into ampoules or vials using the same ingredients and sealed in a container. The active ingredient can be combined with the solid carrier and the resulting mixture can be obtained if desired. Granulating 'and when necessary or necessary, after adding appropriate excipients, the mixture is processed into tablets, pills or capsules (these preparations can be coated with shellac, sugar or both) for oral administration The pharmaceutical composition may also be incorporated into a Pities carrier to allow the active ingredient to diffuse or release in the amount measured.

The pharmaceutical compositions comprise from about 1% to about 95%, preferably from about 2% to about 9%, by weight of active ingredient. The pharmaceutical compositions of the present invention may, for example, be in unit dosage form, such as in the form of ampoules, vials, suppositories, troches, pills or capsules. The formulation can be administered to a human subject to provide treatment for the disease or condition by treating or preventing an effective amount (e.g., preventing, eliminating or reducing the amount of the pathological condition). The preferred dosage of the therapeutic agent to be administered may depend on such variables as the severity of the RSV infection, the overall health of the particular patient, the formulation of the compound/excipient, and the route of administration. Therapeutic Uses of the Compositions of the Invention The pharmaceutical compositions of the invention may be used to treat, ameliorate or prevent lactation. 44 200837079 Diseases Conditions which may be treated or prevented by the pharmaceutical compositions of the present invention include infection with or in the presence of RSV infection Patients at risk, especially if they are infected with RSV, may be at high risk for prevention and treatment: high-risk patients such as infants and young children, who have potential problems (such as chronic lung disease or congenital heart disease) Premature babies and children are at great risk of developing serious diseases such as bronchiolitis and pneumonia after RSV infection. Also, such as immunocompromised adults, especially bone marrow transplant recipients, elderly and high-risk adults with k-pulmonary disease, It may be preferably subjected to prophylactic or therapeutic treatment with the pharmaceutical composition of the present invention. The specific example of this 'X month is a method for preventing, treating or ameliorating a mammal or a plurality of symptoms associated with RSV infection, Including the administration of the anti-RSV recombinant multi-strain antibody of the present invention to the mammal. The use of IT: another specific example is the hair The use of a composition that is resistant to RSV recombination, to ameliorate or prevent the symptoms of the animal, or a plurality of symptoms associated with predicate music. The pet preferably has the mammals of the above specific examples as humans, domestic animals. Or in another embodiment, the method of treating phlegm-suppressing beta, ninth, and preventing or treating one or more of the symptoms associated with Rs v susceptibility. The ceremonial animal preferably has more than 40%, preferably a chronic lung disease. In an alternative embodiment, a bone marrow transplant recipient, an elderly person, and a premature infant in a human or a congenital heart disease, a human being, an immunocompromised adult, especially a high-risk adult of a patient with k-pulmonary disease. 45 200837079 Diagnostic Use Another specific example of the invention is directed to a kit. Kit of the invention 3 An anti-RSv rpAb prepared according to the invention, which may be labeled with a deductible label or unlabeled for non-labeling (d). This kit can be used to identify individuals infected with RSV. The antibody molecules of the invention and related aspects thereof should be noted that the novel antibody molecules disclosed herein are capable of contributing to the prior art by their own capabilities. Accordingly, the invention also relates to any of the antibody molecules disclosed herein, as well as fragments and analogs of such antibodies, wherein the fragments or analogs have at least the CDRs of the antibodies disclosed herein. For example, the inventors of the present invention have discovered that some have been derived from a human donor: a fully human antibody molecule comprising a binding site that, when bound to an antigen, exhibits a highly improved kinetics superior to prior art monoclonal antibodies. feature. Thus, although the disclosure herein is mostly focused on multiple antibody compositions, all of the subject matter associated with the use of multiple antibodies described herein is also related to any of the single antibody molecules disclosed herein, ie, all The disclosure of the various antibody compositions of the invention relating to formulation, dosage, administration, etc., is applicable (with the necessary modifications) to the individual antibody molecules, antibody fragments and antibody analogs disclosed herein, preferably also to the framework sequences. Thus, the invention also relates to an isolated human anti-RSV antibody molecule which extends from the antibody molecule described in Table 5 herein, or a specific binding fragment thereof, or a synthetic or semi-synthetic antibody analog, or The analog comprises at least a complementarity determining region (CDR) of the isolated antibody molecule. Since the antigen-specificity of antibodies is known to depend on the 3D group of CDRs and framework regions. 46 200837079 includes the woven of natural human antibodies, such fragments or analogs often also serve as framework regions for the regions. The expression "isolated antibody molecule" is determined from natural impurities, ie, the same variable region and constant region. It is intended to represent a collection of different antibodies and exhibit the same amino acid sequence (also typically, the antibody molecule, the tablet, the genus or the buccal line are derived from the antibodies listed in Table 8, or include SEQ id Nos) The heavy chain CDR amino acid sequence included in one of 1 ζΐ>ι VN〇S·^44 and the accompanying light bond cdr amine group having SEQIDN0 higher than the amino acid sequence of SEQ ID NOs 144 Acid sequence. This molecule, fragment or analog will include the variable region homology pairs as seen in the 44 identical lines discussed above. As mentioned above, some of the antibody molecules of the invention exhibit extremely high affinity. : The invention therefore also relates to isolated antibody molecules, antibody fragments or synthetic or semi-synthetic antibody analogs comprising the same CDRs as those derived from human antibodies, and which are immobilized on the surface of the sensor at very low density when used Recombinant RSVG protein, which avoids the limitation of mass transport, has a dissociation constant Kd of at most 5〇〇nM for RSV G protein when subjected to surface plasma resonance analysis on Biac〇re3〇〇〇. Antibody molecule, antibody fragment Synthetic or semi-synthetic antibodies typically exhibit up to 4 〇〇 nM 2 lower KD, such as up to 300 nM, up to 200 nM, up to 1 〇〇 nM, up to 1 Μ Μ, up to 900 Μ, up to 8 〇〇 Μ, up to 700 ρΜ, 至夕600 ρΜ, up to 500 ρΜ, up to 400 ρΜ, up to 300 ρΜ, up to 200 ρΜ, up to 1〇〇ρΜ, up to 90 ρΜ and up to 80 ρΜ. Detailed description of Bi〇core measurement is provided in the example 47 200837079 Another specific example of the invention relates to an isolated antibody molecule, antibody fragment or synthetic or semi-synthetic antibody comprising the same antigen binding site as the antigen binding site derived from a human antibody, when used Recombinant Rsv F protein with very low density immobilized on the surface of the sensor to avoid mass transfer limitations, when performing surface plasmon resonance analysis on the BiaCOre 3000, Γ:

The Fab has a dissociation constant of at most 5〇〇nM for the RSV F protein. KD-cleaved antibody molecules, antibody fragments or synthetic or semi-synthetic antibodies typically exhibit a lower Kd of up to 400 nM, such as up to 3〇〇nM Up to 200 rituals, up to 100 nM, up to lnM, up to 9 〇〇pM, up to 8 〇〇PM, up to 700 pM, up to 6 〇〇pM, up to 5 、州, up to 4 〇〇pM, up to 300 pM, up to 200 pM, up to 1 〇〇 _, up to 9 〇 pM, up to 80 pM, up to 70 pM, up to 60 pm, up to 50 pM, up to 4 〇 PM, up to 30 pM, up to 25 _, up to 2 〇 pM, up to 15 _,

Up to 10 pM, up to 9 pM, up to 8 pM, up to 7 pM, up to 6 pM and up to 5 pM. Particularly useful antibody molecules or specific binding fragments or synthetic or semi-synthetic antibody analogs comprise cDRs of human antibodies produced in pure lines 810, 818, 819, like, 825, 827, 85 8 or 894. As mentioned above, such useful antibody molecules of the invention can be formulated in the same manner and used in the same applications as the multi-plant formulations of the invention. Because: 'The present invention relates to antibody compositions, and the inclusions discussed in this section include: knife-specific binding fragments or synthetic or semi-synthetic antibody analogs: pharmaceutically acceptable carriers, excipients, media Agent or diluent. The &> can comprise more than one binding specificity and can, for example, be used to modulate two different antibodies of the invention 48 200837079 and/or specific binding fragments of the invention and/or synthetic or semi-synthetic antibody analogs. The composition may even comprise at least 3 different antibody molecules or antibody fragments and/or synthetic or semi-synthetic antibody analogs, specific binding fragments or synthetic or semi-synthetic antibody analogs of the invention, and thus may comprise a hopper; ", ?, ^^, ^^, ^, ; ^, ^, ^, η, 18, or 30 different antibody molecules and / or fragments and / or a combination of synthetic or semi-synthetic antibody analogs. The composition comprises at least one antibody molecule, fragment or analog of the invention that binds to the RSV F protein and at least one antibody molecule, fragment or analog of the invention that binds to the Rsv g protein. Further, a portion of the invention is encoded by the invention An isolated nucleic acid fragment of an amino acid sequence of at least one of the identified CDR molecules, such as a nucleic acid fragment encoding at least cdr of an antibody produced by one of the pure lines listed in Table 5. The nucleic acid fragment is typically DNA, but It may be rNA. Another specific example is an isolated nucleic acid fragment encoding the CDR sequence of the heavy chain sulfosyl acid sequence described in any of the SEq ID N〇s, or encoding SEQ ID NOs 89-132 Ren - An isolated nucleic acid fragment of the CDR sequence of the light-bonded amino acid sequence described herein. A preferred nucleic acid fragment of the invention encodes a heavy bond CDR amino acid sequence as set forth in any one of SEQ ID NOs 1-44 plus A CDR amino acid sequence having a SEQ ID NO higher than 88 amino acids selected from the group consisting of SEQ ID NOs. 144. Of course, this means that the nucleic acid fragment will encode the above given 7η / A reproducible homologous pair as seen in the 44 identical lines. The nucleic acid fragment may thus comprise a coding sequence contained in seq1NQs: 49 200837079 and/or 1 3 3 - 1 7 6 . Conveniently, the nucleic acid fragment is Introduced into a vector, which is also part of the invention. The vector may be capable of autonomous replication and is typically selected from the group consisting of plastids, bacilli, eosmids, minichromosomes and viruses. Where the vector is a performance vector, it will preferably have the following summary (see also the exemplary carrier in Figure 3):

- at least in the 5, 3, direction and in the operably linked, a promoter for driving the expression of the first nucleic acid fragment discussed above, the first nucleic acid fragment encoding at least one light chain CDR and any necessary framework a nucleic acid sequence encoding a leader peptide; the nucleic acid sequence encoding the constant region of the antibody, as appropriate; and, optionally, the nucleic acid sequence encoding the first terminator, and/or - at 5 In the 3' direction and in the operably linked, at least one promoter for driving the expression of the second nucleic acid fragment of the invention, the second nucleic acid fragment encoding at least one heavy chain CDR and any necessary framework regions; optionally, encoding a nucleic acid sequence of a leader peptide; a second nucleic acid fragment; optionally, a nucleic acid sequence encoding a quenching region; and, optionally, a nucleic acid sequence encoding a second terminator 0. The right vector can be used to stabilize a transformed host cell, which can be subsequently It is especially useful to support the production of recombinant performance products. Therefore, a preferred vector is one which is integrated into the genome of the host cell when introduced into a host cell. Accordingly, the invention also relates to transformed cells carrying the vectors of the invention as discussed in this section and also to stable cell lines carrying such vectors and which exhibit the nucleic acid fragments of the invention as discussed in this section. The recombinant expression product (i.e., the antibody molecule, antibody fragment or analog of the present invention) is secreted or carried on its surface by transformed cells and cells. Example 1 This Bega addition is a collection for illustrating the method of the present invention. a. Negative plasma mother cells from donor blood sorting Peripheral blood mononuclear cells (PBMC) were isolated from donor blood using Lymphoprep (Axis Shield) and gradient centrifugation according to the method described by Wei. The isolated PBMCs were frozen in FCS under 1st generation; 1%% DMS® or used directly. The B cell fraction was labeled with an anti-CD19 antibody and partially isolated from the PBMC using magnetic cell sorting (MACS). PBMC (1×10 6 cells) were incubated with anti-CD19-FITC binding antibody (BD Pharmingen) for 20 min at 4 C. The cells were washed twice in MACS buffer (Miltenyi Biotec) and resuspended therein. Anti-FITC microbeads (Miltenyi Biotec) were mixed with labeled cells and incubated at 4 °C for 15 min. The washing procedure was repeated and the cell-bead suspension was then applied to a LS MACS column (Miltenyi Biotec). CD19 positive cells were eluted from the column according to the manufacturer's instructions and stored in FCS-10% DMSO, or directly subjected to single cell sorting. Based on the performance characteristics of CD19, CD38 and CD45 cell surface proteins, plasma mother cells were selected from CD19+ B cell fractions by fluorescence activated cell sorting (FACS). CD 19 is a b cell marker that is also expressed on plasma cell precursor cells, while CD38 is highly expressed on plasma mother cells and plasma cells. Plasma mother cells apparently had slightly lower CD19 and CD45 expression than the rest of the CD 19+ cells, which allowed the different populations to be isolated. The cells were washed in 51 200837079 FACS buffer (pBS; 1% BSA) and stained with anti-CD19-FITC, anti-CD38-APC and anti-λ-ΡΕ (BD Pharmingen) for 20 min. Λ-light chain staining is included to exclude cells that cannot act as a pcR template (see section c). The stained cells are washed in FACS buffer and resuspended therein. The flow rate of the cells during FACS was set to about 2 事件 events/second and the cell concentration was 5 χ 105 cells/ml to obtain high plasma cell rescue. Use the following thresholds. Each threshold is the child of the former. Threshold 1: FSC/SSC tricks. The lymphocyte population with the highest FSC is selected to ensure live cell sorting. Threshold 2: SSCh/SSCw. This threshold ensures single cell sorting (doublet discrimination). 〇门礼3: Gating events representing plasma blasts in a CD38/CD19 dot plot in the form of CD38/CD19 medium. Threshold 4: Since the pCR program described in section c only amplifies the kappa light chain, the lambda_negative event is gated in the lambda/CDB dot plot. As an alternative to or in addition to the threshold 3, plasma maternal months may also be identified as CD38 high/CD19 medium in the CD45/CD38 dot plot. This will require staining the cells with anti-CD45-PerCP. The resulting population of single cells that met these four criteria were sorted into 96 1 PCR plates containing sorting buffer (see section e). The cell-containing plates were stored at -80%. Office · Enzyme-linked immunospot method (5)

The percentage of blood staves expressing anti-RS V antibodies in the obtained cell samples (i.e., pBMc, MACS 52 200837079 purified CD 19 + cells or FACS sorted plasmablasts) was estimated using ELISpot. A % plate with a surface of nitrocellulose (Millipore) was coated with a solution of 25 |Lig/ml inactivated rs V Long particles (HyTest). The wells were blocked by incubation with RPMI, 2% milk powder, and allowed to stand at 4 ° C for about 5 h, followed by incubation at 37 ° C for 1 h. The plates were washed and cell samples were added to each well in RPMI medium, and I1 was cultured for 24 hours under standard tissue culture conditions. The secreted R S V specific antibody will bind to the immobilized viral particles surrounding the antibody producing cells. Cells were removed by washing three times in PBS; 0·01〇/〇 Tween20 and washing three times in PBS. Anti-human igG (H+L) (CalTag) conjugated to HRP and anti-human igA (Serotec) in combination with HRP were added and allowed to react with the immobilized antibody for 1 h at 37 °C. The washing procedure was repeated and the chromogen substrate (3_Amine_9_ethylcarbazole dissolved in N,N-DMF (dimethylformamide)) was added. Color development was terminated after 4 min by the addition of h20. A red dot is found at the site where the cells of the antigen-specific antibody are located. c. Homologous VH and VL pair ligation The ligation of the Vh& Vl humer sequence is performed on a single cell obtained as described in section a, facilitating homologous pairing of the vH and vL coding sequences. This procedure utilizes a two-step PCR procedure based on: single-step multiplex overlap extension RT-PCR followed by nested PCR. The primer mix used in the examples of the present invention amplifies only the K light chain. However, primers capable of amplifying the lambda light chain can be added to the multiplex primer mix and the nested PCR primer mix, if desired. If the λ primer is added, the sorting procedure in the a portion is adjusted so that the λ positive cells are not excluded. Figure 53 illustrates the conjugation of homologous Vh and & sequences. 2008 20087979 Original shell 1j. The 96-well PCR plates generated in section a were thawed and the sorted cells were used as templates for multiplex overlap extension RT-PCR. Sorting buffer added to each well prior to single cell sorting contains reaction buffer (single step RT-PCR buffer; Qiagen), RT-PCR primer (see Table 2) and RNase inhibitor (Rnasin, Promega) ). This sorting buffer was supplemented with a single step RT-PCR enzyme mix (25-fold dilution; Qiagen) and dNTP mix (200 μM each) to obtain a specific final concentration in the 20-μ1 reaction volume. The plates were incubated at 55 ° C for 30 min to allow reverse transcription of RNA from each cell. After RT, the plates were subjected to the following PCR cycles: 10 min at 94 °C, 35x (40 sec at 94 °C, 60 (5 min at 40 sec, 5 min at 72 °C), 10 mino at 72 °C. PCR reactions were performed on a H20BIT thermocycler (ABgene) of a Pex Seal Basket on a 24x 96 well plate to facilitate high yield. The PCR plates were stored at -20 ° C after cycling. Table 2: Multiple overlap of RT-PCR extending the primer mixture of primer name final concentration nM sequence of SEQ ID NO: VH group CH-IgG 0.2 GACSGATGGGCCCTTGGTGG 179 CH-IgA 0.2 GAGTGGCTCCTGGGGGAAGA 180 VH-1 0.04 TATTCCCATGGCGCGCCCAGRTGCAGCTGGTGCART 181 VH-2 0.04 TATTCCCATGGCGCGCCSAGGTCCAGCTGGTRCAGT 182 VH-3 0.04 TATTCCCATGGCGCGCCCAGRTCACCTTGAAGGAGT 183 VH-4 0.04 TATTCCCATGGCGCGCCSAGGTGCAGCTGGTGGAG 184 VH-5 0.04 TATTCCCATGGCGCGCCCAGGTGCAGCTACAGCAGT 185 VH-6 0.04 TATTCCCATGGCGCGCCCAGSTGCAGCTGCAGGAGT 186 VH-7 0.04 TATTCCCATGGCGCGCCGARGTGCAGCTGGTGCAGT 187 VH-8 0.04 TATTCCCATGGCGCGCCCAGGTACAGCTGCAGCAGTC 188 54 200837079 LC group CK1 0.2 ATATATATGCGGCCGCTTATTAACACTCTCCCCTGTTG 189 VL-1 0.04 GGCGCGCC ATGGGAATAGCTAGCCGACATCCAGWTGACCCAGTCT 190 VL-2 0.04 GGCGCGCCATGGGAATAGCTAGCCGATGTTGTGATGACTCAGTCT 191 VL-3 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAATTGTGWTGACRCAGTCT 192 VL-4 0.04 GGCGCGCCATGGGAATAGCTAGCCGATATTGTGATGACCCACACT 193 VL-5 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAACGACACTCACGCAGT 194 VL-6 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAATTGTGCTGACTCAGTCT 195

W Α/Τ, R= A/G, S= G/C For the nested PCR step, a 96-well PCR plate (20-μ1 reaction) was prepared in each well using the following mixture to obtain a specific final concentration: 1 x FastStart buffer (Roche), dNTP mixture (200 μΜ each), nested primer mix (see Table 3), Phusion DNA polymerase (0.08 U;

Finnzymes) and FastStart High Fax Enzyme Blend (0.8 U; Roche). As a template for nested PCR, 1 μΐ was transferred from the multiplex overlap extension PCR reaction. The nested PCR plates were subjected to the following PCR cycles, 35x (30 sec at 95 °C, 30 sec at 60 °C, 90 sec at 72 °C), and 10 min at 72 °C. Randomly selected reactions were analyzed on a 1% agarose gel to verify the presence of overlapping stretches of about 1070 bp. The plates were stored at -20 °C until further processing of the PCR fragments. Table 3: Nested primer set primer name Final concentration ηΜ Sequence SEQ ID CK2 0.2 ACCGCCTCCACCGGCGGCCGCTTATTAACACTCTCCCCTGTTGAAGCTCTT 196 PJ 1-2 0.2 GGAGGCGCTCGAGACGGTGACCAGGGTGCC 197 PJ3 0.2 GGAGGCGCTCGAGACGGTGACCATTGTCCC 198 PJ4-5 0.2 GGAGGCGCTCGAGACGGTGACCAGGGTTCC 199 PJ6 0.2 GGAGGCGCTCGAGACGGTGACCGTGGTCCC 200 d. Cognate Vη and V^ For insertion into a screening vector to identify antibodies having binding specificity for RSV particles or antigens, the VhAV1 coding sequence obtained as described in Section 55 200837079, as part C, is expressed as a full length antibody. This involves inserting the VH and VL coding pairs into the expression vector and transforming into host cells. A two-step colonization procedure is employed to generate a spectrum of expression vectors containing linked and VL coding pairs. Statistically, if the expression vector contains more than ten times as many recombinant plastids as the number of homologous pairs and VL PCR products used to generate the screening profile, the probability of presenting all unique gene pairs is 99%. Thus, right to obtain 400 overlapping extension v gene fragments in section c, then 1 produces at least 4 pure lines for screening. Briefly, the linked and vL code pairs from the nested pCR in Part C are confluent (no pairs from different donors are mixed). The PCR fragment was cleaved at the recognition site in the sputum introduced into the pcR product by using w and fine I DNA endonuclease. The decomposed and purified fragments are ligated to the digested mammalian igG expression vector (113) by standard binding procedures. The ligation mixture was electroporated into E. coli and added/added to a 2 χ Τ plate containing appropriate antibiotics and incubated overnight at 37 t:. The amplified vector profile was purified from cells recovered from the plate using standard DNA purification methods (Qiagen). The plastids were prepared to achieve insertion of the promoter _ leader fragment by using &# core 1 endonuclease cleavage. The restriction sites for these enzymes are located between the VH and VL encoding gene pairs. After purification of the vector, the digested bi-directional mammalian promoter 'derivative fragment is inserted into the restriction site by standard ligation procedures. The ligated vector is amplified in a large intestine rod and the plastid is purified using standard methods. The resulting vector of the selection vector was transformed into E. coli by a conventional private sequence. The 56 200837079 venom was combined into a 3 84-well mother plate and stored. The number of colonies arranged is at least 3 times greater than the number of input PCR products, so the probability of the presence of all unique V gene pairs obtained in the ^ portion is 95 〇/〇. e. Screening The colonies arranged in the d portion were inoculated into a medium of a similar 384-well plate and grown overnight. DNA for transfection was prepared from each well in a cell culture plate. One day before transfection, 384-well plates were seeded in 20 μM medium at 3000 cells/well with ch〇 Flp-In cells (Invitrogen). Cells were transfected with DNA using Fugene6 (Roche) according to the manufacturer's instructions. After 2-3 days of culture, the supernatant containing the full length antibody was collected and stored for screening. Screening was performed using the Applied Biosystems 8200 FMATTM system based on a homogeneous capture of FLISA (fluorescence-linked immunosorbent assay) (Swartzman Specialist 1 999, Anal·Biochem. 27 1:143-1 5 1 ) A number of antigenic lines, including granules, recombinant G protein, and biotinylated peptides derived from the RSV antigen, were used for screening. These peptides are derived from the conserved region of the G protein (amino acid 164-176) and the cysteine region (amino acid 171_187, strain Long and 18537) and the extracellular region of the SH protein (amine of the A2 strain) Amino acid 42-64 and amino acid 42-65 of the 18537 strain. The RSV strain was cultured by incubating 300 μΐ 5% w/v polystyrene beads (6·79 μm diameter, Spher〇tech) with 300 μ ΐ virus stock solution (protein concentration: 2〇〇叩/) Long inactivated virus particles (HyTest) are immobilized on the beads. The soluble recombinant G protein (amino acid 66-292 of the 18537 strain sequence) was similarly immobilized directly onto the polystyrene beads, while 57 200837079 captured the biotin-labeled peptide at pre-coating antibiotics at saturation/density. Streptavidin polystyrene beads (6 〇 _8 〇 diameter,

