Ultra high affinity neutralizing antibodies

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<td>DESCRIPTION</td><td>BACKGROUND OF THE INVENTION</td>

The present invention relates to novel ultra-high affinity neutralizing antibodies.

The current frequency of infection caused by resistance or difficulty in controlling microbes has created a need for newer applications to control such organisms, as well as to treat those already infected.

Among the most difficult infectious agents to control and treat are viruses.

For example, respiratory syncytial virus (RSV).

Syncytial leading cause of acute respiratory diseases in young children admitted to hospitals and the leading cause of lower respiratory tract infection in young children. A major obstacle to producing an effective vaccine against such agents as RSV has been the issue of safety. Conversely, some value in the use of immunoglobulins against such viral agents has been shown. For example, studies have shown that highly titrated RSV immunoglobulin was effective in both prophylaxis and therapy for RSV infections in animal models.

An alternative application has been the development of antibodies, especially neutralizing monoclonal antibodies, with high specific neutralizing activity. Another disadvantage to this pathway has been the need to produce human antibodies more than those of the mouse or rat and thus minimize the development of responses to human anti-mouse or anti-rat antibody, potentially resulting in other immune pathology.

An alternative application has been the production of human murine chimeric antibodies in which the

ES 2 349 348 T3 genes encoding mouse heavy and light chain variable regions have been coupled to genes for human heavy and light chain constant regions to produce chimeric, or hybrid, antibodies.

In some cases, mouse CDRs have been grafted onto human constant and framework regions with some of the mouse framework amino acids that are replaced by correspondingly placed human amino acids to provide a "humanized antibody." [Queen, US Pat. Nos. 5,693,761 and 5,693,762]. However, such antibodies contain intact mouse CDR regions and have been found with mixed efficacy, producing affinities often no greater than 10 to 10 M

A humanized anti-RSV antibody with good affinity has been prepared and is currently being marketed. [See: Johnson, US Pat. No. 5,824,307]

The production of ultra-high affinity antibodies should be desirable from the standpoint of both the neutralizing ability of said antibody as well as the more practical aspects of the need to produce less antibody to achieve a desirable degree of clinical efficacy, thereby cutting production costs and / or allowing a higher degree of clinical efficacy for administration in the same volume.

BRIEF COMPENDIUM OF THE INVENTION

The present invention provides isolated high affinity neutralizing immunoglobulin according to claim 1, and compositions according to claim 26. The invention further provides uses of said compositions, drugs and antibodies to treat or prevent a disease caused by RSV as described. detailed in claims 27-39.

ES 2 349 348 T3

The present invention relates to high affinity neutralizing antibodies and active fragments thereof that have affinity constants of at least 10¹⁰ M^-1, and even 10¹¹ M^-1, and more specifically to said neutralizing monoclonal immunoglobulin, including antibodies and / or fragments thereof, where the antibody and / or fragment has human constant regions.

The present invention solves the aforementioned problems by providing high affinity neutralizing antibodies without the presence of intact mouse CDR regions causing Human Anti-Mouse Antibody (HAMA) reactions and with activity sufficiently high affinity neutralizer to reduce cost and overall production efficiency.

One aspect of the present invention relates to high affinity neutralizing antibodies with affinity constants of at least 10¹⁰ M^-1, and even 10¹¹ M^-1, and with specificity towards specific antigenic determinants, such as those presented by proteins expressed by viruses.

An object of the present invention is to provide said high affinity neutralizing antibodies with specificity towards an RSV F antigen, such as where said antigens are expressed by RSV infected cells in a mammal, especially a human.

The high affinity neutralizing immunoglobulin, including the antibodies and / or active fragments thereof, of the present invention, and active fragments thereof, are specific for the F antigen expressed by said RSV and also expressed on the surfaces of infected cells. with RSV (the presence of

ES 2 349 348 T3 said antigen on the cell surface causes fusion of cells in the syncytium).

A high affinity neutralizing immunoglobulin, including the antibodies and / or active fragments thereof, of the present invention binds to the same epitope of an RSV F antigen as the antibody whose variable light chain has the sequence of SEQ. ID. NO: 1 (shown in Figure 1A) and whose heavy chain variable chain has the sequence of SEQ. ID. N °: 2 (shown in Figure 1B).