Kisker). The coating mixture was incubated overnight and washed twice in pBs. The beads were resuspended in 50 ml of bovine serum albumin containing pBs (PBS/BSA) and 5 μM goat anti-human IgG Aiexa 647 conjugate (Molecular probes). The 1 〇 μΐ resuspended coating mixture was added to the 2 〇 丨 antibody-containing supernatant in a FMAT-compatible 384-well plate and cultured for about 12 h, after which the fluorescence at the surface of the beads in the individual wells was measured. Fluorescence events are considered positive if the fluorescence intensity is at least 6 standard deviations above the background baseline. Pure lines that are initially compatible are recovered from the original master and collected in the new plate. DNA was isolated from such pure lines and dna sequencing for the v gene was submitted. Compare these sequences and select all unique pure lines. Further verify the selected pure line. Briefly, hlO6 Freestyle (9) cells (lnvitrogen) were transfected with 7 of the selected pure line of DNA and 0.3 pg of pAdVAntage plastid (Promega) in 2 ml of Freestyle medium (Invitr〇gen) according to the manufacturer's instructions. Two days later, the supernatant was clarified by FLISA and/or ELISA using different antigens for preliminary screening and recombinantly purified F protein and E. coli produced prion protein fragment (amino acid 127-203 of the sequence of 1 8537 strain) IgG performance and activity were tested. The antibody supernatant was tested in serial dilutions and the pure lines were fractionated according to antigen activity. f. Pure line repair When using the multiplex PCR method as described in section c, v-base 58 200837079 is expected to undergo a certain degree of cross-priming due to high homology between the family (d) and the v gene family. Cross-priming introduces amino acids that are not naturally produced in the immunoglobulin framework, resulting in a number of factors, such as structural changes and increased immunogenicity, which result in reduced therapeutic activity. In order to eliminate these disadvantages and to ensure that the selected pure line reflects the natural humoral immune response, the mutation is corrected during the so-called pure line repair. In the first step of the pure line repair procedure, the Vh sequence is amplified with a primer set containing a sequence corresponding to the vH gene from which the pure line of interest is derived, thereby correcting any mutation caused by cross-initiation. The PCR fragment was cloned and Asclylated and re-ligated into the mammalian expression vector digested with Xh〇I/ASG1 using the f-knitting procedure (Table 3). The ligated vector is amplified in E. coli and the plastid is purified by standard methods. The sequence was sequenced to verify that the calibration was used τ and only the NheI/N〇tI digestion vector was used to prepare it for insertion of the light bonds. In the second step, the entire light chain is subjected to PCR amplification using a primer set containing a sequence corresponding to the VL gene from which the pure line of interest is derived, thereby correcting any mutation caused by cross-initiation. The PCR fragment was digested with NheI/NotI and ligated to the vH-containing vector prepared above. The ligation product was amplified in E. coli and the plasmid was purified by standard methods. The light chain is then sequenced to verify the correction. In the case where the K constant region of the selected pure line contains a mutation introduced during gene amplification as described in section c, it is replaced with an unmutated constant region. This was done in abundance PCR in which the % repaired gene (amplified without a constant region) was fused to a constant region with the correct sequence (obtained in a separate pcR: 59 200837079. The entire sequence was amplified and colonized) To the above Vh-containing vector and to sequence the repaired light chain to verify the correction. ^ g. Generation of multiple cell lines The production of multiple strains of recombinant multi-strain antibodies is a multi-step procedure, including the production of each The single vH& VL gene sequence represents an individual expression cell line of unique antibodies. Multiple cell lines are obtained by mixing individual cell lines and dispensing the mixture into ampoules to generate a multi-study cell bank (pRCB) or master cell bank. (pMCB), a plurality of working cell banks (pWCB) can be produced by amplifying cells from a research cell bank or a master cell bank. Usually, a plurality of cell lines derived from prcb are directly used without producing pWCB. The individual steps of the method are as follows: Transfection and selection of pen mammalian cell lines using the Flp-In CHO cell line (Invitrogen) as the starting cell line. In the cytoplasmic cell line, the parental Flp_In CHO cell line was sub-selected by limiting dilution and several pure lines were selected and amplified. Based on the growth behavior, a pure line CHO_Flp-In ( 019 ) was selected as the starting cell line. CHO-Flp- The In ( 019 ) cell line was cultured in adherent cells in HAM-F12 with 10% fetal calf serum (FCS). The selected and repaired VH of each of the f-containing fractions was used in a T175 flask using Fugene 6 (Roche). Individual plastid preparations of the VL-encoded pair were co-transfected with Flp recombinase-encoded plastids to approximately 19χ106 CHO-Flp-In (019) cells (see WO 04/061 104 for details). Cells were pancreatic after 24 h. Protease treatment and transfer to 2 layers (1260 cm2) cell factory 200837079 (Nunc). Add 48〇pg/mi geneticin (Geneticin) after 48h transfection, and culture in the presence of geneticin to recombine cells The selection was made. A pure line appeared approximately two weeks later. The pure lines were counted and the cells were trypsinized and subsequently cultured as a mixture of pure lines expressing one of the RSV-specific antibodies. g-2 for no blood; α(^αρ〖αΗ〇η ) will be individually adhered Cell cultures expressing anti-RSV antibodies were trypsinized, centrifuged and transferred to separate shake flasks (25 〇ml) in appropriate serum-free medium (Excell302, JRH Biosciences; 500 pg/ml geneticin) In the anti-aggregation agent (1:25〇) and 4 mM L-glutamic acid, it was 1.15χ1〇6 cells/ml. Track growth and cell morphology for several weeks. After 4-6, the cell line typically shows good and stable growth behavior with a doubling time of less than 3 〇 h, and then the individual cell lines that are adapted are frozen in multiple ampoules. The individual antibodies that were expressed during the adaptation were purified from the supernatant using the method described in section 1). The purified antibodies are used to characterize antigen specificity and biochemical properties as described below. The characteristics of the pen 3 cell line ^ characterize all individual cell lines in terms of antibody production and proliferation. This following test was performed: Generation: The production of recombinant antibodies from individual expression cell lines was followed by kappa-specific EUSA during adaptation. The goat anti-human Fc purified antibody (Ser〇tec) used in the carbonate buffer (ΡΗ 9.6) was coated with an ELISA plate overnight. The plates were washed 6 times with 61 200837079 Wash Buffer (PBS; 0.05% Tween 20) and blocked by incubation for 1 h in wash buffer containing 2% skim milk. The cell culture supernatant was added and the culture was extended for 丨h. The plate was washed 6 times with a wash buffer and a second antibody (goat anti-human K HRP,

Serotec) and repeat the culture. After vigorous washing, the ELISA was developed with a τμβ matrix and the reaction was stopped by the addition of E^SO4. The plates were read at 450 nm. Furthermore, the total amount of expression was measured using intracellular staining and the homogeneity of the cell population associated with the performance of the recombinant antibody was determined. 5xl〇5 cells were washed in cold FACS buffer (PBS; 2% FCS) and then fixed by incubation in CeUFix (BD-Bi〇SCiences) for 20 min. The cells were centrifuged into pellets and permeabilized in ice-cold methanol (permeab for 10 min and washed twice in FACS buffer. Fluorescently labeled antibody was added to the suspension (goat F (ab ') 2 fragment, anti-human IgG (H+L)_pE,

Beckman Coulter). After 20 min on ice, the cells were washed in faCS buffer and resuspended, followed by FACS analysis. Proliferation: Take an aliquot of the cell suspension two to three times a week with vi_ Cell XR (cell survival rate (ceU viabinty) analyzer, Beckman

Coulter) analysis determined cell number, cell size and viability. The doubling time of the cell culture was calculated using the number of cells obtained from Vi-Cell measurements. G-4 Characterization of antigen specificity of individual antibodies The antigen and epitope specificity of the individual expressed antibodies were evaluated to produce anti-RSv rpAbs with well-characterized specificity. As described in Section 62 200837079, antibodies identified with a single rsv antibody during screening were assessed by FLISA, ELISA, and surface plasma resonance (SPR; Biacore).

Original (recombinant G protein, recombinant or purified F protein) or its peptide fragment (conserved region of g protein subtypes A and B and cysteine core motif, and extracellular domain of sh protein subtypes A and B) The binding specificity is used to verify these antibodies. Antigen determinant specificity was determined in an ELISA by competition with commercially available antibodies that were well characterized, some of which are shown in Table 4. Not all of the antibodies shown in Table 4 are required to characterize each of the antibodies of the invention, and it is also possible to use other antibodies or antibody fragments (with respect to the antigen, antigenic site and/or antigenic determining characteristics to which they bind) ). Briefly, the immobilized antigen (RSV Long particles, HyTest), which is used for antigen-based blocking of the anti-sputum or antibody fragment, is cultured in a large excess, that is, the concentration is empirically determined to produce 75% of the maximum binding. 1〇〇 times the concentration (Ditzel et al., J. Mol Bi〇1 1997, 267 684 695). After washing, individual antibody pure lines were incubated with various concentrations of blocked antigen and any bound human IgG was detected using a goat anti-human hrp conjugate (Ser〇teC) according to standard ELISA protocols. Characterization of epitope specificity is further characterized by the use of saturation at 1 degree (according to experience by <blocking and detecting antibodies for pair-wise competition between different antibody lines). Direct amine coupling (Biac〇re) and a purified purified F or G protein is used as an antigen. In the epitope based on ELISa and Biacore, Φ, # > ^_ & ^ reduces the binding of the epitope after blocking Non-competitive binding comparison. 63 200837079 Table 4: Monoclonal antibody MAb/Fab antigen epitope locus (aa) for epitope mapping of anti-F and anti-G antibodies Reference 131-2a F FI Fla 1, 2 9C5 F FI Fla 5 92-llc F FI Fib 1,2 102-10b F FI Flc 1,2 133-lh FC F2 1,2,3 130-8f FC F2 (241/421) 1,2,3, 4 143-6c FA/II F3 U,3 palivizumab FA/II (272) 8 1153 FA/II (262) 3,4 1142 FA/II 3 1200 FA/II /(272) 2,4 1214 FA/II (276) 3,4 1237 FA/II (276) 3,4 1129 FA/II (275) 3,4 1121 FA/II 3 1112 FB/I (389) 3,6 1269 FB/I (389 3,6 1243 FC (241/421) 3,6 Fab 19 FA/II (266) 7 R SVF2-5 F IV (429) 4 Mabl9 F IV (429) 12 7.936 FV (432-447) 13 9.432 F VI (436) 13 63-10f G(A) Gil GCRR (A171-187) 1,2 130- 6d G(A) G12 (A174-214) 1,2,9 131-2g G (A+B) G13 (150-173) 1,2,9 143-5a G (A+B) G5a 2 L9 G ( A+B) Al/Bl Conservative (164-176) 14,15 8C5 G ND 5 1C2 G(A) ND GCRR (A172-188) 10,11 3F4 G(A) ND 10,11 4G4 G(A) ND GCRR (A172-188) 10,11 The "antigen" column indicates the RS V-associated protein to which Mab/Fab binds, and if subtype specificity is known, this is indicated by (). "Antigenic determinant (aa)" column 64 200837079 indicates the name of the epitope recognized by Mab/Fab, and () indicates the amino acid position at which the RSV escape mutant is produced, or the peptide/protein fragment to which it binds. The numbered references (Ref.) given in Table 4 correspond to the following: 1. Anderson et al., J. Clin·Microbiol. 1986, 23: 475-480 〇 2. Anderson et al., J. Virol· 1988, 62: 1232-4238 〇 3. Beeler & van Wyke Coelingh, J. Virol. 1989, 63:2941-2950 ° 4. Crowe et al., JID 1998, 177: 1073-1076. 5. Sominina et al., Vestn Ross Akad Med Nauk 1995, 9:49-54. 6. Collins et al., Fields Virology, p. 1313-1351. 7. Crowe et al., Virology 1998, 252: 373-375 o 8. Zhao & Sullender, J. Virol. 2004, 79: 3962-3968. 9. Sullender, Virology 1995, 209: 70-79. 10. Morgan et al., J. Gen. Virol. 1987, 68:2781-2788 ° 11. McGill et al., J. Immunol. Methods 2005, 297: 143-152. 12· Arbiza et al., J. Gen. Virol. 1992, 73:2225-2234. 13. Lopez et al. J. Virol 1998, 72:6922-6928. 14· Walsh et al., J. Gen. Virol. 1989, 70: 2953-2961. 15. Walsh et al., J. Gen. Virol. 1998, 79: 479-487. In addition, the characteristics of the antibody-derived lines were determined by FACS in combination with human laryngeal epithelial HEp-2 cells (ATCC CLL-23) infected with different RSV strains (Long and B1) 65 200837079. Briefly, HEp-2 cells were cultured in serum-free medium at a ratio of 0.1 Pfu/cell with RSV Long (ATCC No. / (VR-26) strain or Rsv β1 (ATCC No. VR-1400) virus strain) After 24 h (Long virus strain) or 48 h (B1 virus strain). After separation and washing, the cells were distributed in 96-well plates and diluted with individual anti-RS V antibodies at 37 ° C (4 ρ Μ 2 〇〇 μΜ) The i匕 was cultured together. The cells were fixed in P/o formaldehyde and detected by 4. The armpit was cultured with goat F(ab)2 anti-human IgG-PE conjugate (Beckman c〇uher) to detect Binding to antibody on the cell surface. Similar analysis of binding to HEp_2 cells in mock infection. Also selected by Eus A for the pure line test identified as the specificity of Shibai G and recombinant human Frattalkine (fractalkine) Cross-activity of (CX3CL 1 ; R&D systems) Anti-human CX3CL1/Flatake monoclonal antibody (R&D (10)) was used as a positive control. Characteristics of binding kinetics of individual antibodies: Very low density of recombinant antigen immobilized on the surface of the sensor to avoid mass transfer restrictions, Βίκ〇κ 3000 (Βί ^ 〇ΓβΑΒ, υρρ Fo

Surface plasmon resonance analysis was performed on Sweden to perform the kinetic analysis of the antibody j of the present invention. This analysis was performed using Fab fragments prepared from the pure lines of individual antibodies using the immunoPure Fab Formulation Kit (). Briefly, a total of 2 (1) resonance unit (RU) recombinant protein f or a total of 5 〇 RU recombinant protein g, bound to the surface of the CM5 wafer, was used in accordance with the manufacturer's instructions using an amine coupling kit (Biac〇re). Fab fragments were injected in serial dilutions onto the surface of wafer 66 200837079 to initiate an optimum concentration that would not produce a RUmax value of more than 25 when tested on wafers with immobilized proteins. The binding rate constant (ka) and the dissociation constant (kd) were estimated by (10)4·丨 software (BIAcore) using a predetermined 1:1 (Langmuir) binding and dissociation model.

- By performing a kinetic analysis of the Fab fragment, it is ensured that the data obtained truly reflects the binding affinity for the RSV protein. If an intact antibody is used, the data should reflect the binding affinity (avidity) and cannot be easily interpreted as a meaningful measure of the quasi-breaking f of the binding characteristics of the antibody to the antigen. G-6疋Specific antibodies to the biochemical properties of individual antibodies. Common phenomena of heterogeneous antibodies and recombinant proteins. Antibody modification typically occurs during performance, such as post-translational modifications such as n-glycosylation, protein hydrocracking, and N- and C-terminal heterogeneity' resulting in size or charge heterogeneity. Alternatively, methionine oxidation and deamikamine modification may occur during subsequent short or long term storage. Because of the need to specify such parameters for therapeutic antibodies, multiple cell lines are analyzed prior to production. Methods for characterizing purified individual antibodies (see section i) include SDS_PAGE (reduction and non-reduction conditions), weak cation exchange chromatography (5) X), size exclusion chromatography (SEC) and Rp_HpLc (reduction and non-reduction conditions) ). The sds_page scores under reducing and non-reducing conditions: and SEC indicate that the purified antibodies are indeed intact, with trace amounts of disrupted and aggregated forms. Analysis of the enthalpy of the purified antibody yields a curve with a single peak or a < ^ chromatogram with multiple peaks indicating the selectivity of these particular antibodies. Detailed analysis of the production of sigma peaks in sputum analysis by Ν terminal sequencing and/or generation of light chain or heavy chain abnormal migration in SDS gels or 67 200837079 Complete N-terminal of antibody preparations producing unusual RP-HPLC curves And the heterogeneity caused by the difference in oligosaccharide characteristics was analyzed. In addition, enzymatic treatment and subsequent SDS-PAGE analysis were used to analyze the presence of additional N-glycosylation sites in the variable chain of the selected antibody. G-7 establishes multiple cell lines for anti-RSV recombinant multi-strain antibody production from a collection of selected cell lines to select subgroups to produce multiple cell lines and multiple research/primary cell banks (pRCB/pMCB) ). The selection parameters can be determined based on the use of multiple antibodies to be produced from a plurality of cell lines and the performance of individual cell lines. The following parameters are usually considered: Cell line characteristics; to optimize the stability of multiple cell lines, the car father has a doubling time between 21 and 30 hours and beyond! Individual cell lines of Peck/Cell/Day antibody production capacity. Reactivity; carefully consider the antigen/antigen site and epitope against which the anti-RSV rpAb should be reactive. Protein chemistry; preferably, the anti-RSV rpAb preferably includes antibodies having well-defined biochemical characteristics. Each individual cell line expressing each of the recombinant anti-RSV antibodies was thawed and expanded in serum-free medium in a shaking flask at 37 C to achieve at least 4 各 of each pure line with a population doubling time of 2 1 3 4 1 〇 8 cells. The survival rate of the car is between 93% and 96%. A plurality of cell lines were prepared by mixing 2 x 1 〇 6 cells from each cell line. A plurality of cell lines were distributed in frozen ampoules containing 5·6χ1〇7 cells and frozen. This collection of vials with multiple cell lines is called a multi-study study/primary cell bank (pRCB/PMCB), and multiple working cell banks (pWcB) can be generated as follows. · 2008 200879079 by one from PRCB/pMCB The ampoule was expanded to achieve a sufficient number of cells to establish a multi-plant cell bank (pWCB) of approximately 200 ampoules with the same cell density as the prcb/PMCB ampoule. Mold fungi and sterility were tested on samples from the cell bank. h· Recombinant multi-strain anti-RSV antibodies are expressed in a 5 a liter bioreactor (B. Braun Biotech International,

Recombinant multi-strain anti-RSV antibody batches were produced in Melsimgen, Germany. Briefly, vials from pRCB or p WCB were thawed and expanded in a shake flask (Corning). The cells were cultured in ExCell 3〇2 medium with G418 and an anti-aggregation agent at 37 ° C, 5% c〇2. The bioreactor was inoculated with 0.66 cells/ml suspended in 3 1 ExCell 302 medium without G418 and anti-aggregation agent. The number of cells/viable cells were monitored by mother day counting by CASY or viCeU. At h, 2000 ml of ExCell 03 medium was added and a temperature drop of 3 7 ° C to 32 ° C was performed after 92 h. After 164 h, the cell culture supernatant was collected and subjected to purification as described in section i). i. Purification of individual anti-RSV antibodies and multiple anti-RSV antibodies using MabSelect SuRe column (protein A) pairs such as g.g_2 and h

The antibodies (all IgG1 subtypes) shown in the section were subjected to affinity purification. Individual antibodies interact with immobilized protein A at pH 7.4, and the contaminating proteins are washed away from the column. The bound antibody was then applied from the column/valley by lowering the pH to 2.7. The fractions containing the antibody (measured by absorbance at 28 〇nm) were pooled and the buffer was changed to 5 mM sodium acetate, 150 mM NaCl (pH 5) using a 〇25 column and at -20 °C. Store. 69 200837079 j. In-vitro neutralization test y-/Preparation of live use in vitro The human laryngeal epithelial HEp-2 cells (ATCC CLL-23) were seeded in 175 cm2 flasks in 7 cells/flask. RSv L〇ng (ATCc No. 乂11-26), RSV B1 (ATCC No. VR-1400) or RSVB Wash/18537 (Advanced Biotechnologies) strain was used in 3 ml serum-free medium at a ratio of p1 pfu/cell. Perform cell infection. Cell infection was performed for 2 h at 37 C, 5% C〇2, followed by the addition of 37 (4) complete MEM medium. The cells are cultured until visible cytopathic effects. The cells were smeared by scraping and the medium and cells were ultrasonicated for 2 sec and aliquoted, frozen instantaneously in liquid nitrogen and stored at _8 〇 °c. J-2 plaque reduction neutralization test (pRNT) HEp-2 cells were seeded at 2 x 10 4 cells/well in 96-well culture plates and cultured overnight at 37 ° C, 5% C 〇 2 . Test substances were diluted in serum-free MEM and pre-incubated with RSV for 3 〇 min at 37t:T with or without complement (from rabbit complement serum, Sigma). This mixture was applied to a single layer of HEp-2 cells and cultured for 24 h at 37 t, 5% C02. The cells were fixed with 80% acetone, 20% PBS for 2 〇 min. After washing, the biotin-labeled goat anti-RSV antibody (AbD Serotec) in PBS with 1% BSA was added (1:200) and cultured at room temperature for 1 匕. After washing, HRP-avidin (avidin) was added and cultured for 3 min. Plaques were formed by incubation with 3_amino-9-ethyl sulfonate (AEC) substrate for 25 min (RSV Long) or 45 min (RSVBl). At Bioreader (BiQ_Sys

Counting plaques in GmbH). Calculate the EC" value (the number of induced plaques is reduced by 70 200837079 to be used for potency comparison when appropriate. 5 〇% of the required effective concentration) The j-3 fusion inhibition assay is performed as usual for the fusion inhibition test, before infection. In the bloodless HEp_2 cells, the duration is ι·5匕. Basically, as in the plaque reduction and neutralization test, the RSV is added to the monolayer in the addition of the test substance clear medium.

The supernatant was exempted and the test substance was added to complete MEM medium with or without complement (from rabbit complement serum, Sigma). The plates were incubated overnight and treated as described above for the plaque reduction neutralization test. The j- 4 microneutralization test, in addition to the PRNT and fusion inhibition assays described in sections j-2 and j-3, was performed using a microneutralization assay based on RSV protein detection for rsv neutralization and fusion inhibition assays. For the neutralization test, the test substance was diluted in serum-free MEM and pre-incubated with RSV in 96-well culture plates at room temperature in the absence or with complement (from rabbit complement serum). 〇min. Trypsin-treated HEp 2 cells were added at 1.5 cells/well and at 37. (:, 5% eh culture for 2_3 days. The cells were washed and fixed with 80% acetone, 20% PBS for 15 minutes at 4 ° C and dried. Then the plate was blocked with PBS with 0.5% gelatin at room temperature. 30 MIN and a mixture of murine monoclonal antibodies against RSV protein at room temperature (]^(:^劣)3,

Novocastra) (diluted 1:200 in pbs with 0.5% gelatin and 0.5% Tween-20) for 2 h. After washing, multiple rabbit anti-mouse immunoglobulin HRP conjugates (P0260; DakoCytomation) (diluted 1:1000 in PBS with 0.5°/〇 gelatin and 0.5% Tween-20), 71 200837079 :to! Wen Xia Pei # 2 h. The plate was washed and developed by the addition of o-phenylenediamine. The plate was stopped by adding h2Scm kiosk and the plates were read at 49 〇 _ in the Qingshou board. The fusion inhibition assay was performed on the thiol t as a microneutralization test, except that the virus was added to the cells and cultured at 37 ° C, 5% C 〇 2 for 5 h, and then added to the diluted MEM. Test substance. The plate was cultured for 2 to 3 days under Π, 5/〇 C〇2 and developed as described above. k. In vivo protection test Mouse functional toxicity model Female BALB/c mice aged 7-8 weeks were intraperitoneally inoculated with G.2 ml antibody preparation one day before the study. The placebo-treated mice were similarly intraperitoneally inoculated (M ml PBS buffer. On study day, mice were anesthetized with inhaled "isofluorane" and intranasally inoculated 5 〇 y 1 〇 Human 10 pfu of Rsv strain A2 or cell lysate (pseudo-inoculation). Animals are allowed to inhale the inoculum for 30 seconds while remaining upright until fully recovered from anesthesia. After five days of dysfunction, overdose of pentobarbital Sodium kills the mice. The blood is obtained by bled blood from the infraorbital blood vessels to prepare serum. The lungs are removed and homogenized with hot sand in 2.5 ml buffer. The lung homogenate is centrifuged to make the sand and Cell debris was pelleted and the supernatant was aliquoted and stored at -70 ° C. The viral load was determined by quantifying the number of RS V RNA copies in the lung samples using reverse transcriptase (RT_) PCR. According to the manufacturer's instructions, RNA was extracted from lung homogenate samples using the MagNA Pure LC Total Nucleic Acid Kit (R〇che Diagn〇stics) automated extraction system using LightCycler instruments and reagents (Roche 72 200837079)

Diagnostics) and primers and fluorophore-labeled probes specific for the N gene of RSV subtype A, as described by Whiley et al. (J. Clinical Microbiol 2002, 40: 4418-22), with a single tube Instant rT-PCR to perform RSV RNA detection. A sample having a known number of copies of rsV RNA was similarly analyzed to obtain a standard curve. The quality of different cytokines and chemokines in lung tissue samples was determined by using a multi-analyte profile 'MAP' in a commercially available multiplex immunoassay using Rules-Based Medicine (Austin, TX). K-2 cotton rat function model 1 day before the study, 6-8 week old female cotton rats (Brassica chinensis ((10))) were intraperitoneally inoculated with 〇·5 ml antibody preparation or pacifier (PBS). After 24 hours, the animals were lightly anesthetized with isoflurane and administered intranasally with 1 〇7 pfu of RSV strain A2 or control matrix (simulated inoculum). A total volume of 丨〇〇 μ1 inoculum was administered and distributed equally across the two nostrils. After the intranasal dysfunction is completed, keep the animals in an upright position for a minimum of 30 seconds to allow complete inhalation of the inoculum. Five days after the dysfunction, the animals were sacrificed by lethal intraperitoneal injection of pentobarbital and bled by heart puncture. A clear sample was obtained and frozen at -80 ° C and each animal was dissected under sterile conditions to remove lung and nasal tissue. Tissue samples were homogenized and the supernatant was stored in aliquots at -80 °C. The viral load in the tissue samples was determined by quantifying the number of Rsv RNA replicas based on a Taq-Man assay based on the method of Van Elden et al. (J Clin Microbiol 2003 41(9): 4378. 4381). Briefly, 73 200837079 RNA was extracted from lung homogenate samples using the Rneasy (Qiagen) method according to the manufacturer's instructions. The extracted RNA was reverse transcribed into cDNA and then subjected to the use of a Superscript III Platinum single-step quantitative RT-PCR system (Invitrogen) using primers specific for the N gene of RS V subtype A and a housekeeping group. p CR amplification. A sample having a known RSV concentration was similarly analyzed to obtain a standard curve. Example 2 In this example, the isolation, screening, selection and construction of a homologous VH and VL pair containing a full length antibody having anti-RSV specificity are described. Donors A total of 89 donors were recruited from employees who were hospitalized in the Department of Paediatrics at Hvidovre Hospital (Denmark) during the RSV season and their parents. An initial blood sample of 8 m丨 was taken, CD19+ B cells (Example i, part a) were purified and screened for the presence of anti-RSV antibodies using EUSp〇t (Example 1, part b) and analyzed by FACS The frequency of plasma cells is measured. Eleven donors were found to be positive when screening the initial blood samples and 450 ml of the second blood sample was collected from ten of them. According to the Example, & part of the plasma mother cells were subjected to single cell sorting. The ELISpot was partially performed on one of the CD19-positive B cells.

The ELISpot frequency in the second blood donation was identified as having four donors of RSV-specific, m-cell (sand + and 1 gA+) between o' and 〇.6% of the total plasma cell population. These frequencies are considered to be sufficiently high for the continuation of the linkage of the homologous VH 74 200837079 and v L pairs. Isolation of homologous VH and VL coding pairs Nucleic acid encoding antibody profiles were isolated from single-cell sorted plasma cells from five donors by multiplex overlap extension RT-PCR (Examples i, C). Multiple overlap extension RT_PCR forms a physical link between the heavy chain variable region gene fragment (νΗ) and the full-length light chain (Lc). An experimental protocol was designed to amplify antibody genes of all VH gene families and kappa light chains by using two primer sets (one for Vh amplification and one for B. amplification). After reverse transcription and multiplex overlap extension PCR, the ligated sequences were subjected to a second PCR amplification using nested primer sets. Each donor was individually treated and produced by the multiple overlap extension RT_pCR to 2,450 heavy products. The homologous linkages generated from each donor, the vH and vL coding pairs, were confluent and inserted into the scorpion animal IgG expression vector (Fig. 3) as described in section d of the Examples. The resulting spectra were transformed into E. coli and fixed in twenty 384 well plates and stored. The spectral composition consists of a pure line between lxl〇6 and 3.6X106 per donor. Screening The supernatant containing the IgG antibody was obtained from CHO cells transiently transfected with DNA prepared from the fine sputum of the mother board. The supernatant was selected as described in the Examples section. About _ a preliminary qualifier is sequenced and aligned. Most of them belong to a cluster of two or more members, but there is also a pure system that separates only a single copy sequence (singlet〇n). The representative pure lines from each group, single copy sequence were subjected to the validation assay as described in the section i, e). &, in the v疋 feature excludes many preliminary qualifiers, this 75 200837079 due to inappropriate sequence characteristics, such as unpaired cysteine, non-conservative mutations (which may be PCR errors), insertion of multiple codons and / or missing and truncated. A total of 85 unique pure lines were verified. These pure lines are summarized in Table 5. Each pure line number specifies a specific VH and V1 pair. The IGHV and IGKV gene families are described for each pure line and the framework regions (FR) of the selected pure lines are designated. The amino acid sequence of the complementarity determining region (CDR) of the antibody expressed by each pure line is shown, wherein CDRH1, CDRH2, CDRH3 represent the CDR regions 1, 2 and 3 of the heavy chain and CDRL1, CDRL2 and CDRL3 represent the CDR regions of the light chain 1, 2 and 3. The complete variable heavy and light chain sequences can be created from the messages in Table 5. Further details of the individual blocks of Table 5 are given below. The names of the IGHV and IGKV gene families were designated according to the official HUGO/IMGT nomenclature (IMGT; Lefranc & Lefranc, 2001, The Immunoglobulin FactsBook, Academic Press). Numbering and comparison according to Chothia (Al-Lazikani et al. 1997 J. Mol. Biol. 273: 927-48). Pure line 809 has 2 codon insertions at 5' of CDRH1, which may be translated into an extended CDR loop. The pure line 821 has one codon deletion at position 31 in CDRH1. The "Ag" column indicates an RSV-associated protein recognized by an antibody produced from a designated pure line as judged by ELISA, FLISA, and/or Biacore. "+" indicates that the pure line binds to RSV particles and/or cells infected with RSV, but the antigen has not been identified. The "antigenic determinant" table is not determined by the antigenic site or epitope recognized by the antibody produced by the specified pure guanidine (see Table 4 and Table 76 200837079). "U" indicates that the epitope is unknown. UCI and UCII refer to unknown clusters and rafts. Antibodies belonging to such clusters have similar activity characteristics, but specific epitopes have not yet been identified. Some antibodies recognize complex epitopes such as A and C. The epitope represented by () is only determined in the ELISA.

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%L 200837079 / J% 趄Conserved Conserved A/II B/I/Fl A/II UU Fl&C UCI A&C/IV A&CU (FI) GCRRA A/工工Centr. dom GCRRA GCRR (A/II Conserved GCRRA GCRRA GCRRA GCRRA GCRRA UUUS' rn ^ 0 CP» 00 ΕηΗΕηΕηΕηΗΕηΗΕηΗΕηΕηΕηΕηΕηΒΗΗΕηΕηΗΕηΗΗΕηΗΕη 1 1 1 1 O 1 ω 1 1 1 1 1 1 1 II i 1 1 1 1 1 1 1 1 1 1 CUIlF^^lfUIIIIIIIIIIIIIIIIIIII co cu i 〇 <; cu cu cu 〇i cu ou ic^cucociiaiaiaiaiiijcuc/icuatC^coai QHlS^lSiJi-^iHEHSQHfui-^ljutnii-tljMiiCCUEMCnCu^C/ltHE-· CC;:I::ZiCO;2:ti:Z:tH〇 E~<|2:WWC/)C/3C/3COWCOC/)C/3{/lWHC/)C/DC/3 >Η〇>Η(ΛΡί;>Η>ΗΕΐ4>Ηω>Ηϋ3>Η>Η(<ζ|-^<>Η>Ηϋΐ>Η>Η(-^>Η>Ηω ο ο ο ο ο aw οσοσκ οοσοοοο οοοοοοοο os 〇ι〇ο〇ι〇ισοο» Οοο〇ισ〇ι〇ι〇ι〇ι〇ι〇οοοο〇ι〇ί oooouuuoouaoooooouououououo CM Q m C0(/)HC0E-iDC:H>H>HWa3C/DC/l(/)E-ipC!cy3WC0E-iC0WWC0EHWC ^ ω ^^ο>ΗΡι3αω〇ίωκραωα〇ι〇ιαα<ς<;ω〇ί〇<ι;< a WZE-tS^SSEnE-fEHSWWE-i^WE-iitE-HSEHE-tE-'EHCOE-iW C/ 5C/3(/)C/D(/)C/DC/DC/]C/DC/]C/3C/)C/3C/}cy3C/3C/3WC/DC/}C/)a)C/ 3(/)C/DC/)C/3 ΓΟ tH 0 PO 〇〇<CPCS<CwSCCECS<;C<(iCf<rt:<<CiiC<CC<f=a:fiCS l:Hllllll art lllllllllll ^llllll IP^llllll^lllllllllll^llllll IOIIIIIISIIIIIIIIIIISIIIIII 1 a....... 1 1 1 1 1 i 1 1 1 1 1 ^ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CO 1 1 1 1 1 1 1 1 | 1 | 1 1 1 1 1 I IWIIIIIi^llllllllllpcicOlliaDII {/)p^Ocow>HQco>HC〇c〇(y)aD^:Si^c〇BS0>HSSc〇(yDfii;w OhJOrcCaiEHOco^cnOCE -i^QcowOcoOHqwcoaDc^cow μ^μμ>μμιη>μμιη>μμμμμμ>>μμμ>μμ coaDE-iHcnQQQc^tzjWWWSQOOOOWWHOOWOW ααασααα〇ι〇ια〇ι〇ια〇ια〇ί〇ιααα〇ι〇ι〇ι〇ι〇 Οο cowaDWcowcnc^cncoc/acocowscowcoaDcnwcocowcnww Ddpci^aa〇i〇ipii^piaaaciPic^〇^^^p^pc;cdciaaaK IGKV gene i 7 7 1 1 · i 7 1 ? 1 i ^ V i 7 7 ii 7 * i ^ i ? HcMrOrHOO^^^^THdrHddrHtHdrHrHPO 守^τΗ^ΟοΗ^Η Ο 00 ta (3⁄4 QO cn S><Offi>l-^ffiQt-lStH>H>>H[u>H>MQfl4>H>HM>>Ha)l-^QQQQQ!LDQ[i3QQQQQQ<;QQQaiQQQQQ>[£lQ 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Θ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 O 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ΙΙΙΙβΙΙΙΙΙΙΙ;Η||||||||| Art III l^tMlOlllllll^lllllllll^Olll 1 CU 1 >-· 1 1 1 1 i 1 lWl>hSl 1 IHI<C>H| 1 1 O >>-· 1 I 1 1 1 1 Pm rt5 1 ff: g: U 1 1 1 CU 1 Q >H 1 1 1 >H Eh >H >-( 1 1 1 1 1 1 >H t, ω tM 〇1 1 1 0 1 Q 1 1 1 S 〇^ >-* ω 1 1 1 U4 1 1 >HW>lSWtM 1 >[rjt4tMpi>H 1 1 h4 HPCWdffil 1 1 < 1 | 1 1 1 >H > Q >-· C〇1 1 | Q | tn>H>〇(^e}pI; | 〇〇ωω>Η(ϊ: I is: W 〇&gt ;-.>-( >nii >iw ip^>hh>s>ci4(cC ii hacker ω σ) r< O CO 1 1 0>H 1 S〇CO>〇>CU l 1 1 M SOSOS 1 1 &lt ;CCUt4C<COCOWSHP^fcCi3E-i〇iiCWEH>HtMEH S E-ι woi tM(^>s>wco〇co〇it-^<CSiHeiMe)fiCQ>Ha) 00CT;〇rt:OfcO^>H〇 SQ〇>-'SO<>QtnO^EHWW〇^S «Λ(^οι^>-·ο〇2ΕΗΐ=ί:ΕΗϊΗ〇αοω>〇>Ηα2ε〇>ΗΜ>Ηϋω P^〇H&gt ;〇Di:〇WC3<l;MM>aDtnSi2:ai&4〇WQKE-iCi4a)〇OOffiO^〇KWU<CltC;<;Qfeff:WEHhq[£|Ct4〇〇c〇0> ^MQE- iQ〇l>QQ>MEHQ^Q0Q〇〇IQQWW〇PC:WrE: (^:SW(^DC;CliPiPC;!^EHD^〇i;〇C;ttii^〇£i〇tiP£i$PiiC^Hfr :pc:C/3Wff: OUUUUOOOOUOUUU UOOUOOUOOCJUUO 臣Q m OOOHWOWWOOOE-tOOOQOWOOQOQOcnwO (OODWCOWCOCOWi^i^^Wi^OiCO^^COWWpCiSWi^COWW woQE-'CucMi^CMCMaoE-ioioQwoiPMQQoaoacucMfi; fcCCfiCD^SE-'SSCCWS.-qw^^C ^icCHr^fcCffi^Sn:^ >H>H>Htl>H>HDC>H>H>-lCn^>H>-H>HiH>H>H>H>H>HiH>H>H>-l>H>-l>HMPC!<C>HEHpM2iW0DiCl4>H«O>HS>H^tlQ>H^>H>-tQWωωΕΗ^ΕΗΗΕ-<ΕΗ^ΕΗΕΗ«ΗΕ-<^Ε-ιΕ-ιΕΗ^^ΕΗΕΗΕ-·ΗΕΗΕΗΗ (itQiHQOcnOOOOOQOOWOOOSUeiOOOOOiH OOOQWQWOODQSQSSOSSWOOSSSZC^WW QC〇Q^Q〇ffil^W[£l〇^SHe)Qffi>HQQQ>H>H>HQffiCu!2: 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <C 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ^ >-· <d J 1 ID^I 1 <:(!!< 1 (<>>!(<> 1 >η>ι<<<<lt ;Εη 1 1 w cnaaDQ>H>H[u>Ha3QSQc/3SW(/)w>HC〇aiwwc/3〇>H>H〇£: rH S 8 cv, Γ0 acEHUwwooowKKcowaaniscnoKKwwwwwwa: cS〇o>n^pM&gt ;H〇cyD〇w〇^〇0〇>He)e)e)〇〇0w>HE-t >H>-irr:>>HWffi>HSl-^SS>1^M>H>HKtM>-lCtliHCO>HiMSCO IIIP^QllllltCQIIIIIIIIIIIte)^l IIIOCIOIIIISIIIII0IIIIIIOQI QHE-iCOSWScnfcCEHOClE-t 1 iHHWWE-iWWpiiSWCOCOO OH 00 ^ <M 1 P0 Ο Ο 1 rH n(J\CO^rn^CDCO^(DCO<X> n 〇OOOCOOOOO 1 rH 00 ΓΟγΗτΗ^ΟΊΟΟτΗγΗΗγΗΟ^ΟΓ-ΓΟΙ liPOl'lll'l 1*111*1 IIIIIPOII 1 on 00 04 1 L〇^ί-ίΓΗ ΟατΗ^ΟΟΓΗτΗ^Ρ ΟΟτ-ΙτΗτ-ίτΗ I Γ0 oo ^ ^vor-oocncNPO^Lnr-oocriO^HcO'sriovDoocrirHCMooLnkDcxicri THtHrHrHtHCNJCNCNCNlCNCNJCNOOrOOOPOrOPOOOPO^r^^1^^^^ 〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇CX)CX )〇〇〇〇〇〇〇〇CX)〇〇〇〇〇〇〇〇〇〇〇〇CDC〇<X>〇〇〇〇〇〇