It is an objective of the present invention to provide ultra-high affinity neutralizing antibodies that basically have the framework regions of the immunoglobulin described in Figure 1 (with the same specificity of that immunoglobulin, which is an anti-RSV antibody structure) but where the immunoglobulins, including antibodies and active fragments thereof, of the present invention contain one or more CDRs (complementarity determining regions) whose amino acid sequences are independent of those in the dominated reference antibody, although said sequences may, in some cases, differ by no more than one amino acid and this may be limit to a difference in only one of said CDR regions.

The novel immunoglobulins of the present invention will differ from the antibody of Figure 1 (hereinafter the "basic antibody or" reference antibody or "reference immunoglobulin") only in the sequences of one or more of the CDRs and, in the most preferred embodiment these differences occur only at CDRs L2, L3, H1 and H3.

ES 2 349 348 T3

Especially preferred embodiments of the present invention have the framework sequences

<td>described</td><td>in</td><td>the</td><td>Figure</td><td>1, which</td><td>they have like this the sequences</td>

<td>variables</td><td>of</td><td>the</td><td>chain</td><td>heavy</td><td>and light described in the</td>

<td>Figures 3,</td><td> 4,</td><td> 5,</td><td>6 and 7.</td><td></td><td></td>

In one embodiment, the high affinity neutralizing antibodies of the invention include a human constant region.

In a preferred embodiment, a high affinity RSV neutralizing antibody of the invention, including active fragments thereof, with an affinity constant (K_to) of at least as high as 10¹⁰ M¹, and even 10¹¹ M^-1, is a recombinant immunoglobulin, such as an antibody or active fragment thereof, that includes a human constant region and framework regions for heavy and light chains in which at least a portion of the framework is derived from a human antibody (or from a consensus sequence of a human antibody framework), an example of such framework regions described for the antibody sequences of Figure

1.

In one embodiment, the entire framework is derived from a human antibody (or a human consensus sequence).

In another highly specific embodiment, a high affinity RSV neutralizing antibody, with an affinity of at least 10¹⁰ M^-1, is a recombinant antibody that has a human constant region, one or more CDRs that are derived from a non-human antibody in which at least one of the amino acids in at least one of the CDRs is changed and in which all or a portion of the framework is derived from a human antibody (or a consensus sequence of a human antibody framework).

ES 2 349 348 T3

In a separate embodiment, a humanized neutralizing immunoglobulin that binds to the same epitope as the basic or reference antibody or immunoglobulin whose variable chains are shown in Figure 1, and that has an affinity of at least 10¹¹ M^-1, includes at least one of the following amino acids at the following positions of the CDRs: an alanine at position 2 of CDR H1, a phenylalanine at position 6 of CDR H3, a phenylalanine at position 3 of CDR L2 and a phenylalanine at position 5 of CDR L3. Other embodiments comprise other single amino acid substitutions at these locations.

It is another object of the present invention to provide compositions comprising the immunoglobulins described herein in which said structures are suspended in a pharmacologically acceptable diluent or excipient.

It is a still further objective of the present invention to provide compositions and medicaments for preventing and / or treating respiratory syncytial viruses, where it is intended that the composition / medicament be administered to a patient at risk thereof, or afflicted with it, containing said composition or drug an immunoglobulin of the invention, such as where said antibodies exhibit the specificity and affinity properties described herein for the immunoglobulins of the invention.

DEFINITIONS

The term "antigen" refers to a structure, often a polypeptide or protein, present on the surface of a microorganism, such as a virus, for which an antibody has affinity and specificity.

ES 2 349 348 T3

The term "antigenic determinant" refers to a specific binding site on an antigenic structure for which an immunoglobulin, such as an antibody, has specificity and affinity. Thus, a particle, such as a virus, may represent an antigen but may have a number of different separate sites on its surface, such as where the virus has a number of different surface proteins and each represents a potential binding site. different for an immunoglobulin.

The term "immunoglobulin" refers to a protein or polypeptide that has specificity and affinity for an antigen or antigenic determinant. This term includes antibodies, commonly represented as tetrameric, as well as active fragments thereof, said fragments having specificity and affinity for antigens or antigenic determinants. Thus, immunoglobulin as used herein includes antibodies and all active fragments thereof.

The term "antibody" refers to a protein or polypeptide that has an affinity for an antigenic determinant, usually one found on the surface of a microorganism, especially a virus. Just like an antibody it is commonly composed of 4 chains and is therefore tetrameric.