6L 200837079

(Α/ΙΙ) GCRRA U Centr. dom GCRRA GCRRA FI B/I/Fl Conserved FI A/II GCRRA (FI) Conserved Conserved (FI) UCI Conserved U UCII UCII UUA/II U (FI) GCRR GCRRA [^〇[ jL4〇〇C)[jM[jui〇[i||i4〇|jM〇〇[j4[l4C)|iitL(&4t4 + tl (i4 〇〇cn cn 00 ΗΕηΒΗ^ηΕ-ιΕηΗΕηΕ-ιΕ-ιΕηΗΕ- 'ΕηΕηΕ-'Ε-'Ε-ιΕ-ι&ηΗΕ-'Ε-ιΕηΗΕ-' Guest OiHnqwPuIS 窆H>h 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 ΙΙΙΙΙΙΙΙΙΗΙΙΐμίΙΗΙΙΙΙ£ϊ;ΙΙ&ιΙΙΙ COOtCUCUOtiliCliCliD-iCU 1 (XiCUCUdidiCIiaiaiDjai II OidiWCU MO^SE-iCiqOIScnWQHSiS&HWSWSWWWCn s CO 〇CO SHKOQM^<CS>HWSrt:S〇iHQSS>H>Ht4Di:S^^S w cn >h οα〇ιασ〇ι〇ικ^σσ〇ικωσ〇ι〇ι^〇ισοωαΰ οοσο OS Θ 〇i Ο» SS〇l〇i〇ia〇i〇l〇i〇i〇l〇l〇 〇l〇〇lCH〇l〇〇S〇i ouooououooououuuoooouoououo CNJ 3 LT) Q WWE-t<WWO)E-iCOWEHE-iEHEHE-<WCncnE-iCOWCOE-<HWWC/D ω^>$ο<< Ωσ〇ΐ4〇ιω is called cuowoif^o oK^ <c σ ω (Oi^Sc〇(y3>HS!^EHcnSE-<Q(<WWSEHE-tc〇2:c〇SEH〇c; MW cncncncncnc/icnE-icniOinciiE-icnuicnuicncncncncnuiui/ia^cn CME-i<CfcO<Cf=C<<(iC<C»=Crt3f=i<C<C^CiiC<C<C<C<<C 0 <1 QOQOf^Ei^QiCfiJQicCQOOfiCQiiCOsCiiC^ QWCt-qi^ 00 τΗ Q ^ CM fcCWSCCQQSCSCCSKClcCSSCCZSSCrtlSCiC mouth mouth qqql_q(jjl_ql_q>>(_qHqHq>1_q(Jl ΙΕ-ι|ΙΙΕ-·^;ΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ^;Ι Ι^ΙΙΙ^;>-ι|| |||||||||||||||>ι| ιοιιιοωιιιιιιιιιιιιιιιιιιιi IQIIIQSIIIIIIIIIIIIIIIIII^I 1 1 1 1 1 ^ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 till IWISICOD^ IIIIIIISIIIIIIIIIIEhI SS>nwSQffiSSWWHS5sQc〇s^icocow>i^a3cocc:co >i>cncna:^^^)c^<icn(n^:t^cna:cnpi:E-i<<cnB-i^f ^i-^c^ Mt-^>>MH^t-^MMM>HIHMt-iMHM>MMM>> MJM SWQWCcOCOQOtHWLDQcOWOiOCjJWEHBHWWEHC/DW 〇〇〇〇〇|〇|〇|〇|〇|〇|〇〇 |〇|〇〇〇1〇|〇|〇|〇|〇〇|〇ο ο ο σ (^p^〇i A〇ctac;Di〇ittacc:a〇iaaa〇ia(xaapc:a(xapcifi IGKV gene 丄ι^ιΛιιλιιι Λιιιλιιιιιιιι Λ CMjqPO^CMCNj^t-liHrO^^OOrOtH^rHrOTHTHrHPOOOrHCN 〇cn EC Pi P o cn 窆:SI2: 2: SS: S 窆: S 萁 3: customer isss > H > H > Hty > HM > § > HWHq > a) fl < Wffi > H > H (-PPC! C / DiHCliai ^ M >QQQQQQQQ〇QQQQQQaQQQKQ>HQClQQQ 1 1 1 1 1 1 S 1 1 1 1 1 i 1 1 1 1 1 1 1 1 1 1 till 1 1 1 1 1 1 O 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 ΙΙΙΙΙΙΩΙι-^ΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ^Ι llltMll>H|cnillllllllllllllll lli>-i||>H|(/)lbllllllllllllllQl ΙΙΙ>Η|&Μ>Η|^Ι^^ΙΙΙΙΙΙΙΙΙΙΙ I 1 S ItxjlSlOCUI ^tj^Ol 1 1 tj t-^ 1 1 t. 1 t4 1 Pm 2 O ΡΜ>ιωιαα3ΐω>Η>-'^ι iq>h>^i>h>hi>h i>hm(^ >H Cn CO 1 1 S 1 >H CO 〇C: C tM ^ Cx. 1 1 ^ tM tq UH ◦ ϋ)θ〇Ι SC〇CUW>H〇?HCljffie)^rr!E-iQ>H>H | M ^ >H > til W 0 1 SC〇C;|S>HCUW Ci;(liWiHpC:〇〇>H&g t;H[tjC〇Q>H〇J >Ρ00ωΡχ4Ρ>〇(Λ〇>Η>〇[£1>-<Ο3:μΐ^Ο〇ΜΜ>ωΖΟ [£|WfiCW>H>>WEHCO 〇CiE-i〇><〇W^E-'WCOMQ ^ Q ^: 〇Ο^Η〇ΕΗ〇ΜΚ>ωωω>Η^Ε-'(ΛΜ(Λ>Ηεοσΐι<ΕΗ cu 〇(3⁄4 ^〇 c〇Cnc〇CQWSQS: WWEHi-^QW&Ha3E-<Qa3> cri <>h iq ffia,〇>npc:HM0>>H^^〇hqiH>H>HW>H>H>- I〇>a>-'I^S 〇O0D^>-'l^^i^JO>QE-'^M^t4C£|>H〇>H>H〇>Q>|i , 2 〇D^CUMJe)aiSCDW>H〇EHEHi^ mouth COCDP^fiCQSH^ Et i-^ O tP WQ<;CUQQWSOQQOt-qQQ〇riCWE-tWC0SQ Eh Et P ^oiPiPdpClPCj^SE-'^^aiOciPci^OciaOi^ffiOiPciffJi ^PiiOtiCti υοοοουοουυουυουυουυοουοοοου (N ffi (3⁄4 QL〇OQOOOOOOOOOOOWOWOOOWOOWCDE-tcoO fc[x4li,tJC34Hq>t4>>>Cx4tMt^>l-q>>>Kq>bMHq>^^^ pcjtqi^wi^swec/ Iwwi^i^wwcowcowcowi^wm in m cC auaCUWOPOQQQOICLjWQGjQQQiliPaCU Q EI CU a cn<CCco<C<COCEHf4:Cf3:<!2;<SO<C&lt ;CPci<Cc〇W<CaDS<: >Π>Η>Η>Η>Η>ί>Η>Η&4>Η>Η^>Η>ΗίΗ>Η>Η(/1>Η>- Ι>-Ι>Η>Η >H >-l >H >H >H tl HEnCU<CEHE-'E-'&4!^^HEHaif=5:WEH^«E-ipciME- Icn^^HEH nCSHQJSSWtyi^SeSHC/iSWSHHQSScOSQEHS ooswoe)e)〇c〇w>oo〇Ei^c〇se)QwoowQ〇o SS^QSSOJOOE-tS^WWHQOOQOSCoOQcoS <fflt〇rf:c〇>H( OEHQQWQc〇ffiOSQQO^W>ffi Q ^ ^ in 1 1 ! 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.... .1 1 1 1 1 1 1 1 1 1 1 1 1 1 <C<CU〇4〇Pi:C)CU>HZrt:(CCCLt 1 ^ 1 in ffi O 1 Z < 1 S 1 1 C Sw>Cncy)waD>wswco>naww^a)wQ〇sffiwQi^wMEhJMMIHM>MIHMM>MMMMMM>MMS mmmm H^[SPC:>3:S:ODc:>PMC〇^pcico>W>><: (-qffi>HW>〇c:cn^ ffi 00 ptj 8 - ΕΗ0Επ^<0<;Η00Η0ΕΗ>Η^ΪΗ00^0ω^?Η0ω>Ηϋ >ΗΗ^>Η>Η>Η>Η>Η ^^>Η>Η>ΗίΗ>Η>Η>Η>Η>ΗίΗμ-ί^[^Ι>>~l S 13⁄4 >H ι S ι ι ι ι ι ι ι & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ σ ;TirHCX)OOOOC^r〇OOOOOCT\ n〇Cnr〇〇r〇n〇0„ n〇〇(Ti00 fT)rHUDLDrH1-iCM^〇P000<NTHMDYP0lOP0r0Cs3 / ^\;ripr〇r^P〇TH *llllllllllll;lllll *l on ^pllll ^rHrHLOrHt-irOi-HrOOOCOiHTH^OO^POrOrO^OO^^rOCM^rH « OiHCsiroLnvDr'Oocri^Hro'^r'OocnorH^cnorH'srLOVDi^oocr» LDLDLnLOLOiDLnLnLn^vo^Dko^^r^r -r'r'Oooooooo oooooooo CDOOOOOOOOOOOOOOOOOOOOOOOOOOOOCnOOOOOOOOCOCXJOOOOOOOOOO 08 200837079

Dom U Conserved UCI Centr. dom A2 aa42-64 Ο O + O Pm O ® cn 00 8 00 Pm Pd Pm (x4 (jq Pm Eh Η H Fh Η Η H >h pc: ^ μι ω ^ 1 1 1 1 1 1 1 1 1 ! 1 1 1 1 1 CU P-ι CU 1 C^i Di Eh H >h ^ ^ >h WOW 〇ws WK Port ZBQQS 〇C DC C >-· >h >h OO 〇〇〇〇〇〇S β β 〇l 〇l 〇uouuuuo CM ^ LD Q ω C/3 W CO HH (7) ω $ ohq 4 σ (3⁄4 ^ ^ SBSH Et w κη mmm Wt mm < O <1 m <c <3 < C h^l O ^ OO PO Η 8 00 C\JSQO c〇ccc η θ θ θ θ θ Θ Cti ffi Q >h S ffi ^ 1 2: 1 Η 1 1 1 1 >i 1 S 1 1 1 1 O 1 〇1 1 1 1 S 1 Q 1 1 1 1 1 1 1 1 1 1 \ cC \ m \ ii H ffi ^ pc: SC/D w cn J Cc: > O CQ Eh MJMJ >> M 'z :n (j ω ω ω zn σ ο o Di ο a pc: ww 〇cn W σ) co <CW <; CO fCC fO; < Di pc: a pci aaa IGKV gene cr» oo r- l〇l〇^ 00 CN3 rH <NT-1 rH u/ 1 1 1 1 1 1 V. fH CNJ rH CN 00 00 ο η X Λ Q 〇 Cn ^ ^ ^ ^ ^ 3: ^ tli >>h>h ffi M Eh QQQQQPQ 1 1 1 1 1 1 1 1 1 1 1 1 1 ^ I 1 1 1 1 1 W 1 1 1 1 1 Pm >H 1 1 1 1 IQ > 1 1 1 1 1 O iiiii ω Geisha丨丨丨1 O IS 1 1 1 1 o Q 1 1 1 tM >-1 Q HI 1 1 >H ^ C >丨1 Pi: (_q >H qwii co 〇o > ^ Pm Q CO > 〇CD 〇CU >H ^ C3 >HOSHM hJ ω w cu >ho <ca pc: 〇〇H tM >< M CU 〇CO CM MO <; ^} C > Q cti Di Ou, Eh ct: M <><C<C〇<3<; uouoooo CN S Λ Q IT) Q Ο Ο Ο Ο WQ 〇^ ^ ^ cr: ^ σ tM >>>> HC/3 (3⁄4 w (7) (7) ¥ 〇Q <C s Q Dj W ^ 2! into >H >H >H >H >H >H >HBQQ h >;Ί U4 Eh s Fi WM E-ι HO] ffi <C ^ H 0 s OC cn C3 〇OOOOO CO o WQ ω QQ Λ Q 1 1 b 1 1 1 1 1 1 pc; 1 1 1 1 〇| &gt H CO >H DC: 1 〇4 S ω ^ OD ^ ffi s Μ > Μ Μ M > (-^ H > Yan >> (7) H aa λ 8 - DC KW ffi KOKM ssss ^ s >H Eh ^ Ο O >-· O tl >H <;>-l>H>H>H 1 1 1 1 1 1 1 iiiii ω i MWSHQC/D Ω CD HOL〇Ο ro np〇tH po oo 7* 1 1 1 1 1 Λ 1 rH p〇po no ro ^ tH 〇tH c\] ro to <Ti CTt CTi &lt Ti (Ti (NL 〇 cxd oo oo oo oo < Τΐ 200837079) The amino acid sequence from top to bottom in the block called CDRH1 is in the same subsequence in SEQ ID NOs: 201-285. The amino acid sequence from top to bottom in the column designated CDRH2 is in the same subsequence in SEQ ID NOs: 286-370. The amino acid sequence from top to bottom in the block called CDRH3 is in the same subsequence in SEQ ID NOs: 371-455.

The amino acid sequence from top to bottom in the block called CDRL1 is in the same subsequence in SEQ ID NOs: 456-540. The amino acid sequence from top to bottom in the column designated CDRL2 is in the same subsequence in SEQ ID NOs: 541-625. The amino acid sequence from top to bottom in the block called CDRL3 is in the same subsequence in SEQ ID NOs: 626-710. Characterization of antigen specificity During the validation period, the antigenic Terry nature of the pure line was determined to some extent by binding to viral particles, soluble G and F proteins, and fragments of G proteins. 〆 For a pure line with anti-F reactivity, further evaluate the specificity of the individual antibodies expressed by the pure line to determine the antigenic site and, if possible, the epitopes that are bound by the individual pure lines (see Example g, g_4 part) ). Figure 4: shows the antigenic determinant specificity of the antibodies obtained from the pure line 8〇1 using Biacore analysis. This analysis shows that when the antibody ρ is blocked by 133_lh or palivizumab (the antigenic sites c and π, respectively) before the antibody 801 is injected into the cell, the antibody 8〇ι can be detected. High-level combination. When compared to the binding of the non-competitive & 8 〇 1 antibody, the competition = 82 200837079 antibody binding decreased a little. However, this reduction is much lower than the direct competition of the binding site and is more likely due to steric hindrance. Blocking protein F (antigenic site F1) with the 9c5 antibody prior to injection of antibody 8〇1 into the cells showed that the binding of antibody 80 1 to the F protein was almost completely inhibited. It is therefore concluded that antibody 8w binds to protein f at or near the site of F1. For pure lines with anti-G reactivity, the specificity of the individual antibodies expressed by the pure line is further evaluated to determine whether the individual antibodies bind to the central domain, conserved region or GCRR of the G protein, and also whether the epitope is conserved or sub-determined. Type specificity. This was carried out by ELISA and/or FUSA using the following G protein fragment: G(B): residue 66_292 of RSV strain 1 8537 (expressed in EM344 CHO cells) G (B) fragment: RSV strain 18537 Residues 127-203 (expressed in E. coli) GCRR a : Residues of RSV strain Long, 171-187 (synthesized with a cysteine bridge formed selectively) GCRR B : Residues of RSV strain 18537 -187 (synthesized with a cysteine bridge formed selectively) G Conserved: Residues 164-176. Other epitope assays were also performed against the G-active pure line by a competition assay as described in section g-4 of Example 1. In addition, one of the pure lines identified in the screening program as described in section e of Example 1 produced an SH-specific antibody. In addition, many of the pure lines bind to one or more of the 4 RSV strains tested, but the antigen has not yet been determined. 83 200837079 All validated antigens are specific for 5 of them. Experience 镫#彡+ 々, data summary in the table ^ pure line no - with human laryngeal epithelial cells ", household", ... heterosexual pure,,, and second and called no - and human fluta hunger (CX3CL1) knot =856 Characteristics of 疋 Binding Kinetics > : 纟面 a #Resonance determines the binding affinity of recombinant My antigen for many antibodies. The sputum & 仃4 assay prepared by enzymatic decomposition of the full length antibody was used. Data for a number of high affinity antibody lines with Kd values in the periam of Pimol to Nym ((10) 〇^r) are presented in Table 6. The Fab fragment derived from the commercially available pelizumab (Synagis) was similarly analyzed for reference. Pure phylogenetic binding constants and affinity

Generation of cell lines expressing the pure lines of individual antibodies 84 200837079 To generate stable individual expression cell lines each expressing a unique antibody from a single VH and V1 gene sequence, select the pure lines 73 5, 736, 744, 793, 795 corresponding to Table 5 , 796, 799, 800, 801, 804, 810, 811, 812, 814, 816, 817, 818, 819, 824, 825, 827, 828, 829, 830, 831, 835, 838, 841, 853 > Subgroup of unique cognate VH and Vl coding pairs of 855, 856, 857, 858, 859, 861, 863, 868, 870, 871, 880, 881, 884, 885, 886, 888, 894, and 955 . The complete sequence of 44 selected pure lines (except for 828, 885 and 955 (designated above) (DNA and deduced amino acids) is shown in SEQ ID NOs 1-176. 44 pure lines are produced by the following VH Characterized by sequence, which is listed in SEQ ID NOs 1-44: Pure Line 735:

QVQLQESGPGLVKPSETLSLTCTVSNGAIGDYDWSWIRQSPGKGLEWIG

NINYRGNTNYNPSLKSRVTMSLRTSTMQFSLKLSSATAADTAVYYCARD

VGYGGGQYFAMDVWSPGTTVTVSS Pure 736:

C QVQLVESGGGVVQPGGSLRLSCTASGFTFSTYGMHWVRQAPGKGLEW

VAFIRYDGSTQDYVDSVKGRFTISRDNSKNMVYVQMNSLRVEDTAVYY CAKDMDYYGSRSYSVTYYYGMDVWGQGTTVTVSS Pure 744:

QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYMHWVRQAPGQGLE

WMGWINTSSGGTNYAQKFQGRVTMTRDTSISTAHMELRRLRSDDTAVY

YCAREDGTMGTNSWYGWFDPWGQGTLVTVSS Pure 793:

QVQLVESGGGLVKPGGSLRLSCAASGFPFGDYYMSWIRQAPGKGLEWV

AYINRGGTTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAGDTALYYCA

RGLILALPTATVELGAFDIWGQGTMVTVSS Pure 795: 85 200837079

• QVQLQESGPGLVKPSQTLSLTCTVSGASISSGDYYWSWIRQSPRKGLEWI GYIFHSGTTYYNPSLKSRAVISLDTSKNQFSLRLTSVTAADTAVYYCARD VDDFPVWGMNRYLALWGRGTLVTVSS Pure 796:

QVQLVESGGGVVQPGRSLRLSCAASGFSFSHFGMHWVRQVPGKGLEW

VAIISYDGNNVHYADSVKGRFTISRDNSKNTLFLQMNSLRDDDTGVYYC

AKDDVATDLAAYYYFDVWGRGTLVTVSS Pure 799:

QVQLVESGGGVVQPGRSLKLSCEASGFNFNNYGMHWVRQAPGKGLEW

VAVISYDGRNKYFADSVKGRFIISRDDSRNTVFLQMNSLRVEDTAVYYCA

RGSVQVWLHLGLFDNWGQGTLVTVSS Pure Line 800: /

QVQLVESGGAVVQPGRSLRLSCEVSGFSFSDYGMNWVRQGPGKGLEWV

AVIWHDGSNKNYLDSVKGRFTVSRDNSKNTLFLQMNSLRAEDTAVYYC

ARTPYEFWSGYYFDFWGQGTLVTVSS Pure System No. 801:

QVQLVESGGGVVQPGRSLRLSCAASGFPFNSYAMHWVRQAPGKGLEW

VAVIYYEGSNEYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYC

ARKWLGMDFWGQGTLVTVSS pure system number 804:

EVQLVESGGGLVRPGGSLRLSCSASGFTFSNYAMHWVRQAPGKRLEYVS

ATSTDGGSTYYADSLKGTFTISRDNSKNTLYLQMSSLSTEDTAIYYCARR

FWGFGNFFDYWGRGTLVTVSS l Pure system 810:

QVQLVQSGAEVKKSGSSVKVSCRASGGTFGNYAINWVRQAPGQGLEW

VGRIIPVFDTTWAQKFQGRVTITADRSTNTAIMQLSSLRPQDTAMYYCL

RGSTRGWDTDGFDIWGQGTMVTVSS Pure 811:

QVQLVQSGAVVETPGASVKVSCKASGYIFGNYYIHWVRQAPGQGLEW

MAVINPNGGSTTSAQKFQDRITVTRDTSTTTVYLEVDNLRSEDTATYYC

ARQRSVTGGFDAWLLIPDASNTWGQGTMVTVSS Pure 812:

QVQLVQSGAEMKKPGSSVKVSCKASGGSFSSYSISWVRQAPGRGLEWV

GMILPISGTTNYAQTFQGRVIISADTSTSTAYMELTSLTSEDTAVYFCARVF

REFSTSTLDPYYFDYWGQGTLVTVSS 86 200837079 Pure 814:

QVQLVESGGGVVQPGKSVRLSCVGSGFRLMDYAMHWVRQAPGKGLD

WVAVISYDGANEYYAESVKGRFTVSRDNSDNTLYLQMKSLRAEDTAVYF

CARAGRSSMNEEVIMYFDNWGLGTLVTVSS Pure 816:

EVQLLESGGGLVQPGGSLRLSCVASGFTFSTYAMTWVRQAPGKGLEWV

SVIRASGDSEIYADSVRGRFTISRDNSKNTVFLQMDSLRVEDTAVYFCANI

GQRRYCSGDHCYGHFDYWGQGTLVTVSS Pure Line 817:

QVQLVESGGGVVQPGRSLRLSCAASGFGFNTHGMHWVRQAPGKGLEW

LSIISLDGIKTHYADSVKGRFTISRDNSKNTVFLQLSGLRPEDTAVYYCAK

DHIGGTNAYFEWTVPFDGWGQGTLVTVSS Pure 818:

QVTLRESGPAVVKPTETLTLTCAFSGFSLNAGRVGVSWIRQPPGQAPEWL

ARIDWDDDKAFRTSLKTRLSISKDSSKNQVVLTLSNMDPADTATYYCAR

TQVFASGGYYLYYLDHWGQGTLVTVSS pure line 819:

QVQLQESGPGLVKPSQTLSLTCTVSSGAISGADYYWSWIRQPPGKGLEW

VGFIYDSGSTYYNPSLRSRVTISIDTSKKQFSLKLTSVTAADTAVYYCARD

LGYGGNSYSHSYYYGLDVWGRGTTVTVSS Pure 824:

QVQLQESGPGLVKPSETLSLTCTVSGGSIGNYYWGWIRQPPGKGLEWIG

HIYFGGNTNYNPSLQSRVTISVDTSRNQFSLKLNSVTAADTAVYYCARDS

SNWPAGYEDWGQGTLVTVSS Pure 825:

QVQLVQSGAEVKKPGASVKVSCKVSGYTFTSNGLSWVRQAPGQGFEW

LGWISASSGNKKYAPKFQGRVTLTTDISTSTAYMELRSLRSDDTAVYYCA

KDGGTYVPYSDAFDFWGQGTMVTVSS Pure 827:

QVQLVQSGAEVKKPGASVKVSCRVSGHTFTALSKHWMRQGPGGGLEW

MGFFDPEDGDTGYAQKFQGRVTMTEDTATGTAYMELSSLTSDDTAVYYC

ATVAAAGNFDNWGQGTLVTVSS Pure 829:

QVTLKESGPALVKATQTLTLTCTFSGFSLSRMSRMSVSWIRQPPGKALEWL 87 200837079

ARIDWDDDKFYNTSLQTRLTISKDTSKNQVVLTMTNMDPVDTATYYCA

RTGIYDSSGYYLYYFDYWGQGTLVTVSS Pure System No. 830:

QVQLVQSGAEVKVPGASVKVSCKASGYTFTTYGVSWVRQAPGQGLEW

MGWISAYNGNTYYLQKLQGRVTMTTDTSTSTAYMELRGLRSDDTAMY

YCARDRVGGSSSEVLSRAKNYGLDVWGQGTTVTVSS Pure No. 831:

QVQLVQSGAEVKKPGASVKVSCKASANIFTYAMHWVRQAPGQRLEWM

GWINVGNGQTKYSQRFQGRVTITRDTSATTAYMELSTLRSEDTAVYYCA

RRASQYGEVYGNYFDYWGQGTLVTVSS Pure 835:

QVQLVQSGAEVKRPGASVKVSCKASGYTFISYGFSWVRQAPGQGLEW

MGWSSVYNGDTNYAQKFHGRVNMTTDTSTNTAYMELRGLRSDDTAVY

FCARDRNVVLLPAAPFGGMDVWGQGTMVTVSS Pure 838:

QVQLVESGGGVVQPGTSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWV

AVISYDGNKKYYADSVKGRFTISRDNSKNTLYLQVNSLRVEDTAVYYCA

AQTPYFNESSGLVPDWGQGTLVTVSS Pure 841:

QVQLVQSGAEVKKPGASVKVSCKASGYTFISFGISWVRQAPGQGLEWM

GWISAYNGNTDYAQRLQDRVTMTRDTATSTAYLELRSLKSDDTAVYYCT

RDESMLRGVTEGFGPIDYWGQGTLVTVSS Pure 853:

EVQLVQSGAEVKKPGQSLKISCKTSGYIFTNYWIGWVRQRPGKGLEWM

GVIFPADSDARYSPSFQGQVTISADKSIGTAYLQWSSLKASDTAIYYCARP

KYYFDSSGQFSEMYYFDFWGQGTLVTVSS Pure 855:

QVQLVQSGPEVKKPGASVKVSCKASGYVLTNYAFSWVRQAPGQGLEW

LGWISGSNGNTYYAEKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFC

ARDLLRSTYFDYWGQGTLVTVSS Pure 856:

QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGFSWVRQAPGRGLEW

MGWISAYNGNTYYAQNLQGRVTMTTDTSTTTAYMVLRSLRSDDTAMY

YCARDGNTAGVDMWSRDGFDIWGQGTMVTVSS 88 200837079 Pure 857:

EVQLLESGGGLVQPGGPLRLSCVASGFSFSSYAMNWIRLAPGKGLEWVS

GISGSGGSTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

EPWIDIVVASVISPYYYDGMDVWGQGTTVTVSS Pure 858:

QVQLVQSGAEVKKPGSSVKVSCKASGGSFDGYTISWLRQAPGQGLEWM

GRVVPTLGFPNYAQKFQGRVTVTADRSTNTAYLELSRLTSEDTAVYYCAR

MNLGSHSGRPGFDMWGQGTLVTVSS Pure 859:

QVQLVESGGGVVQPGRSLRLSCAVSGSSFSKYGIHWVRQAPGKGLEWV AVISYDGSKKYFTDSVKGRFTIARDNSQNTVFLQMNSLRAEDTAVYYCA f TGGGVNVTSWSDVEHSSSLGYWGLGTLVTVSS Pure 861:

QVQLVESGGGVVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEW

VAFIWNDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

VKDEVYDSSGYYLYYFDSWGQGTLVTVSS Pure Department No. 863:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWV

SSISASTVLTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAK

DYDFWSGYPGGQYWFFDLWGRGTLVTVSS Pure 868:

QVQLQESGPGLVTPSETLSVTCTVSNYSIDNAYYWGWIRQPPGKGLEWI ( GSIHHSGSAYYNSSLKSRATISIDTSKNQFSLNLRSVTAADTAVYYCARDT

^ ILTFGEPHWFDPWGQGTLVTVSS Pure Line 870:

QVQLQESGPGLVKPSETLSLTCTVSGDSISNYYWSWIRQPPGKGLEWIGE

ISNTWSTNYNPSLKSRVTISLDMPKNQLSLKLSSVTAADTAVYYCARGLF

YDSGGYYLFYFQHWGQGTLVTVSS Pure System No. 871:

QVQLVESGGGVVQPGRSLRVSCAASGFTFSNYGMHWVRQAPGKGLEW

VAVIWYDDSNKQYGDSVKGRFTISRDNSKSTLYLQMDRLRVEDTAVYYC

ARASEYSISWRHRGVLDYWGQGTLVTVSS Pure Line 880:

QITLKESGPTLVRPTQTLTLTCTFSGFSLSTSKLGVGWIRQPPGKALEWLA 89 200837079

LVDWDDDRRYRPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAH

SAYYTSSGYYLQYFHHWGPGTLVTVSS Pure 881:

EVQLVESGGGVVQPGGSLRLSCEVSGFTFNSYEMTWVRQAPGKGLEWV

SHIGNSGSMIYYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCA

RSDYYDSSGYYLLYLDSWGHGTLVTVSS Pure 884:

QVQLVQSGAEVRKPGASVKVSCKASGHTFINFAMHWVRQAPGQGLEW

MGYINAVNGNTQYSQKFQGRVTFTRDTSANTAYMELSSLRSEDTAVYYC

ARNNGGSAIIFYYWGQGTLVTVSS Pure 886:

QVQLVESGGGVYQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEW

VAVISNDGSNKYYADSVKGRFTISRDNSKKTMYLQMNSLRAEDTAVYFC

AKTTDQRLLVDWFDPWGQGTLVTVSS Pure 888:

QLQLQESGPGLVKPSETLSLTCTASGGSINSSNFYWGWIRQPPGKGLEWI

GSIFYSGTTYYNPSLKSRVTISVDTSKNQFSLKLSPVTAADTAVYHCARH

GFRYCNNGVCSINLDAFDIWGQGTMVTVSS Pure 894:

QVQLVESGGGVVQPGKSLRLSCAASGFRFSDYGMHWVRQAPSKGLEW

VAVIWHDGSNIRYADSVRGRFSISRDNSKNTLYLQMNSMRADDTAFYYC ARVPFQIWSGLYFDHWGQGTLVTVSS These VH amino acid sequences are also increased in SEQ ID NOs 45-88 colonize the germline sequence encoding a nucleic acid listed in the following: pure line No. 735: caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacgtgcactgtgtct aatggcgccatcggcgactacgactggagctggattcgtcagtccccagggaagggactggagtggattgggaa cataaattacagagggaacaccaactacaacccctccctcaagagtcgagtcaccatgtccctacgcacgtccac gatgcagttctccctgaagctgagctctgcgaccgctgcggacacggccgtctattactgtgcgagagatgtaggc tacggtggcgggcagtatttcgcgatggacgtctggagcccagggaccacggtcaccgtctcgagt Homogenous No. 736: caggtgcagctggtggagtctgggggaggcgtggtccagcctggggggtccctgagactctcctgtacagcgtct ggattcaccttcagtacctatggcatgcactgggtccgccaggctcccggcaaggggctggaatgggtggcattta 200837079 Tacggtatgatggaagtactcaagactatgtagactccgtgaagggccgattcaccatctccagagacaattccaag aatatggtgtatgtgcagatgaacagcctgagagttgaggacacgggggtctattgtgcgaaagacatggatta ctatggttcgcggagttattctgtcacctactactacggaatggacgtctggggccaagggaccacggtcaccgtct cgagt pure 744 No: caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct ggatacaccttcagcggctattatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatgg atcaacactagcagtggtggcacaaactatgcgcagaagtttcagggcagggtcaccatgaccagggacacgtc catcagcacagcccacatggaactgaggaggctgagatctgacgacacggccgtgtattattgtgcgagagagg acggcaccatgggtactaatagttggtatggctggttcgacccctggggccagggaaccctggtcaccgtctcga gt Homogenous No. 793:, caggtgcagctggtggagtctgggggaggcttggtcaagcctggggggtccctgagactctcctgtgcggcctct ggattccccttcggtgactactacatgagctggatccgccaggctccagggaagggactggagtgggttgcatac attaatagaggtggcactaccatatactacgcagactctgtgaagggccgattcaccatctccagggacaacgcca agaactccctgtttctgcaaatgaacagcctgagagccggggacacggccctctattactgtgcgagagggctaat tctagcactaccgactgctacggttgagttaggagcttttgatatctggggccaagggacaatggtcaccgtctcga gt Homogenous No. 795: caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctct ggtgcctccatcagcagtggtgattattactggagttggatccgtcagtctccaaggaagggcctggagtggattgg gtacatcttccacagtgggaccacgtactacaacccg tccctcaagagtcgagctgtcatctcactggacacgtcca agaaccaattctccctgaggctgacgtctgtgactgccgcagacacggccgtctattattgtgccagagatgtcgac gattttcccgtttggggtatgaatcgatatcttgccctctggggccggggaaccctggtcaccgtctcgagt, pure line No. 796: \ - caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctct ggattcagcttcagtcactttggcatgcactgggtccgccaggttccaggcaaggggctggagtgggtggcaatta tatcatatgatgggaataatgtacactatgccgactccgtaaagggccgattcaccatctccagagacaattccaag aacacgctgtttctgcaaatgaacagcctgagagatgacgacacgggtgtgtattactgtgcgaaggacgacgtg gcgacagatttggctgcctactactacttcgatgtctggggccgtggcaccctggtcaccgtctcgagt pure line No. 799: caggtgcagctggtggagtctgggggcggcgtggtccagcctgggaggtccctgaaactctcttgtgaagcctct ggattcaacttcaataattatggcatgcactgggtccgccaggcaccaggcaaggggctggagtgggtggcagtt atttcatatgacggaagaaataagtattttgctgactccgtgaagggccgattcatcatctccagagacgattccagg aacacagtgtttctgcaaatgaacagcctgcgagttgaagatacggccgtctattactgtgcgagaggcagcgtac aagtctggctacatttgggactttttgacaactggggccagggaaccctggtcaccgtctcgagt pure line number 800: 91 200837079 caggtgcagctggtggagtctgggggagccgtggtccagcctgggaggtccctgagactctcctgtgaagtgtct ggattcagtttcagtgactatggcatgaactgggtccgccagggtccaggcaaggggctggagtgggtggcagtt atatggcatgacggaagtaataaaaattatctagactccgtgaagggccgattcaccgtctccagagacaattccaa gaacacattgtttctgcaaatgaacagcctgagagccgaagacacggctgtatattactgtgcgaggacgccttac gagttttggagtggctattactttgacttctggggccagggaaccctggtcaccgtctcgagt pure line No. 801: caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattccccttcaatagctatgccatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtg atatattatgaagggagtaatgaatattatgcagactccgtgaagggccgattcaccatctccagagacaattccaa gaacactctgtatttgcaaatggatagcctgagagccgaggacacggctgtctattactgtgcgaggaagtggctg gggatggacttctggggccagggaaccctggtcaccgtctcgagt pure line No. 804: gaggtgcagctggtggagtctgggggaggcttggtccggcctggggggtccctgagactctcctgttcagcctct ggattcaccttcagtaactatgctatgcactgggtccgccaggctccagggaagagactggaatatgtttcagctac tagtactgatggggggagcacatactacgcagactccctaaagggcacattcaccatctccagagacaattccaag aac Homogenous acactgtatcttcaaatgagcagtctcagtactgaggacacggctatttattactgcgcccgccgattctgggga tttggaaacttttttgactactggggccggggaaccctggtcaccgtctcgagt No. 810: caggtgcagctggtgcagtctggggctgaggtgaagaagtccgggtcctcggtgaaggtctcctgcagggcttct ggaggcaccttcggcaattatgctatcaactgggtgcgacaggcccctggacaagggcttgagtgggtgggaag gatcatccctgtctttgatacaacaaactacgcacagaagttccagggcagagtcacgattaccgcggacagatcc acaaacacagccatcatgcaactgagcagtctgcgacctcaggacacggccatgtattattgtttgagaggttccac ccgtggctgggatactgatggttttgatatctggggccaagggacaatggtcaccgtctcgagt Homogenous No. 811: caggttcagctggtgcagtctggggctgtcgtggagacgcctggggcctcagtgaaggtctcctgcaaggcatct ggatacatcttcggcaactactatatccactgggtgcggcaggcccctggacaagggcttgagtggatggcagtta tcaatcccaatggtggtagcacaacttccgcacagaagttccaagacagaatcaccgtgaccagggacacgtcca cgaccactgtctatttggaggttgacaacctgagatctgaggacacggccacatattattgtgcgagacagagatct gtaacagggggctttgacgcgtggcttttaatcccagatgcttctaatacctggggccaggggacaatggtcaccgt ctcgagt Homogenous No. 812: caggtgcagctggtgcagtctggggctgagatgaagaagcctgggtcctcggtgaag gtctcctgcaaggcttct ggaggctccttcagcagctattctatcagctgggtgcgacaggcccctggacgagggcttgagtgggtgggaatg atcctgcctatctctggtacaacaaactacgcacagacatttcagggcagagtcatcattagcgcggacacatccac gagcacagcctacatggagctgaccagcctcacatctgaagacacggccgtgtatttctgtgcgagagtctttaga gaatttagcacctcgacccttgacccctactactttgactactggggccagggaaccctggtcaccgtctcgagt Homogenous No. 814: 92 200837079 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaagtccgtgagactctcctgtgtaggctct ggcttcaggctcatggactatgctatgcactgggtccgccaggctccaggcaagggactggattgggtggcagtt atttcatatgatggagccaatgaatactacgcagagtccgtgaagggccgattcaccgtctccagagacaattcaga caacactctgtatctacaaatgaagagcctgagagctgaggacacggctgtgtatttctgtgcgagagcgggccgt tcctctatgaatgaagaagttattatgtactttgacaactggggcctgggaaccctggtcaccgtctcgagt Homogenous No. 816: gaggtgcagctgttggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgtagcctcc ggattcacctttagtacctacgccatgacctgggtccgccaggctccagggaaggggctggagtgggtctcagtc attcgtgctagtggtgatagtgaaatctacgcagactccgtgaggggccggttcaccatctccagagacaattccaa gaacacggtgtttctgcaa atggacagcctgagagtcgaggacacggccgtatatttctgtgcgaatataggccag cgtcggtattgtagtggtgatcactgctacggacactttgactactggggccagggaaccctggtcaccgtctcgag t Homogenous No. 817: caggtgcagctggtggagtctgggggaggcgtggtccaacctgggaggtccctgagactctcctgtgcagcctct ggattcggcttcaacacccatggcatgcactgggtccgccaggctccaggcaaggggctggagtggctgtcaatt atctcacttgatgggattaagacccactatgcagactccgtgaagggccgattcaccatctccagagacaattccaa gaacacggtgtttctacaattgagtggcctgagacctgaagacacggctgtatattactgtgcgaaagatcatattgg ggggacgaacgcatattttgaatggacagtcccgtttgacggctggggccagggaaccctggtcaccgtctcgag t Homogenous No. 818: caggtcaccttgagggagtctggtccagcggtggtgaagcccacagaaacgctcactctgacctgcgccttctctg ggttctcactcaacgccggtagagtgggtgtgagttggatccgtcagcccccagggcaggccccggaatggcttg cacgcattgattgggatgatgataaagcgttccgcacatctctgaagaccagactcagcatctccaaggactcctcc aaaaaccaggtggtccttacactgagcaacatggaccctgcggacacagccacatattactgtgcccggacacag gtcttcgcaagtggaggctactacttgtactaccttgaccactggggccagggaaccctggtcaccgtctcgagt l Homogenous No. 819: caggtgcagctgcaggagtcgggcccaggactggtgaagcct tcacagaccctgtccctcacctgcactgtctct agtggcgccatcagtggtgctgattactactggagttggatccgccagcccccagggaagggcctggagtgggtt gggttcatctatgacagtgggagcacctactacaacccgtccctcaggagtcgagtgaccatatcaatagacacgt ccaagaagcagttctccctgaagctgacctctgtgactgccgcagacacggccgtgtattactgtgccagagatct aggctacggtggtaactcttactcccactcctactactacggtttggacgtctggggccgagggaccacggtcacc gtctcgagt Homogenous No. 824: caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctct ggtggctccatcggaaattactactggggctggatccggcagcccccagggaagggacttgagtggattgggcat atctacttcggtggcaacaccaactacaacccttccctccagagtcgagtcaccatttcagtcgacacgtccaggaa ccagttctccctgaagttgaactctgtgaccgccgcggacacggccgtgtattactgtgcgagggatagcagcaac tggcccgcaggctatgaggactggggccagggaaccctggtcaccgtctcgagt 93 200837079 Homogenous No. 825: caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttctg gttacacctttaccagtaatggtctcagctgggtgcgacaggcccctggacaagggtttgagtggctgggatggat cagcgctagtagtggaaacaaaaagtatgccccgaaattccagggaagagtcaccttgaccacagacatttccac gagcacagccta Homogenous catggaactgaggagtctgagatctgacgatacggccgtatattactgtgcgaaagatggggg cacctacgtgccctattctgatgcctttgatttctggggccaggggacaatggtcaccgtctcgagt No. 827: caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcagggtttcc ggacacactttcactgcattatccaaacactggatgcgacagggtcctggaggagggcttgagtggatgggattttt tgatcctgaagatggtgacacaggctacgcacagaagttccagggcagagtcaccatgaccgaggacacagcca caggcacagcctacatggagctgagcagcctgacatctgacgacacggccgtatattattgtgcaacagtagcgg cagctggaaactttgacaactggggccagggaaccctggtcaccgtctcgagt C Homogenous No. 829: caggtcaccttgaaggagtctggtcctgcgctggtgaaagccacacagaccctgacactgacctgcaccttctctg ggttttcactcagtaggaatagaatgagtgtgagctggatccgtcagcccccagggaaggccctggagtggcttgc acgcattgattgggatgatgataaattctacaacacatctctgcagaccaggctcaccatctccaaggacacctcca aaaaccaggtggtccttacaatgaccaacatggaccctgtggacacagccacctattactgcgcacggactggga tatatgatagtagtggttattacctctactactttgactactggggccagggaaccetggtcaccgtctcgagt Homogenous No. 830: caggtgcagctggtgcagtctggagctgaggtgaaggtgcctggggcctcagtgaaggtctcctgcaaggcttct ggttacacctttaccacttacggtgtcagctgggtgcggcaggcccctggacaagggcttgagtggatgggttgga tcagcgcttacaatggtaacacatactatctacagaagctccagggcagagtcaccatgaccacagacacatccac gagcacagcctacatggagctgcggggcctgaggtctgacgacacggccatgtattactgtgcgagagatcgtgt tgggggcagctcgtccgaggttctatcgcgggccaaaaactacggtttggacgtctggggccaagggaccacgg (tcaccgtctcgagt Homogenous No. 831: caggttcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagttaaggtttcctgcaaggcttctg caaacatcttcacttatgcaatgcattgggtgcgccaggcccccggacaaaggcttgagtggatgggatggatcaa cgttggcaatggtcagacaaaatattcacagaggttccagggcagagtcaccattaccagggacacgtccgcgac tacagcctacatggagctgagcaccctgagatctgaggacacggctgtgtattactgtgcgaggcgtgcgagcca atatggggaggtctatggcaactactttgactactggggccagggaaccctggtcaccgtctcgagt Homogenous No. 835: caggtgcagctggtgcagtctggagctgaggtgaagaggcctggggcctcagtgaaggtctcctgcaaggcttc aggttacacctttatcagctatggtttcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatgg agcagcgtttacaatggtgacacaaactatgcacagaagttccacggcagagtcaacatgacgactgacacatcg acgaacacggcctacatggaactcaggggcctgagatctgacga Homogenous cacggccgtgtatttctgtgcgagggatcgc aatgttgttctacttccagctgctccttttggaggtatggacgtctggggccaagggacaatggtcaccgtctcgagt 94 200837079 Number 838: caggtgcagctggtggagtctgggggaggcgtggtccagccggggacttccctgagactctcctgtgcagcctct ggattcaccttcagtacgtttggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atatcatatgatggaaataagaaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaa gaacacgctgtatctgcaagtgaacagcctgagagtcgaggacacggctgtgtattactgtgcggcccaaactcc atatttcaatgagagcagtgggttagtgccggactggggccagggcaccctggtcaccgtctcgagt Homogenous No. 841: caggtgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct ggttacacctttatcagttttggcatcagctgggtgcgacaggcccctggacaaggacttgagtggatgggatggat cagcgcttacaatggtaacacagactatgcacagaggctccaggacagagtcaccatgactagagacacagcca cgagcacagcctacttggagctgaggagcctgaaatctgacgacacggccgtgtactattgcactagagacgagt cgatgcttcggggagttactgaaggattcggacccattgactactggggccagggaaccctggtcaccgtctcga gt Homogenous No. 853: gaagtgcagctggtgcagtctggagcagaggtgaaaaagccggggcagtctctgaagatctcctgtaaga cttct ggatacatctttaccaactactggatcggctgggtgcgccagaggcccgggaaaggcctggagtggatgggggt catctttcctgctgactctgatgccagatacagcccgtcgttccaaggccaggtcaccatctcagccgacaagtcca tcggtactgcctacctgcagtggagtagcctgaaggcctcggacaccgccatatattactgtgcgagaccgaaata ttactttgatagtagtgggcaattctccgagatgtactactttgacttctggggccagggaaccctggtcaccgtctcg agt Homogenous No. 855: caggttcagctggtgcagtctggacctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct ggttatgtgttgaccaactatgccttcagctgggtgcggcaggcccctggacaagggcttgagtggctgggatgga tcagcggctccaatggtaacacatactatgcagagaagttccagggccgagtcaccatgaccacagacacatcca cgagcacagcctacatggagctgaggagtctgagatctgacgacacggccgtttatttctgtgcgagagatcttctg cggtccacttactttgactactggggccagggaaccctggtcaccgtctcgagt Homogenous No. 856: caggtgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct ggttacaccttttccaactacggtttcagctgggtgcgacaggcccctggacgagggcttgagtggatgggatgga tcagcgcttacaatggtaacacatactatgcacagaacctccagggcagagtcaccatgaccacagacacatcca cgaccacagcctacatggtactgaggagcctgagatctgacgacacggccatgtatt actgtgcgagagatggaa atacagcaggggttgatatgtggtcgcgtgatggttttgatatctggggccaggggacaatggtcaccgtctcgagt pure line No. 857: gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggcccctgaggctctcctgtgtagcctct ggattcagctttagcagctatgccatgaactggatccgcctggctccagggaaggggctggagtgggtctcaggt attagtggtagcggtggtagcacttactacggagactccgtgaagggccggttcaccatctccagagacaattcca agaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagagccgt 95 200837079 ggatcgatatagtagtggcatctgttatatccccctactactacgacggaatggacgtctggggccaagggaccac ggtcaccgtctcgagt pure line No. 858: caggttcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcctct ggaggatccttcgacggctacactatcagctggctgcgacaggcccctggacaggggcttgagtggatgggaag ggtcgtccctacacttggttttccaaactacgcacagaagttccaaggcagagtcaccgttaccgcggacagatcc accaacacagcctacttggaattgagcagactgacatctgaagacacggccgtatattactgtgcgaggatgaatct cggatcgcatagcgggcgccccgggttcgacatgtggggccaaggaaccctggtcaccgtctcgagt pure line No. 859: caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccttgagactctcctgtgca Gtgtct ggatccagcttcagtaaatatggcatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atatcgtatgatggaagtaaaaagtattccagactccgtgaagggccgattcaccatcgccagagacaattccca