The term "neutralizing immunoglobulin" or "neutralizing antibody" refers to the ability of the immunoglobulins, including antibodies, of the present invention to reduce the replication of microorganisms, especially viruses, in organisms as well as in cell cultures. An indication of such ability is the data from the microneutralization tests described hereinafter.

ES 2 349 348 T3 memory. Said structure normally has both variable and constant regions whereby the variable regions are largely responsible for determining the specificity of the antibody and will comprise complementary determining regions (CDRs ).

The term "complementarity determining region or" CDR refers to variable regions of either H (heavy) or L (light) chains that contain amino acid sequences capable of specifically binding antigenic targets. These CDR regions explain the basic specificity of the antibody for a particular antigenic determinant structure. Such regions are also called "hypervariable regions."

The term "active fragment" refers to a portion of an antibody that itself has high affinity for an antigenic determinant and contains one or more CDRs that account for such specificity. Non-limiting examples include Fab, F (ab) '₂, heavy-light chain dimers and single chain structures, such as a complete light chain or complete heavy chain.

The term "specificity" refers to the ability of an antibody to preferentially bind to one antigenic site against a different antigenic site and does not necessarily imply high affinity.

The term "affinity" refers to the degree to which an antibody binds to an antigen to shift the balance of antigen and antibody toward the presence of a complex formed by their binding. Thus, where an antigen and antibody are combined in relatively equal concentration, a high affinity antibody will bind to the available antigen to shift the balance toward high concentration of the resulting complex.

ES 2 349 348 T3

The term "affinity constant" refers to an association constant used to measure the affinity of an antibody for an antigen. The higher the affinity constant, the higher the affinity of the immunoglobulin for the antigen or antigenic determinant and vice versa. An affinity constant is a binding constant in units of reciprocal molarity. This constant is easily calculated from the index constants for association-dissociation reactions as measured by standard kinetic methodology for antibody reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the amino acid sequence of the variable regions of the light and heavy chain of an anti-RSV antibody where the CDR regions are underlined while the non-underlined residues form the framework regions of the variable regions of each chain. In this antibody, the CDRs are derived from a mouse anti-RSV antibody while the framework regions consist mostly of sequences derived from a human antibody. For each CDR, the locations where the amino acid substitutions were used to achieve the high affinity CDRs and the antibodies described herein are in bold. According to the description herein, such substitutions were only at CDRs L2, L3, H1 and H3. Figure 1A shows the variable region of the light chain and Figure 1B shows the variable region of the heavy chain of the light and heavy chains, respectively. Constant region sequences are not shown. These sequences are present in the basic clone (see Table 2), designated IX-493 throughout this description (i.e., SWSG- meaning a serine (S) at position

ES 2 349 348 T3 key (see tables 1 and 3) of CDR H1 of high affinity, a tryptophan (W) in the key position of CDR H3 of high affinity, a serine (S) in the key position of CDR L2 of high affinity and a glycine (G) at the key position of high affinity CDR L3). For the purposes of this description, this is the "reference antibody.

Figure 2 shows affinity comparisons for a particular set of beneficial or high affinity clones. The clonal designations are to the left of the legend to the right of the drawing together with the substitutions indicated in shown to the right of by ELISA (OD at 560 nm Clone L1FR represents those of the reference antibody the CDRs H1, H3, L2 and L3 the legend. Measurements are displayed on the left axis).

results for the structure of Figure 1.

Figure 3 shows the heavy and light chain variable regions for the preferred embodiment of clone 1 (Table 2) of the invention described herein. CDR regions are underlined while amino acid differences to the antibody of Figure 1 are indicated in bold. Thus, this preferred (i.e. high affinity) antibody has several of the high affinity CDRs (Table 3) present that result in higher affinity (above 10¹⁰ M^-1) than the basic or reference antibody.

Figure 4 shows the heavy and light chain variable regions for the preferred embodiment of clone 2 (Table 2) of the invention described herein. CDR regions are underlined while amino acid differences to the antibody of Figure 1 are indicated in bold. Thus, this preferred (i.e. high affinity) antibody has several of the high affinity CDRs (Table 3) present that

ES 2 349 348 T3 give rise to higher affinity (above 10¹⁰ M^-1) than the basic or reference antibody.