Homogenous gaacacggtttttctgcaaatgaacagcctgagagccgaggacacggctgtctattactgtgcgacaggaggggg tgttaatgtcacctcgtggtccgacgtagagcactcgtcgtccttaggctactggggcctgggaaccctggtcaccg tctcgagt No. 861: caggtgcagctggtggagtctgggggaggcgtggtccagcctggggggtccctgagactctcctgtgcagcgtc tggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcattt atatggaatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaa gaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtattactgtgtgaaagatgaggtc tatgatagtagtggttattacctgtactactttgactcttggggccagggaaccctggtcaccgtctcgagt Homogenous No. 863: gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacgtttagctcctataccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaagta ttagtgctagtactgttctcacatactacgcagactccgtgaagggccgcttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgagtagcctgagagccgaggacacggccgtatattactgtgcgaaagattacgatttt tggagtggctatcccgggggacagtactggttcttcgatctctggggccgtggcaccctggtcaccgtctcgagt Homogenous No. 868: caggtgcagctgcaggagtcggg cccaggactggtgacgccttcggagaccctgtccgtcacttgcactgtctct aattattccatcgacaatgcttactactggggctggatccggcagcccccagggaagggtctggagtggataggc agtatccatcatagtgggagcgcctactacaattcgtccctcaagagtcgagccaccatatctatagacacgtccaa gaaccaattctcgttgaacctgaggtctgtgaccgccgcagacacggccgtatattactgtgcgcgcgataccatc ctcacgttcggggagccccactggttcgacccctggggccagggaaccctggtcaccgtctcgagt Homogenous No. 870: caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccttgtccctcacctgcactgtctca ggtgactccatcagtaattactactggagttggatccggcagcccccagggaagggactggagtggattggagaa atatctaacacttggagcaccaattacaacccctccctcaagagtcgagtcaccatatctctagacatgcccaagaa 96 200837079 ccagttgtccctgaagctgagctctgtgaccgctgcggacacggccgtatattactgtgcgagagggcttttctatg clonal number 871 acagtggtggttactacttgttttacttccaacactggggccagggcaccctggtcaccgtctcgagt: caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagagtctcctgtgcagcgtc tggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagt tatatggtatgatgacagtaataaacagtatggagactccgtgaagggccgattcaccatctccagagacaattcc Homogenous a agagtacgctgtatctgcaaatggacagactgagagtcgaggacacggctgtgtattattgtgcgagagcctccga gtatagtatcagctggcgacacaggggggtccttgactactggggccagggaaccctggtcaccgtctcgagt No. 880: cagatcaccttgaaggagtctggtcctacgctggtgagacccacacagaccctcacactgacctgcaccttctctg ggttctcactcagcactagtaaactgggtgtgggctggatccgtcagcccccaggaaaggccctggagtggcttg cactcgttgattgggatgatgataggcgctacaggccatctttgaagagcaggctcaccgtcaccaaggacacctc

Homogenous caaaaaccaggtggtccttacaatgaccaacatggaccctgtggacacagccacatattactgtgcacacagtgcc tactatactagtagtggttattaccttcaatacttccatcactggggcccgggcaccctggtcaccgtctcgagt No. 881: gaggtgcagctggtggagtctgggggaggcgtggtacagcctggaggctccctgagactctcctgtgaagtctcc ggattcaccttcaatagttatgaaatgacctgggtccgccaggccccagggaaggggctggagtgggtttcacac attggtaatagtggttctatgatatactacgctgactctgtgaagggccgattcaccatctccagagacaacgccaag aactcactatatctgcaaatgaacagcctgagagtcgaggacacggctgtttattactgtgcgaggtcagattactat gatagtagtggttattatctcctctacttagactcctggggccatggaaccctggtcaccgtctcgagt Homogenous No. 884: caggtgcagctggtgcagtctggggctgaggtgaggaagcctggggcctcagtgaaggtttcctgcaaggcttct ggacatactttcattaactttgctatgcattgggtgcgccaggcccccggacaggggcttgagtggatgggatacat caacgctgtcaatggtaacacacagtattcacagaagttccagggcagagtcacctttacgagggacacatccgc gaacacagcctacatggagctgagcagcctgagatctgaagacacggctgtgtattactgtgcgagaaacaatgg gggctctgctatcattttttactactggggccagggaaccctggtcaccgtctcgagt Homogenous No. 886: caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctg agactctcctgtgcagcctct ggattcagcttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atatcaaatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaa gaaaacgatgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtatttctgtgcgaagacaacaga ccagcggctattagtggactggttcgacccctggggccagggaaccctggtcaccgtctcgagt Homogenous No. 888: cagctgcagctgcaggagtcgggcccaggactggtgaagccatcggagaccctgtccctcacctgcactgcctct ggtggctccatcaacagtagtaatttctactggggctggatccgccagcccccagggaaggggctggagtggatt gggagtatcttttatagtgggaccacctactacaacccgtccctcaagagtcgagtcaccatatccgtagacacgtc caagaaccagttctccc1: gaagctgagccctgtgaccgccgcagacacggctgtctatcactgtgcgagacatgg 97 200837079 cttccggtattgtaataatggtgtatgctctataaatctcgatgcttttgatatctggggccaagggacaatggtcaccg tctcgagt

Number Clone No.894: caggtgcagctggtggagtctgggggaggcgtcgtccagcctggaaagtccctgagactctcctgtgcagcgtct ggattcagattcagtgactacggcatgcactgggtccggcaggctccaagcaaggggctggagtgggtggcagt tatctggcatgacggaagtaatataaggtatgcagactccgtgaggggccgattttccatctccagagacaattcca agaacacgctgtatttgcaaatgaacagcatgagagccgacgacacggctttttattattgtgcgagagtcccgttcc agatttggagtggtctttattttgaccactggggccagggaaccctggtcaccgtctcgagt same inbred, the complete amino acid sequence of the light chains (i.e. comprising a light chain constant region and variable region) having also in SEQ ID NOs: 89-132 as The following amino acid sequences are listed: Pure 735:

EIVLTQSPATLSLSPGERATLSCRASQSVNSHLAWYQQKPGQAPRLLIYNT

FNRVTGIPARFSGSGSGTDFTLTISSLATEDFGVYYCQQRSNWPPALTFGG

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Department No. 736:

DIQMTQSPSSLSASVGDRVTFTCRASQRISNHLNWYQQKPGKAPKLLIFG

ASTLQSGAPSRFSGSGSGTDFTLTITNVQPDDFATYYCQQSYRTPPINFGQ

GTRLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Series 744:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG

ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSSLSTWTFG

QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNRGEC Pure Series 793:

DIQMTQSPSSLSASVGDRVTITCRASQSITGYLNWYQQKPGKAPKLLIYA

TSTLQSEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTLTFGGGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC 98 200837079 Pure 795:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIHG

ASTGATGTPDRFSGSGSGTDFTLTISTLEPEDFAVYYCQQYGRTPYTFGQG

TKLENKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Department No. 796:

DIVMTQTPLSLSVTPGQPASISCRSSQSLLRSDGKTFLYWYLQKPGQSPQP

LMYEVSSRFSGVPDRFSGSGSGADFTLNISRVETEDVGIYYCMQGLKIRR

TFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC Pure Department No. 799:

DIQMTQSPSTLSASVGDRVTFSCRASQSVSSWVAWYQQKPGKAPKLLISE

ASNLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYHSYSGYTFGQ

GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Line 800:

AIQLTQSPSSLSASVGDRVTLTCRASQGITDSLAWYQQKPGKAPKVLLYA

ASRLESGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYSKSPATFGPG

TKVEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure System No. 801:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGFNYVDWYLQKPGQSPQ

LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALETP

LTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC Pure System No. 804:

EIVLTQSPGTLSLSPGGRATLSCRASQSVSSGYLAWYQQKPGQAPRLLIY

GASGRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYFGSPYTFGQ

GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNRGEC 99 200837079 Pure 810:

NIQMTQSPSAMSASVGDRVTITCRASQGISNYLVWFQQKPGKVPKRLIYA

ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNISPYTFGQGT

KLETKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure No. 811:

DIVMTQSPDSLAVSLGERATINCRSSETVLYTSKNQSYLAWYQQKARQPP

KLLLYWASTRESGVPARFSGSGSGTDFTLAISSLQAEDVAVYYCQQFFRS

PFTFGPGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC Pure System No. 812:

EIVLTQSPGTLSLSPGERVTLSCRASQSVSSSYIAWQQKPGQAPRLVIYA

ASRRATGVPDRFSGSGSATDFTLTISRLEPEDLAVYYCQHYGNSLFTFGPG

TKVDVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Line 814:

DIQMTQSPSTLSASVGDRVTITCRASQSIGSRLAWYQQQPGKAPKFLIYD

ASSLESGVPSRFSGSGSGTEFTLTISSLQPEDLATYYCQQYNRDSPWTFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure 816:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGRYYVDWYLQKPGQSPH

LLIYLASNRASGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCMQGLHT

PWTFGQGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC Pure Line 817:

EIVMTQSPATLSASPGERATLSCWASQTIGGNLAWYQQKPGQAPRLLIYG

ASTRATGVPARFSGSGSGTEFTLAISSLQSEDFAVYYCQQYKNWYTFGQG

TKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC 100 200837079 Pure 818:

DIQMTQSPSSLSASVGDRVTITCRASQTIASYVNWYQQKPGRAPSLLIYA

ASNLQSGVPPRFSGSGSGTDFTLTISGLQPDDFATYYCQQSYSYRALTFG

GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNRGEC pure line 819:

EIVLTQSPATLSLSPGERATLSCRASQSVSSSLAWYQQTPGQAPRLLIYDA

SYRVTGIPARFSGSGSGIDFTLTISSLEPEDFAVYYCQQRSNWPPGLTFGGG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure System No. 824:

AIQLTQSPSSLSASVGDTVTVTCRPSQDISSALAWYQQKPGKPPKLLIYG

ASTLDYGVPLRFSGTASGTHFTLTISSLQPEDFATYYCQQFNTYPFTFGPG

TKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure No. 825:

DIVMTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQP

PKLLIHLASTREYGVPDRFSGSGSGTDFALIISSLQAEDVAVYYCQQYYQT

PLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC Pure No. 827:

DIQMTQSPSSLAASVGDRVTITCRASQFISSYLHWYQQRPGKAPKLLMY

AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTNPYTFGQ

GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNPYPREAKVQWKV dnalqsgnsqesvteqdskdstyslsstltLskadyekhkvyacevthq GLSSPVTKSFNRGEC Pure Department No. 829:

DIQMTQSPSSLSASVGDRVTITCRASQSIASYLNWYQQKPGKAPKLLIYA

ASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSYSTRFTFGPGT

KVDVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC 101 200837079 Pure System No. 830:

DIQMTQSPSTLSASVGDRVTITCRASQSVTSELAWYQQKPGKAPNFLIYK

ASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPYTFGQG

TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure Department No. 831:

DIQMTQSPSTLSASVGDRLTITCRASQNIYNWLAWYQQKPGKAPKLLIY

DASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSLSPTFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure 835:

DIQLTQSPSFLSASLEDRVTITCRASQGISSYLAWYQQKPGKAPKLLLDA

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPRTFGQG

TKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Series 838:

DIQMTQSPSSLSASVGDRVSITCRASQGISNYLAWYQQKPGKVPKLLIYA

ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPQTFGQG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure Line 841:

DIVMTQSPDSLAVSLGERATINCRSSQSVLYSSNNKNYLAWYQQKPGQPP

KLLVYWASTRASGVPDRFSGSGSGTDFTLTLSSLQAEDVAVYYCQQFHST

PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC Pure Series 853:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIY

GASSRAAGMPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSPLTFG

GGTEVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNRGEC 102 200837079 Pure 855:

DIQMTQSPSSVSASVGDRVTITCRASQAISNWLAWYQQKPGKAPKLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQADTFPFTFGPG

TKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Series 856:

DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSNDGNTYLDWYLQKPGQSP

QLLIYTFSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRIEF

PYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC Pure Department No. 857:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHRNEYNYLDWYLQKPGQSPQ

LLIYWGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLQT

PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC Pure Series 858:

DIQMTQSPSSVSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIFD

ATKLETGVPTRFIGSGSGTDFTVTITSLQPEDVATYYCQHFANLPYTFGQG

TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNPYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure Series 859:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKVPKLLVFA

ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNSAPLTFGGG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure 861:

DIQMTQSPSSLSASVGDRVTITCRASQIIASYLNWYQQKPGRAPKLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPIFTFGPG

TKVNIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC 103 200837079 Pure Department No. 863:

EIVLTQSPATLSLSPGERATLSCRTSQSVSSYLAWYQQKPGQAPRLLIYDA

SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWLTFGGGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC Pure Series 868:

EIVMTQSPATLSVSPGERATLSCRASQSIKNNLAWYQVKPGQAPRLLTSG

ASARATGIPGRFSGSGSGTDFTLTISSLQSEDIAVYYCQEYNNWPLLTFGG

GTKVEIQRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Line 870:

DIQMTQSPPSLSASVGDRVTITCRASQRIASYLNWYQQKPGRAPKLLIFA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQSYSTPIYTFGQ

GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC Pure Department No. 871:

DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIFD

ASNLESEVPSRFSGRGSGTDFTFSISSLQPEDIATYFCQQYDNFPYTFGQG

TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure Line 880:

DIQMTQSPSSLAASVGDRVTITCRASQTIASYVNWYQQKPGKAPNLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQSYSFPYTFGQG

TKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC Pure 881:

DIQMTQSPSSLSASVGDRVTITCRASQTIASYVNWYQQKPGKAPKLLIYA

ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPRLTFGG

GTKVDITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC 104 200837079 Pure Department No. 884:

DIQMTQSPSSLSASVGDRVTITCRSSQTISVFLNWYQQKPGKAPKLLIYA

ASSLHSAVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQESFSSSTFGGGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC Pure Department No. 886:

EIVMTQSPATLSVSPGETATLSCRASQSVSSNLAWYQHKPGQAPRLLIHS

ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNMWPPWTFG

QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNRGEC Pure Series 888:

DIVMTQSPLSLPVTPGAPASISCRSSQSLLRTNGYNYLDWYLQKPGQSPQ

LLIYLGSIRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLQTSI

TFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC Pure 894:

EIVMTQSPATLSVSPGERATLSCRASQSVGNNLAWYQQRPGQAPRLLIYG

ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDKWPETFGQG

TKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC The nucleic acid fragments encoding the light chain in such pure lines have the following nucleic acid sequences also provided in SEQ ID NOs: 13 3-176:

Clone No 735 Μ: gaaattgtgttgacacagtctccagccaccctgtccttgtctccaggagaaagagccaccctctcctgcagggc cagtcagagtgttaacagccacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctata atacattcaatagggtcactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcacc atcagcagccttgcgactgaagattttggcgtttattactgtcagcagcgtagcaactggcctcccgccctcacttt cggcggagggaccaaagtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagt ggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacag cacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcg aagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 105 200837079

Clone No No. 736: gacatccagatgacccagtctccatcctccctgtctgcatctgtgggagacagagtcaccttcacttgccgggcc agtcagaggattagcaaccatttaaattggtatcaacaaaagccagggaaagcccctaaactcctgatctttgg tgcatccactcttcaaagtggggccccatcaaggttcagtggcagtggatctgggacagatttcactctcaccat cactaatgtacaacctgacgattttgcaacttactactgtcaacagagttacagaactcccccgatcaacttcgg ccaagggacacgcctggacattaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagca gttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaa ggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacct acagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagt cacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 744: gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcc agtcagagtgttagcagcagctacttagcctggtatcagcagaaacctggccaggctcccaggctcctcatcta tggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctc accatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatgatagctcactttctacgtgga cgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatct gatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagta cagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgc ctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 793: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggc aagtcagagcattaccggctatttaaattggtatcagcagaaaccagggaaagcccctaaactcctgatctatg ctacatccactttgcaaagtgaggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcacca tcagcagtcttcaacctgaagattttgcaacttactactgtcaacagagttataataccctcactttcggcggagg gaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaa atctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgga taacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccca tcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 795: gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcc agtcagagtgttagcagcagctacttagcctggtatcagcagaaacctggccaggctcccaggctcctcatac atggcgcatccaccggggccactggcaccccagacaggttcagtggcagtgggtctgggacagacttcactct caccatcagtacactggagcctgaagattttgcagtgtattactgtcagcaatatggtaggacaccgtacactttt ggccaggggaccaagctggagaacaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagt ggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacag cacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcg 106 200837079 aagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt Clone No No. 796 ·· gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtcta gtcagagcctcctgcgaagtgatggaaagacgtttttgtattggtatctgcagaagccaggccagtctccccaa cccctaatgtatgaggtgtccagccggttctctggagtgccagataggttcagtggcagcgggtcaggggcag atttcacactgaacatcagccgggtggagactgaggatgttgggatctattactgcatgcaaggttt gaaaattc gtcggacgtttggcccagggaccaaggtcgaaatcaagcgaactgtggctgcaccatctgtcttcatcttcccg ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagc aaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtct acgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No 799:

gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccttctcttgccgggcc agtcagagtgttagtagttgggtggcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctctga ggcctccaatttggaaagtggggtcccatcccggttcagcggcagtggatccgggacagaattcactctcacc atcagcagcctgcagcctgaagattttgcaacttattactgccaacagtatcatagttactctgggtacacttttgg ccaggggaccaagttggaaatcaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcac ctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

It Clones No No. 800: gccatccagttgacccagtctccatcgtccctgtctgcatctgtaggcgacagagtcaccctcacttgccgggcg agtcagggcattaccgattctttagcctggtatcagcagaaaccagggaaagcccctaaggtcctgctctatgct gcttccagattggaaagtggggtcccatccaggttcagtggccgtggatctgggacggatttcactctcaccatc agcagcctgcagcctgaagactttgcaacttattactgtcaacagtattctaagtcccctgcgacgttcggccca gggaccaaggtggaaatcagacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 801: gatattgtgatgacccagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtcta gtcagagcctcctaaatagtaatggattcaactatgtggattggtacctgcagaagccagggcagtctccacaa ctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagatt ttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctagaaactccg ctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcc atctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaa 107 200837079 ggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctac gcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 804: gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccagggggaagagccaccctctcctgcagggcc agtcagagtgttagcagcggctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatcta tggtgcatccggcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctc accatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtattttggctcaccgtacacttttgg ccaggggaccaagctggagctcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcac ctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 810: aacatccagatgacccagtctccatctgccatgtctgcatctgtaggagacagagtcaccatcacttgtcgggc gagtcagggcattagtaattatttagtctggtttcagcagaaaccagggaaagtccctaagcgcctgatctatgc tgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacaa tcagcagcctgcagcctgaagattttgcaacttattactgtctacagcataatatttccccttacacttttggccagg ggaccaagctggagaccaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No 811 M: gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaggtc cagtgagactgttttatacacctctaaaaatcagagctacttagcttggtaccagcagaaagcacgacagcctc ctaaactactcctttactgggcatctacccgggaatccggggtccctgcccgattcagtggcagcggatctggg acagatttcactctcgccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaattttttagga gtcctttcactttcggccccgggaccagactggagattaaacgaactgtggctgcaccatctgtcttcatcttccc gccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc aaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacag caaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtc tacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 812: gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagttaccctctcttgcagggcca gtcagagtgttagcagcagttacatagcctggtaccagcagaagcctggccaggctcccaggctcgtcatctat gctgcatcccgcagggccactggcgtcccagacaggttcagtggcagtgggtctgcgacagacttcactctca ccatcagtagactggagcctgaagatcttgcagtgtattactgtcagcactatggtaactcactattcactttcggc cctgggaccaaggtggatgtcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcag ttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaag 108 200837079 gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcaccta cagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtc acccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 814: gacatccagatgacccagtctccctccaccctgtctgcatctgtcggagacagagtcaccatcacttgccgggc cagtcagagtattggtagccggttggcctggtatcagcagcaaccagggaaagcccctaaattcctgatctatg atgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatcagggacagaattcactctcacc atcagcagcctgcagccggaggatcttgcaacttattactgccaacagtacaatagagattctccgtggacgttc ggccaagggaccaaggtggaaatcaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatga gcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtg gaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagc acctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcga agtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 816: gatattgtgatgacccagtctccactctccctgcccgtcaccccaggagagccggcctccatctcctgcaggtct agtcagagcctcctgcatagtgatggacgctactatgtggattggtacctgcagaagccagggcagtctccaca cctcctgatctatttggcttctaatcgggcctccggggtccctgacaggttcactggcagtggatcaggcacagat tttacactgaaaatcagcagagtggaggctgaggatgttggcgtttattactgcatgcaaggtctacacactcctt ggacgttcggccaggggaccaaggtggacatcaagcgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaag gacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacg cctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 817: gaaattgtaatgacacagtctccagccaccctgtctgcgtccccaggggaaagagccaccctctcctgttgggc cagtcagactattggaggcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatg gtgcatccaccagggccactggtgtcccagccaggttcagtggcagtgggtctgggacagagttcactctcgcc atcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataaaaactggtacacttttggccagg ggaccaagctggagctcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttga aatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgg ataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacag cctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccc atcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 818 ·· gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggc aagtcagaccattgccagttacgtaaattggtaccaacaaaaaccagggagagcccctagtctcctgatctatg ctgcatctaacttgcagagtggggtcccaccaaggttcagtggcagtggatctgggacagacttcactctcacc atcagcggtctgcaacctgacgattttgcaacttattactgtcaacagagttacagttatcgagcgctcactttcgg cggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc 109 200837079 agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcac ctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 819: gaaattgtgttgacacagtctccagccaccctgtcgttgtccccaggggaaagagccaccctctcctgcagggc cagtcagagtgttagcagctccttagcctggtaccaacagacacctggccaggctcccaggcttctcatctatga tgcgtcctacagggtcactggcatcccagccaggttcagtggcagtgggtctgggatagacttcactctcaccat cagcagcctagagcctgaagattttgcagtttactattgtcagcagcgtagcaactggcctccggggctcactttc ggcggggggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatga gcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtg gaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagc acctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcga

Agtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 824: gccatccagttgacccagtctccatcctccctgtctgcatctgttggagacacagtcaccgtcacttgccggccaa gtcaggacattagcagtgctttagcctggtatcagcagaaaccagggaaacctcctaagctcctgatctatggt gcctccactttggattatggggtcccattaaggttcagcggcactgcatctgggacacatttcactctcaccatca gcagcctgcaacctgaagattttgcaacttattactgtcaacagtttaatacttacccattcactttcggccctggg accaaagtggatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaa tctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggat aacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcc tcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccat cagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 825: gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtc cagccagagtgttttatacaactccaacaataagaactacttagcctggtatcagcagaaaccaggacagcct cctaagctcctcattcacttggcatctacccgggaatacggggtccctgaccgattcagtggcagcgggtctgg gacagatttcgctctcatcatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacaatattatcaa actcctctaacttttggccaggggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcc cgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggc caaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca gcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaag tctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 827: gacatccagatgacccagtctccatcctccctggctgcatctgtaggagacagagtcaccatcacttgccgggc aagtcagttcattagcagctatttacattggtatcagcaaagaccaggcaaggcccctaaactcctgatgtatgc tgcctccactttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatc agcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacactaacccatacacttttggccag no 200837079 gggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 829 ·· gacatccagatgacccagtctccatcctccctatctgcatctgtaggagacagagtcaccatcacttgccgggc aagtcagagcattgccagctatttaaattggtatcagcagaaaccagggaaagcccccaaactcctgatctat gctgcatccagtttgcatagtggggtcccatcaagattcagtggcagtggatctgggacagatttcactctcacc atcagcagtctgcaacctgaagattttgcaacttactactgtcaacacagttacagtactcgattcactttcggccc tgggaccaaagtggatgtcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggt ggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctac

Agcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtca cccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 830: gacatccagatgacccagtctccttcgaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggc cagtcagagtgttactagtgagttggcctggtatcagcagaaaccagggaaagcccctaacttcctgatctata aggcgtctagtttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacc atcagcagcctgcagcctgatgattttgcaacttattactgccaacagtataatagttttccgtacacttttggccag gggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 831: gacatccagatgacccagtctccttccaccctgtctgcatctgtaggcgacagactcaccatcacttgccgggcc agtcagaatatttataactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctatga cgcctccactttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagagttcactctcacca tcagcagcctgcagcctgatgattttgcgacttattactgccaacaatataatagtttgtctccgacgttcggccaa gggaccaaggtggaaatcaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 835: gacatccagttgacccagtctccatccttcctgtctgcatctttagaagacagagtcactatcacttgccgggcca gtcagggcattagcagttatttagcctggtatcagcaaaaaccagggaaagcccctaagctcctgctcgatgct gcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagagttcactctcacaat 111 200837079 cagcagcctgcagcctgaagattttgcaacttattactgtcaacagcttaatagttaccctcggacgttcggccaa gggaccaaggtggacatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 838: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcagcatcacttgccgggc gagtcagggcattagcaattatttagcctggtatcagcagaaaccagggaaggttcctaagctcctgatctatgc tgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatc agcagcctgcagcctgaggatgttgcaacttattactgtcaaaagtataacagtgcccctcaaacgttcggcca agggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggt § ggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctac \ agcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtca cccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 841: gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaggtc cagccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaaccaggacagcct cctaagctgctcgtttactgggcatcaacccgggcatccggggtccctgaccgattcagtggcagcgggtctgg gacagatttcactctcaccctcagcagcctgcaggctgaagatgtggcagtttattactgtcagcagtttcatagt actcctcggacgttcggccaagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatctt cccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagag gccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagga cagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtg i t

Clone No No. 853: gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcc agtcagagtgttagcagcaactacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatcta tggtgcatccagcagggccgctggcatgccagacaggttcagtggcagtgggtctgggacagacttcactctc accatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtaactcaccgctcactttcg gcggagggaccgaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgag cagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagca cctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 855: gacatccagatgacccagtctccatcttctgtgtctgcatctgtaggagacagagtcaccatcacttgtcgggcg 112 200837079 agtcaggctattagtaactggttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgc tgcatccagtttgcaaagtggggtcccatcaagattcagcggcagtggatctgggacagatttcactctcactat cagcggcctgcagcctgaggattttgcaacttactattgtcaacaggctgacactttccctttcactttcggccctg ggaccaaagtggatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttga aatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgg ataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacag cctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccc atcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 856: gatattgtgatgacccagactccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtct agtcagagcctcttggatagtaatgatggaaacacctatttggactggtacctgcagaagccagggcagtctcc acagctcctgatttatacattttcctatcgggcctctggagtcccagacaggttcagtggcagtgggtctggcact gatttcacactgaaaatcagcagggtggaggccgaggatgttggagtttattactgcatgcaacgtatcgagttt

ccgtacacttttggccaggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccg ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagc aaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtct acgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 857: gatattgtgatgacccagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtcta gtcagagcctcctgcatagaaatgagtacaactatttggattggtacttgcagaagccagggcagtctccacag ctcctgatctattggggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagat tttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaaactctacaaactcct cggacgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcc atctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaa ggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctac gcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 858: gacatccagatgacccagtctccatcctccgtgtctgcatctgtgggagacagagtcaccatcacttgccaggc gagtcaagacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatcttcg atgcaaccaaattggagacaggggtcccaacaaggttcattggaagtggatctgggacagattttactgtcacc atcaccagcctgcagcctgaagatgttgcaacatattactgtcaacactttgctaatctcccatacacttttggcca ggggaccaagctggagatcaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggt ggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctac agcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtca cccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 859: 113 200837079 gacatccagatgacccagtctccatcttccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcg agtcagggcattaggaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctggtctttgct gcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatca gcagcctgcagcctgaggatgttgcaacttattactgtcaaaggtataacagtgccccgctcactttcggcggag ggacgaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 861: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggc aagtcagatcattgccagctatttaaattggtatcagcagaaaccaggcagagcccctaagctcctgatctatg ctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcacca tcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccccatattcactttcggc cctgggaccaaggtgaatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcag ttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcaccta cagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtc acccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 863: gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcaggacc agtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatga tgcttccaatagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcacca tcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagtgactggctcactttcggcggagg gaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaa atctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgga taacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccca tcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 868: gaaattgtaatgacacagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggc cagtcagagtattaaaaacaacttggcctggtaccaggtgaaacctggccaggctcccaggctcctcacctctg gtgcatccgccagggccactggaattccaggcaggttcagtggcagtgggtctgggactgacttcactctcacc atcagcagcctccagtctgaagatattgcagtttattactgtcaggagtataataattggcccctgctcactttcgg cggagggaccaaggtggagatccaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcac ctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 114 200837079

Clone No No. 870: gacatccagatgacccagtctcctccctccctgtctgcatctgtgggagacagagtcaccatcacttgccgggc aagtcagaggattgccagctatttaaattggtatcagcagaaaccagggagagcccctaagctcctgatctttg ctgcatccagtttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagacttcactctcacca tcagtagtctgcaacctgaagattatgcgacttactactgtcaacagagttacagtactcccatctacacttttggc caggggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagca gttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaa ggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacct acagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagt cacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 871: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggc f gagtcagggcattagcaactatttaaattggtatcaacagaaaccagggaaagcccctaagctcctgatcttcg 'atgcatccaatttggaatcagaggtcccatcaaggttcagtggacgtggatctgggacagattttactttctccat cagcagcctgcagcctgaagatattgcaacatatttctgtcaacagtatgataatttcccgtacacttttggccag gggaccaagctggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacc catcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 880: gacatccagatgacccagtctccatcctccctggctgcatctgtaggagacagagtcaccatcacctgccgggc aagtcagacgattgccagttatgtaaattggtatcaacagaaaccagggaaagcccctaatctcctgatctatg ctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcacca tcagcagtctgcaacctgaagattttgcatcttacttctgtcaacagagttacagtttcccgtacacttttggccagg ^ ggaccaagctggatatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttga aatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgg ataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacag cctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccc atcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 881: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggc aagtcagaccattgccagctatgtaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctat gctgcatccaatttgcaaagtggggtcccttcaaggttcagtggcagtggatctgggacagatttcactctcacc atcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtgtccctcggctcactttcg gcggagggaccaaggtggacatcacacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgag cagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagca cctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa 115 200837079 'gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt Clone No No 884: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccggtca agtcagaccattagcgtctttttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgcc gcatccagtttgcacagtgcggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatc agcagtctgcaacctgaagattctgcaacttactactgtcaagagagtttcagtagctcaac tttcggcggaggg accaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaa tctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggat aacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcc tcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccat cagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 886: f gaaattgtaatgacacagtctccagccaccctgtctgtgtctccaggggaaacagccaccctctcctgcagggc 'cagtcagagtgttagcagcaacttagcctggtaccaacataaacctggccaggctcccaggctcctcatccata gtgcatccaccagggccactgggatcccagccaggttcagtggcagtgggtctgggacagagttcactctcac cataagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataatatgtggcctccctggacgttcg gccaagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgag cagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagca cctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Clone No No. 888: gatattgtgatgacccagtctccactctccctgcccgtcacccctggagcgccggcctccatctcctgcaggtcta gtcagagcctcctgcgtactaatggatacaactatttggattggtacctgcagaagccagggcagtctccacag ctcctgatctatttgggttctattcgggcctccggggtccctgacaggttcagtggcagtggctcaggcacagattt I tacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaatctctacaaacttcgat caccttcggccaagggacacgactggagattaaacgaactgtggctgcaccatctgtcttcatcttcccgccatc tgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagt acagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaag gacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacg cctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

Number Clone No 894: gaaattgtaatgacacagtctccagccaccctgtctgtgtctccgggggaaagagccaccctctcctgcagggc tagtcagagtgttggcaacaacttagcctggtaccagcagagacctggccaggctcccagactcctcatctatg gtgcgtccaccagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcacc atcagcagcctgcagtctgaggattttgcagtttattactgtcagcagtatgataagtggcctgagacgttcggcc aggggaccaaggtggacatcaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcag ttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcaccta 116 200837079 cagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtc acccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt pure lines in all 44 of the above discussion, the encoded antibodies include the same constant IgG heavy chain, having the following amino acid sequence (SEQ ID NO: 178 ):

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

This heavy chain gene sequences (genomic sequence) KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK encoding the following amino acid sequence (SEQ ID NO ·· 177): a ^ tgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggc cctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcg gcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccag cagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttg gtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccg gctatgcagtcccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgcccca ctcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccag gccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctga cctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactc ccaatcttctctctgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccaggtaagccagccca ggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgg gtgctgacacgtccacctccatctctt cctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaa aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagac cctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtaca agtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggtgggacccgt ggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacc tctgtccctacagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaaga accaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatggg cagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctca ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccact acacgcagaagagcctctccctgtccccgggtaaatga In this sequence, the double underlined represent exons. Furthermore, since the digested PCR-producing 117 200837079 encoding the variable heavy chain site in the IgG expression vector is introduced into the X/z6>I digested expression vector to generate the nucleotide agt encoding the initial Ser (bold line) ). The VH and VL coding pairs discussed above are selected based on the binding specificity, epitope localization, antigen diversity and sequence diversity of the various antigens and peptides in ELISA and/or FLISA. If an error is identified, the selected homologous V gene pair is subjected to a pure line repair (Example 1, part f). Individual expression constructs were co-transfected with Flp recombinase expressing plastids into the CHO-Flpln receptor cell line (Invitrogen), followed by avidin selection of the integrants. Transfection, selection and adaptation to jk-free culture were performed as described in sections g-1 and g-2 of Example 1. Individually expressing cell lines stably transfected and adapted to serum-free suspension culture are frozen in multiple ampoules to produce a cell bank of cell lines that form individual antibodies. Example 3 An in vitro neutralization experiment has been performed on a combination of a single antibody pure line and a purified antibody. All antibody mixtures described below consist of a number of individual anti-RSV antibodies of the invention which are combined into a mixture using equal amounts of different antibodies. Single antibody test

First, the neutralizing activity of each antibody was measured by PRNT in the presence of complement against the RSV subtype A and B strains as described in section j-2 of Example 1. The EC50 values of many purified antibodies are shown in Table 7. Interestingly, although most anti-F antibodies exhibited viral neutralizing activity individually, EC5G values could not be determined for most anti-RSV protein g antibodies, indicating that these antibodies cannot neutralize the virus individually. A blank area indicates that the analysis has not been performed. ND 118 200837079 * indicates that the EC" value cannot be determined by PRNT due to very low neutralizing activity or lack of neutralizing activity. Table 7: Purified anti-RSV protein F and protein G antibodies against RSV subtypes A and B EC50 value anti-F antibody mixture __EC_jfc (Hg/ml) subtype A subtype B 793 G 2.52 800 F 0.15 0.16 810 F 0.06 0.14 816 G ND 818 F 1.86 0.21 819 F 0.18 824 F 0.03 0.02 825 F 0.12 0.04 827 F 0.16 0.10 831 F 0.08 1.66 853 G 1.49 855 G 1 6.35 ND 856 G ND 858 F ND 868 G ND 880 F 0.38 0.40 888 --^__ 0.14 894 0.08 0.07 Ability to make anti-RSV protein strains Comparison of neutralization test. Neutralization test or PRNT amount neutralization test for mixture of P F antibody and Rs v of subtype A and B. Use of palivizumab (also anti-F antibody). The trace amount of sputum described in section j of 1 is used to estimate the neutralizing ability. In the preliminary experiment, milizumab was used to compare two antibody mixtures containing five and eleven 119 200837079 anti-F antibodies, anti-F (I) and anti-F(II). Anti-F(I) is derived from pure lines 810, 818, 819, 825 And the antibody composition obtained by 827. Antibody 8 1 0 and 8 1 9 bind antigenic site A/II, antibody 8 1 8 binding site B/I or F1, antibody 825 binds to complex antigen overlapping with sites A and C And the antigen 827 binds to another complex epitope (see Table 5). The anti-F(II) line is derived from the pure lines 735, 800, 810, 818, 819, 825, 827, 863, 880, 8 84 and 894. The obtained antibody consists of anti-F(II) containing multiple binders binding to some defined antigenic sites: antibodies 810, 819 and 863 bind A/II, antibodies 800 and 818 bind F1 (or B/I) Antibody 827 and 825 bind to the above complex epitope, antibodies 735 and 894 belong to the unknown cluster (UC) I, antibody 880 belongs to UCII, and 884 binds to another currently unknown epitope (see Table 5). As shown in Figure 5, the composition anti-F(I) and anti-F(n) were more effective than palivizumab in neutralizing the two subtypes of RSV strain. Table 5 also shows the combination of five antibodies. (Anti-F(I)) appears to be more effective than a combination of eleven antibodies (anti-F(II)). The anti-F(I) mixture contains some of the most potent individual neutralizing antibodies with the specificity of the different epitopes identified to date. The anti-F(II) mixture contains the same five highly potent antibodies, and it also contains other binders that bind to some defined epitopes and the antibodies themselves also exhibit broader neutralizing activity. It is therefore possible that the activity of these highly potent antibodies is weakened in the anti-F(II) combination due to competitive binding to neutralizing epitopes on the F protein. However, because when considered in vivo, 'there may be effects other than neutralizing effects associated with individual antibodies, such as increased phagocytosis, increased antibody-dependent cellular cytotoxicity (ADCC), 120 200837079 anti-inflammatory effects, complement activation, and escape The likelihood of a mutant is reduced, so this result should not be considered as a mixture of five antibodies than a mixture of eleven antibodies when used in vivo. Since the initial experiment, in-vitro tests and pure line combinations have been improved, and some F-specific antibody combinations that are highly effective in the presence of complement have been identified. The EC" value (effective concentration required to reduce the number of induced plaques by 50%) of other anti-F antibody compositions indicates that the neutralizing potency series is in Table 8. What is the exact number of t-series t-series? For the RSV strain subtype A, most of the F-specific antibody combinations tested were more effective than palivizumab. A mixture of anti-G antibodies