Figure 5 shows the heavy and light chain variable regions for the preferred embodiment of clone 3 (Table 2) of the invention described herein. CDR regions are underlined while amino acid differences to the antibody of Figure 1 are indicated in bold. Thus, this preferred (i.e. high affinity) antibody has several of the high affinity CDRs (Table 3) present that result in higher affinity (above 10¹⁰ M^-1) than the basic or reference antibody.

Figure 6 shows the heavy and light chain variable regions for the most preferred embodiment of the clone (Table of the invention described herein.

CDR regions are underlined while amino acid changes against the antibody of Figure 1 are indicated in bold.

Thus, this most preferred (i.e., highest affinity) antibody has several of the high affinity CDRs (Table 3) present that result in higher affinity (for

<td>over</td><td>from 10¹¹</td><td>M^-1) that</td><td>the</td><td>antibody</td><td>basic</td><td>or</td><td>of</td>

<td colspan="2">reference).</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>The</td><td>Figure</td><td>7 sample</td><td>the</td><td colspan="2">variable regions</td><td>of</td><td>the</td>

<td>chain</td><td>heavy and</td><td colspan="2">light for the</td><td>realization</td><td colspan="2">preferred</td><td>of the</td>

clone 23 (Table 4) of the invention described herein. CDR regions are underlined while amino acid changes against the antibody of Figure 1 are indicated in bold. Thus, this preferred (i.e., highest affinity) antibody has several of the high affinity CDRs (Table 3) present that result in higher affinity (above 10¹¹ M^-1) than the basic or reference antibody).

ES 2 349 348 T3

Figure 8 shows the results of the microneutralization experiments on several of the ultra-high affinity antibody clones described herein. The amino acids present at the key positions of the high affinity complementarity determining regions (see Table 3) are shown on the right in order H1, H3, L2 and L3 (as also shown in Table 2 where the clones are simply listed but the table compares to this figure counting the actual amino acids used at critical positions as described in the Table and in the legend to the right of this figure). Thus, for clone 4D2-7, there is an alanine (A) at the key position of the high affinity CDR H1, a phenylalanine (F) at the key position of the high affinity CDR H3, a phenylalanine (F) at the key position of the high affinity CDR L2 and a phenylalanine (F) at the key position of the high affinity CDR L3. Briefly, approximately 25,000 HEp-2 cells were added to each of the wells of a 96-well plate along with RSV and a given concentration of the antibody (the antibody concentration per cell is shown on the abscissa— See Example 2 for exact details). After 5 days of growth, cells were fixed, treated with biotinylated anti-F MAb, then bound to the avidin-peroxidase complex, and the ability of the bound peroxidase to react with thionitrobenzoic acid was determined by measuring OD at 450 nm. The amount of F protein present was an indicator of the extent of viral replication thereby resulting in more substrate reaction by peroxidase and increased uptake. Therefore, the lower the OD 450 value, the greater the neutralizing capacity of the indicated concentration of the given antibody. As used herein, IX-493 (SWSG—

ES 2 349 348 T3 meaning a serine (S) in the key position of the CDR H1 of high affinity, a tryptophan (W) in the key position of the CDR H3 of high affinity, a serine (S) in the key position of the high affinity CDR L2 and a glycine (G) at the key position of the high affinity CDR L3) is the "reference antibody (also indicated as LIFR and IX493L1 FR)."

Figure 9 shows the results for the microneutralization assays for several of the antibodies described herein but in which only the Fab fragments were used for the neutralization of antibody replication. As used herein, IX-493 (SWSG) is the reference Fab fragment and is derived from the Medi-493 antibody (see: Johnson et al. (1997) -ref. 23).

Figure 10 shows the results of microneutralization for an RSV-specific antibody compared to similar experiments for Fab fragments of the same antibody. Here, Medi 493 represents the antibody while IX-493 LIFR represents the Fab fragment of this antibody. The other lines are for Fab fragments of various of the antibodies produced in accordance with the present invention (given various letter-digit code designations for internal identification but having nothing to do with their relative efficacy as neutralizing antibodies).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to ultra-high affinity neutralizing antibodies and active fragments thereof having affinity constants of at least

10^{* 10} M^-1. The active fragments of these antibodies are fragments that contain at least one high affinity complementarity determining region (CDR).