The ability of the anti-G antibody mixture to neutralize the RSV strain of subtype A was tested using PRNT as described in section j_2 of Example 1. The EC5 enthalpy values for the anti-G antibody compositions tested are shown in Table 8. Most of the two anti-sputum antibody combinations did not exhibit a significantly increased virus neutralization capacity compared to individual anti-G antibodies. Regardless of concentration, some combinations of two or three anti-G antibodies never reached 100% virus neutralization. However, when other anti-G antibodies are added to the composition, the potency is increased, possibly indicating that there is a synergistic neutralization effect between the anti-G antibodies. Figure 7 shows an example of an increase in potency when multiple @G-specific pure lines are combined. A mixture of anti-F and anti-G antibodies I compared the ability of a mixture of anti-RSV protein F and protein G antibodies to neutralize the Rsv subtype B virus strain to obtain neutralization using palivizumab. Neutralizing ability was evaluated using a microneutralization fusion assay as described in Example j, j_4, and a plaque reduction neutralization assay (Example 1, part j-2). Initially, the anti-F(I)G and anti-F(II)G neutralizing activities of the two antibody mixtures were measured by a microneutralization fusion inhibition assay. Each of these mixtures contains anti-F antibodies against F(1) and anti-F(II) of the above compositions and anti-G antibodies obtained from pure lines 793, 796, 83 8, 84, 856 and 888, wherein antibodies 793, 796, 838 and G Conserved regions of the protein bind, 841, 856 bind to the GCRR of RSV subtype A and 888 binds to the GCRR of the two subtypes (see Table 5). As shown in Figure 6, both the anti-F(I)G and anti-F(II)G were more effective than palivizumab in neutralizing the rsV B1 strain. In addition, the neutralization activity of the two mixtures is approximately equal. Thus, it appears that when anti-F antibodies are combined with many protein G-specific pure lines, the difference in potency previously observed between the two anti-f antibody mixtures is reduced. This may indicate that the neutralizing activity is generally increased when the antibodies recognizing the antigen and the epitope on the broad RSV are combined. A large number of different anti-F and anti-G antibody combinations have been tested with PRNT with or without complement. The ECw values obtained by this test in the presence of active complement are presented in Table 8. All test compositions including anti-F and anti-sputum antibodies did neutralize RSV subtype A and most of these compositions were more effective than palivizumab. X and the like, and the fruit also shows that the antibody having the natural high affinity can be repeatedly obtained from the human i or the body using the antibody selection technique of the present invention. 122 200837079 Table 8: EC50 values for combinations of anti-RSV antibodies against RSV subtypes A and B. A blank area indicates that the analysis has not been performed. ND indicates that the E C 5 enthalpy cannot be determined by PRNT due to very low neutralizing activity or lack of neutralizing activity. Anti-boat EC50 value (pg/ml) in composition number composition subtype A subtype B 1 810, 818, 819, 825, 827 0.19 0.38 2 810, 818, 819, 825, 827, 831,858, 863 , 884, 894, 793, 796, 816, 838, 853, 856, 859, 888 0.34 3 810, 818, 825, 827, 884, 886, 793, 853, 868, 888 0.30 4 810, 818, 825, 827 , 831,858, 884, 886, 793, 796, 816, 853, 856, 868, 888 0.19 5 810, 818, 825, 827, 831,858, 884, 886, 793, 853, 868, 888 0.21 6 810 , 819, 825, 827, 831, 793, 853, 856, 858, 868 0.20 7 810, 811, 817, 819, 825, 827, 831,838, 853, 856, 858, 859, 863, 868 0.18 8 800 801,811,838,853,855 810, 818, 819, 825, 827, 884, 886 0.15 0.29 12 793, 816, 853,856 0.06 13 793, 816, 853, 855, 856 0.03 14 793, 868, 888, 853, 856 0.34 15 793, 796, 818 , 816, 838, 853, 855, 856, 859, 868, 888 0.11 16 810, 818, 827 0.11 0.21 17 810, 818, 825, 827, 858, 886 0.10 0.16 18 810, 818 , 825, 827, 858, 886, 793, 816, 853, 855, 856 0.04 0.15 19 818, 825, 827, 858, 886, 793, 816, 853, 855, 856 0.06 20 810, 818, 819, 825, 827, 858, 793, 816, 853, 855, 856 0.10 21 810, 793, 816, 853, 855, 856 0.04 22 818, 825, 827, 831,858, 886, 793, 816, 853, 855, 856 0.06 123 200837079 23 818, 825, 827, 831,858, 819, 793, 816, 853, 855, 856 0.06 24 818, 827, 831,858, 819, 793, 816, 853, 855, 856 0.06 25 810, 818 , 819, 824, 825, 827, 858, 793, 816, 853, 855, 856 0.07 26 831,818, 819, 824, 825, 827, 858, 793, 816, 853, 855, 856 0.08 27 831,818 , 819, 824, 827, 858, 793, 816, 853, 855, 856 0.05 28 810, 818, 824 0.06 0.04 29 810, 824 0.05 30 818, 824 0.04 31 810,818 0.11 32 824, 793, 816, 853, 855 , 856 0.05 33 810, 818, 819, 824, 825, 827, 858, 894, 793, 816, 853, 855, 856 0.07 0.03 34 810, 818, 819, 824, 825, 827, 894, 793, 816, 853, 855, 856 0.07 35 793,816 5.94 36 855, 856 ND 37 793, 856 ND 38 793, 853 2.35 39 853, 856 0.21 40 793, 853, 85 666. 47 816,856 0.06 48 816,853 0.75 49 816,853,856 0.07 50 810,818, 824,816 0.09 51 810, 818, 824, 853 0.11 52 810, 818, 824, 856 0.10 124 200837079 53 810, 818, 824, 816, 853 0.09 54 810, 818, 824, 816, 856 0.05 55 810, 818, 824, 853, 856 0.08 56 810, 818, 824, 816, 853, 856 0.05 0.03 palivizumab 0.14 Example 4 Virus in the lungs of mice infected with RSV Reduction in amount As described in section k-1 of Example 1, the in vivo protective ability of the combination of purified antibodies against RSV infection of the present invention was also confirmed in the BALB/c mouse model (Taylor et al. 1984. Immunology 52, 137-142; Mejias, et al. 2005. Antimicrob·Agents Chemother. 49: 4700-4707). In Table 9, data from experiments using three different anti-RS V rpAbs consisting of equal amounts of different lines of the antibody of the invention (as described in Table 8) and uninfected control animals from the same experiment and comforted The data of the agent (PBS) treated animals were compared. Each treatment group contained 5 mice and samples were obtained on the fifth day after infection, which is approximately the peak of viral replication in this model. As shown in Table 9, the rpAb combination effectively reduced viral load by at least an order of magnitude when administered prophylactically. The number of copies is presented as the mean standard deviation of the soil. The number of replicates was below or below this limit of detection, i.e., 3·8 logl 0 RNA copies per milliliter for both treatment groups. 125 200837079 Table 9: Viral load measured by RT-PCR (loglORNA replica/ml) of the viral load of the mouse lung after prevention and RSV dysfunction. Uninfected negative PBS 4.84 ± 0.09 anti-RSV rpAb 18 ( 50 Mg/kg ) <3.80 anti-RSV rpAb 18 (5 mg/kg) 4.31 ±0.22 anti-RSV rpAb 9 (50 mg/kg) <3.80 anti-RSV rpAb 9 (5 mg/kg) 4.34 ± 0.40 anti-RSV rpAb 17 (50 mg/kg) 4.04 soil 0.14 anti-RSV rpAb 17 (5 mg/kg) 4.30 ± 0.21 Cytokine and chemokine content in lung samples from RSV-infected mice as in section k-1 of Example 1. As described therein, lung samples from the experimental mouse prevention study were analyzed by a commercially available multiplex immunoassay to determine the levels of different cytokines and chemokines after RSV infection and prevention with antibodies to rpAb 18 (Table 8). Samples from uninfected and untreated animals were also analyzed to obtain baseline data for BALB/c mice. All samples were obtained on the fifth day after infection. The data is presented as the mean standard deviation. The analysis showed that the levels of many cytokines and chemokines were increased in placebo-treated animals, but the lungs of animals treated with approximately 50 mg/kg rpAb had approximately the same content as uninfected control animals. These cytokines and chemokines have been recognized as important markers of RSV infection and subsequent inflammatory responses in humans and mice, including 'interferon (IFN)-丫' interleukin (IL)-IP, IL-4 , IL-6, IL-8 (KC/GROa), IL-10 'macrophage inflammatory protein (ΜΙΡ)-Ια, normal T cell expression and secretion of activation regulators (RANTES, CCL5) and tumor necrosis factor (TNF) )-a. Similar results were obtained from other anti-RSV rpAb combinations. It should be noted that 'mouse does not have a clear homologue of IL-8, but it has a functional homologue of human GROa 126 200837079 (functioning similar to IL-8) known as KC. The kinetics of the inflammatory response and the dose-response relationship for antibody prevention remain to be studied. Table 10: Cytokine and chemokine content in lung tissue of RSV-infected mice Tissue sample (pg/ml) Uninfected control mice Placebo-treated mice were treated with anti-RSV rpAb Mouse IL-Ιβ 270 gentry 570 soil 100 310±140 IL-4 7.7 ±4·7 26 4.6 4.6 8.5 IL-6 6.4 士2.6 22 土12 8.2 士 3.8 IL-10 120士17 320士58 170士41 IFN-γ 20士七·6 420土88 81 土72 KC/GROa (IL-8) 51 士38 290士83 94 士99 MIP-la (CCL3) 39 士16 940士170 160±110 RANTES (CCL5) 60 soil 28 380 soil 32 140 s 66 TNF-a 18 6.1 9.5 士 10 38 地 25 [Simple diagram] Figure 1: (A) Prototypic virus strain Long (subtype A) and 1 Alignment of the amino acid sequences of the entire G protein of 8537 (subtype B). The signal/transmembrane zone is framed by a dashed line. Two variable domains between amino acids 101-133 and 208-299 identified by Cane et al. 1991 J. Gen. Virol. 72:2091 _2096 are underlined. The central fragment of the G protein has been expressed as a fusion protein in E. coli and framed in black. The two amino acid sequences are serialized in SEQ ID NOs: 711 (subtype A) and 712 (subtype B). (B) Alignment of the central segments as shown in (A). The position of the 13-aa conserved region (amino acid residue 164-176) and the G protein cysteine rich region (GCRR) are indicated by parentheses. Square brackets are used to indicate the disulfide bridge in the GCRR (both subtypes are identical). The two amino acid sequences are serialized in SEQ ID NOs: 713 (subtype A) and 714 (subtype B). 127 200837079 Figure 2 · Schematic diagram of multiple overlap extension RT_pCR (A) and colonization step (b). (A) Two sets of primers CH + VH κ8 and VK1_6 + CK1 which are specific for the Vh| ^ gene family, respectively, were used for the first - pcR step. The homologous region between the Vh or VK primers results in the production of overlapping pCR products. In the second step, amplification of the product 1 by nested PCR also includes recognition sites for restriction enzymes that facilitate selection. (8) The generated homologous linkage % and v coding pairs are confluent and are used in the expression vector (e.g., ' Subsequently, a bidirectional promoter is inserted into the ligation site between the ligation and coding sequences to facilitate expression of the full length antibody. The PCR primers used are indicated by horizontal arrows. CH1: heavy chain strange domain i, CL: deuterium localization, lc: light key; Ab: antibody; P1_P2: bidirectional promoter. Figure 3: A schematic representation of the mammalian full-length antibody expression vector 〇〇_vp_53〇. The vector comprises the following elements: Amp and Amp pro = anti-Amipizil: Umpicillin gene and its promoter. Puc start point = puc copy from V start point ° P1 = mammalian promoter that drives light key expression. P2 = a mammalian promoter that drives heavy chain expression. Lead IGHV = genomic (ge_ic) human heavy chain leader. VH = heavy chain variable region coding sequence. Chat = Ma Ma The sequence of the G1 heavy chain constant region of the immunoglobulin isoform. Rabbit B. globin A rabbit β-globulin p〇lyA sequence. κ producer = sequence encoding the sequence LC-light chain coding sequence of the murine κ s). Sv4 such as melon = 猿 disease 毋 40 ~ stop subsequence. FRT = Flp recognizes the target site. Price 〇 = anti-neomycin gene SV40 p〇ly A = prion 4〇 p〇iy a signal sequence. Figure 4 uses Biacore analysis to characterize the epitope specificity of antibodies obtained from pure line 8〇1 (Ab8〇1) obtained 128 200837079. Three antibodies, 9c5 (2), i33_h (3), and palivizumab (), which bind to the antigenic sites η, c, and η, respectively, were tested in the competition for the binding of protein F (pairs). 801 combined. Reference cells are described for binding to non-competitive Ab8〇i (protein F of 〇. The injection time of the four antibodies is indicated by the head. The reaction is expressed in relative resonance units (RU). The long double-headed arrow indicates the magnitude of the non-competitive reaction. And the short double-headed arrow indicates the magnitude of the 9c5 inhibition reaction.

Figure 5, and the in-vitro neutralization results of RS V subtypes a and B strains. The dilution of the anti-F antibody mixture was tested for its ability to neutralize RSVL ngng (a) and RSV B1 (panel B) strains. The antibody mixture obtained from the pure lines of 810, 818, 819, 825 and 827 is represented by a triangle (▲) anti-F(][), and is represented by a square (_) from 735, 800, 810, 818, 819, 825, 827, The antibody mixtures obtained from the pure lines of 863, 8 80, 8 84 and 894 were resistant to F(n). The palivizumab is represented by a man's shape (♦), and a negative-matched negative control (anti-Rhesus D) antibody is represented by a circle (Lu). Absorbance was measured at 490 nm and correlated with rSV replication. Figure ό: shows the results of the RSV fusion inhibition test in vitro. The dilution of the antibody mixture was tested for its ability to neutralize the RSV B 1 strain. The antibody mixture obtained from 810, 818, 819, 825, 827, 793, 796, 83 8, 841, 856, and 888 is expressed as an anti-F(1)G in an empty square (□), and is represented by an empty triangle (Δ) from 735, 8 00, 810, 818, 819, 825, 827, 863, 880, 884, 894, 793, 796, 838, 841, 856 and 888 are purely obtained antibody mixtures against f(II)G. Pallizumab is indicated by a diamond (♦). Absorbance was measured at 490 nm and correlated with RSV replication 129 200837079. Figure 7: shows the results of in-tube neutralization of R s V produced by a combination of anti-G antibody pure lines as measured by Prnt in the presence of active complement. The dilutions of the individual antibody compositions (described in Table 8) were incubated with the RSV strain Long in the presence of rabbit complement and then infected with HEp-2 cells. After 24 hours of culture, the degree of infection was detected using immunodetection of RSV-specific plaques. The anti-RS V rpAb 13 is represented by an empty triangle (Δ), the anti-RSV rpAb 35 is represented by a triangle (▲), the anti-RSV rpAb 36 is represented by a square (), the anti-RSV rpAb 41 is represented by a circle (·), and the square is an empty square ( □) indicates anti-RSV rpAb 45. Data were provided in the form of infection % ± SD compared to the control group. [Main component symbol description] None

130

Claims (1)

200837079 X. Patent application: 1' anti-RSV recombinant multi-strain antibody capable of neutralizing RSV subtypes A and B, and wherein the multi-strain antibody comprises at least one of at least one RSV envelope protein co-specifically bound together Different antibody members of three different epitopes. An anti-RSV recombinant multi-strain antibody according to claim 1, wherein the multi-strain antibody comprises different antibody members that together provide specific reactivity against at least two RSV envelope proteins. 3. An anti-RSV recombinant multi-strain antibody according to claim 1 or 2, wherein the RSV envelope protein is selected from the group consisting of RSV G protein, RSV F protein and RSV SH protein. 4. The anti-RSV recombinant antibody according to any one of claims ir to 3, wherein the anti-envelope protein reactivity is anti-G and anti-f reactivity, and the reactivity is at least two different The anti-G protein and at least one different anti-F protein are provided. 5. The anti-RSV recombinant multi-strain antibody according to claim 4, wherein the first anti-G antibody specifically binds to the conserved epitope on the prion and the second anti-G antibody specifically binds to the prion protein The cysteine S pool (GCRR ) ' and the anti-ρ reactivity is directed against at least one of antigenic sites j, π, IV, V, VI, C or F1. 6. Apply anti-RSV recombinant multi-strain antibody according to item 4 or 5 of the patent garden, wherein at least part of the anti-G reactivity is for cX3C: motif 〇7·If the patent patent garden is 4th to 6th Anti-RSV Recombinant 131 of any of the items 2008 20087979 Multiple antibodies ' wherein the anti-G reactivity is additionally directed against at least one strain-specific epitope. 8. An anti-rsv recombinant multi-strain antibody as claimed in any of claims 4 to 7 wherein the anti-F reactivity is at least against antigenic site n and anti-in situ IV. 9. The application of the anti-recombinant multi-strain antibody of the ninth item of the patent enclosure, wherein the anti-envelope protein reactivity is directed against the protein, or in addition to the scope of claims 4 to 8 Sh protein. The anti-RSV recombinant polyclonal antibody of any one of the preceding claims, wherein the composition of the different antibody members reflects the diversity, affinity and specificity of the rsv envelope antigen in the donor Humoral immune response 0. 11. An anti-Rsv recombinant polyclonal antibody according to any one of the preceding claims, wherein the different anti-systems are derived from one or more nucleic acids that have been produced against a human donor for a humoral immune response. The sequence is encoded, and the multi-strain antibody is a meta-human antibody. 12. The anti-rsv recombinant polyclonal antibody of any one of claims 10 or 5, wherein the different antibody members are comprised of vH and VL pairs originally present in the (these) donor. The anti-rsv recombinant polyclonal antibody of any one of the preceding claims, wherein each of the different members comprises a CDR1, CDR2 and CDR3 region selected from the group of % and the pair given in Table 5. 14. A pharmaceutical composition comprising, as a reactive component, an anti-RSV recombinant multi-strain antibody according to any one of claims 1 to 13 of the patent application, and 132 </ RTI> pharmaceutically acceptable Job agent. 15. A method for preventing, treating or ameliorating a symptom associated with a mammalian or a plurality of plaque Rsv infections, which comprises administering to the animal an effective amount of an anti-recombinant recombination as claimed in claim U-13 A plurality of antibodies or a pharmaceutical composition according to claim 14 of the patent application. &amp; The method of claim 15 wherein the effective amount is up to (10) mg of antibody per kilogram of body weight, such as up to 9 inches, up to 80, up to 70, up to 6 inches, up to 5 inches, up to 4 inches per kilogram of body weight, Up to %, up to 20, to township 10, to 彡9, up to 8, at most 7, up to 6, eve to eve 4 to eve 3, at most 2, at most 1, at most 〇.9, at most 〇.8, at most 0.7 At most 0.6, to? 〇·5, up to 〇4, up to 〇3, up to 0-2 and up to 0.1. mg. ^ 17. The method of claim 15 wherein the effective amount is from 0.01 kg body weight per kg body weight, such as at least 〇〇5, 〇", 〇2, 〇3, 0.4, 0.5, 0.6, 〇 ·7, 〇·8 〇18·If the method of patent application 帛15, the effective amount is between 公斤·ΐ mg and 20 mg antibody per kg body weight. The method of any one of claims 15 to 18, wherein the anti-system is administered at least once a year, such as 1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 13 per year. 14, 15, 16, 17, 18 and 19 times. 20. The method of claim 19, wherein the anti-system is administered at regular intervals during the year in which there is an increased risk of causing RSV infection. 21. The method of claim 20, wherein the regular time 133 200837079 is every week, every two weeks, every month or every two months. 22. The use of an anti-rsv recombinant polyclonal antibody as claimed in any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 of the patent application 'is used for preparation for treatment, improvement or prevention A composition of one or more of the symptoms associated with RSV infection in a mammal. 23. A method of generating a VH and vL coding pair (repert ire), wherein the member reflects a gene pair that causes a humoral immune response caused by RSV infection, the method comprising: a. self-infected by RSV or from Rsv The donor recovered from the infection provides a portion of the cell containing the lymphocyte; b. optionally enriches the B cell or plasma cell in the cell portion; c. obtains a population of isolated single cells comprising the portion of the cell The cells are individually dispensed into a plurality of containers; and d. using a template derived from the isolated single cells, amplifying in a multiple overlap extension RT-PCR program and effecting the linkage of the Vh and Vl coding pairs; The nested PCR (nested PCR) of the VH and VL coding pairs of the linkages is performed as appropriate. 24. A multi-cell cell line capable of exhibiting a recombinant multi-strain anti-Rsv antibody as claimed in any one of claims 1 to 13. 25 · a multi-strain cell line in which each cell is capable of expressing a single % and VL-encoded pair and the multi-strain cell line is generally capable of expressing a collection of ¥^ and ^^编马对', wherein each VH and VL coding pair encodes RSV antibody. 26. A multi-cell cell line as claimed in claim 25, wherein the 134 200837079 and vL code pair collections are produced according to the method of claim 23 of the scope of the patent application. 27. An isolated human anti-RSV antibody molecule selected from the antibody molecules listed in Table 5 herein or a specific binding fragment or synthetic or semi-synthetic antibody analog of the antibody molecule, the binding fragment or analog comprising at least the The complementarity determining regions (CDRs) of the isolated antibody molecule. 28. The antibody molecule, fragment or analog of claim 27, which is derived from the antibodies listed in Table 8, or a heavy chain thereof comprising one of SEq ID N〇s: one of the materials The CDR amino acid sequence and the associated light chain CDR amino acid sequence of SEQ ID NO, which is 88 higher than the amino acid sequence selected from SEq ID NOs. 29. An isolated antibody molecule, antibody fragment or synthetic or semi-synthetic antibody analog comprising the same CDR as the CDRs derived from a human antibody-derived Fab when used at a very low density to immobilize the surface of the sensor to avoid mass Restricted recombinant RSV G protein transmitted, when subjected to surface plasmon resonance analysis on Biacore 3000, the Fab has a dissociation constant kd of up to 500 nM for the RSV G protein. 30. The isolated antibody molecule, antibody fragment or synthetic or semi-synthetic antibody analog of claim 29, wherein the kd is at most 4 〇〇 nM 'such as at most 300 nM, at most 200 nM, at most 1 〇〇 nM Up to 1 nM, up to 900 pM, up to 800 pM, up to 700 pM, up to 600 pM, up to 500 pM, up to 400 pM, up to 3 〇〇pM, up to 2 〇〇pM, up to ΜρΜ, up to 90 pM and up to 80 pM. 3 1 · an isolated antibody molecule, antibody fragment or synthetic or semi-synthetic anti-135 200837079 "匕έ" derived from the antigen binding site of the Fab in human antibodies, the same antigen binding site, when used at very low density Recombinant Rsv F protein on the surface of the sensor to avoid the limitation of Berry transmission, the Fab has up to 5〇〇ηΜ for RSV F protein when performing surface plasmon resonance analysis on Bia(3) generation 3〇〇〇 The dissociation constant κ〇. 32. The isolated antibody molecule, antibody fragment or synthetic or semi-synthetic antibody according to claim 31, wherein the amount is at most 4 〇〇 nM, such as at most 300 nM, at most 2 〇〇 nM, at most 100 nM, Up to i nM, up to 900 pM, up to 800 PM, up to 700 pM, up to 6 〇〇pM, up to 500 pM, up to 400 pM, up to 3 〇〇pM, up to 200 pM, up to 100 pM, up to 90 pM, up to 80 pM, up to 70 pM, up to 60 PM, up to 50 pM, up to 4 〇pM, up to 3 〇pM, up to 25 pM, up to 20 pM, up to 15 pM, up to 1 〇pM, up to 9 pM, up to 8 pM, up to 7 pM , up to 6pM and up to 5pM. 33. The isolated antibody, molecule, antibody fragment or synthetic or semi-synthetic antibody analog according to any one of claims 27-32, which comprises 810, 818, 819, 824, 825, 827, 858 Or the CDR of a human antibody produced in 894 pure line. 34. An antibody composition comprising the isolated antibody molecule, specific binding fragment or synthetic or semi-synthetic antibody analog according to any one of claims 27-33, in combination with a pharmaceutically acceptable carrier , excipients, vehicles or diluents. 35. The composition of claim 34, comprising two different antibody molecules according to any one of claims 27-33 and/or 136 200837079 specific binding fragments and/or synthetic or semi- Synthetic antibody analogs. 36. The composition of claim 34, comprising at least 3 different antibody molecules and/or antibody fragments and/or synthetic or semi-synthetic antibody analogs according to any one of claims 27 to 33, Specific binding fragments or synthetic or semi-synthetic antibody analogs, such as comprising 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23 '24' 25 '26, 27, 28, 29 or 30 different antibody molecules and/or fragments and/or combinations of synthetic or semi-synthetic antibody analogs. The composition of any one of claims 34 to 36, which comprises at least one antibody molecule, fragment or analog that binds to the RSV F protein and which comprises at least one antibody, fragment or analog that binds to the RSV G protein. An isolated nucleic acid fragment encoding at least one amino acid sequence of a CDR as defined in any one of claims 27-33. An isolated nucleic acid fragment encoding at least the CDRs of an antibody produced by one of the pure lines listed in Table 5. 40. An isolated nucleic acid fragment encoding the CDR sequence of the heavy chain amino acid sequence set forth in any one of SEQ ID NOs 1-44. 41. An isolated nucleic acid fragment encoding a CDR sequence of a light chain amino acid sequence set forth in any one of SEQ ID NOs 89-132. 42. An isolated nucleic acid fragment encoding a CDR sequence of a heavy chain amino acid sequence set forth in any one of SEQ ID NOs 1-44 and having an amino acid sequence greater than 88 amino acids selected from SEQ ID NOs. The accompanying light chain CDR amino acid sequence of SEQ ID NO. 137 200837079 43. A nucleic acid fragment according to claims 38-42, which comprises the coding sequence contained in SEQ ID NOs: 45-88 and/or 133-176. 44. A vector comprising the nucleic acid fragment of any one of claims 38-43. 45. If the carrier of claim 44 is applied, it can be reproduced autonomously. 46. The vector of claim 44 or 45, which is selected from the group consisting of a plastid, a bacteriophage, a cosniid, a minichromosome, and a virus. 47. The carrier of claim 44 or 46, which is included in the 5'-3' direction and in an operative connection, at least one of which is used to drive any of claims 38-43. Promoter of the representation of the first nucleic acid fragment - the first nucleic acid fragment encoding at least one light chain CDR and any necessary framework regions; optionally, a nucleic acid sequence encoding a leader peptide; the first nucleic acid fragment; optionally encoding a constant region a nucleic acid sequence; and optionally a nucleic acid sequence encoding a first terminator, and/or in the 53' direction and in an operative linkage, at least one of which is used to drive any of claims 38-43 a promoter of the second nucleic acid fragment, the second nucleic acid fragment encoding at least one heavy chain CDR and any necessary framework regions; optionally, a nucleic acid sequence encoding a leader peptide; the second nucleic acid fragment; optionally encoding a constant region Nucleic acid sequence; and optionally a nucleic acid sequence encoding a second terminator. The vector of any one of claims 44-47, which is incorporated into the host cell genome when introduced into a host cell. A transformed cell carrying a vector according to any one of the claims of the invention, in the scope of the invention. 50. A stable cell line carrying a vector according to any one of claims 44-48 and which exhibits a nucleic acid fragment according to any one of claims 38-43, and as the case may be Its recombinant performance product is secreted or carried on its surface. XI. Schema: as the next page 139