ES 2 349 348 T3

With the advantage of molecular biology methods and recombinant technology, it is now possible to produce antibodies by recombinant means and thereby generate gene sequences that encode specific amino acid sequences found in the polypeptide structure of antibodies.

Said antibodies can be produced either by cloning the gene sequences that encode the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with in vitro assembly of the synthesized chains to form tetrameric structures (H2L2) active with affinity for specific epitopes and determinants

This has allowed the easy production of antibodies having characteristic neutralizing antibody sequences from different species and sources.

Regardless of the source of the immunoglobulins, as they are recombinantly constructed, or as they are synthesized, in vitro or in vivo, using transgenic animals, such as cows, goats, and sheep, using large laboratory or commercial-size cell cultures, In bioreactors by direct chemical synthesis using non-living organisms at any stage of the process, all immunoglobulins have a similar complete three-dimensional structure. In the case of an antibody, this structure is often given as H2L2 and refers to the fact that antibodies commonly comprise 2 light amino acid chains (L) and 2 heavy amino acid chains (H). Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions that interact with the target are called variable regions.

ES 2 349 348 T3 or "V and are characterized by differences in amino acid sequence from antibodies of different

The variable region of the chains either contains amino acid sequences capable of specifically binding antigenic targets. Within these sequences are smaller sequences folded 'hypervariable due to their extreme variability between antibodies or active fragments of differing specificity.

Such hypervariable regions are also called "complementarity determining regions" CDR regions.

These regions

CDRs explain the basic specificity of the antibody for a particular antigenic determinant structure.

The

CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable regions of the heavy and light chain have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all canonical antibodies each have 3 CDR regions, each non-contiguous with the others (graded L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy ( H). Accepted CDR regions have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977). The figures show the numerical scheme, where the CDRs are underlined and the numbers follow the Kabat scheme.

In all mammalian species, antibody polypeptides contain constant (that is, highly conserved) and variable regions, and within the latter there are CDRs and so-called “framework regions.

ES 2 349 348 T3 composed of amino acid sequences within the variable region of the heavy or light chain but outside the

CDRs.

The immunoglobulins described in accordance with the present high (in constant invention provide extremely affinity on the order of 10¹⁰ M for affinity, or K_to, association that describes the antigen as ligands)

RSVs expressed on neutralizing high affinity cell surfaces and by organisms maintain amino acids for as well as homology ¹, and even defined as binding of the infected surface antigen the present of

RSV ₁₀¹¹ M^-1, for a constant antibody and

F of proteins as well as in that of those with RSV.

invention both reduce in crops sufficient

They are the replication of antibodies to RSV antibodies in cell phones while to the recipient antibody sequences to prevent adverse immunological pathology. The last feature is achieved through the use of constant regions similar to those of the recipient organism, most especially one achieved through the use of similar frameworks, if found to be recipient.

amino acids such as a human mammal. This feature of sequences in antibodies

In order to facilitate not the last case, of also identical amino acids, to those from the organism some substitutions can be made in the sequences between the antigen CDRs for specificity.

framework and keep novel which

As the high affinity interaction of the present sayings is used in the present invention and the antibodies show memory, terms such as "antibody and" active fragment or "fragment" are not considered limiting in determining the full scope of the present invention. . Therefore, the

ES 2 349 348 T3 fact that the term "antibody is used rather than" active fragment or "immunoglobulin is not taken as a limitation on the invention or its uses as long as the required properties of specificity and affinity are presented by said structure.

In accordance with the invention described herein, the affinity constants that characterize the affinities of the high-affinity neutralizing antibodies, and active fragments thereof, of the present invention are association constants and were measured by formation kinetics. of the antigen-antibody complex, with the index constant for the association to form the complex indicated as K_assocokay_on and the indicated dissociation constant K_diss okay_offThe measurement of such constants is well within normal skill in the art and details will not be described further herein except for general methodology and specific conditions, where appropriate, as listed in the examples given in the present specification to further describe the invention.

The high affinity antibodies of the present invention can be achieved, as already described, through appropriate engineered antibody gene sequences, i.e. amino acid sequences, by placing the sequences of appropriate nucleotides and the expression of these in a suitable cell line. Any of the desired nucleotide sequences can be produced using the codon-based mutagenesis method, as described, for example, in US Patent Nos. 5,264,563 and 5,523,388. Such procedures allow the production of any and all amino acid residue frequencies at any of the codon positions.

ES 2 349 348 T3 desired within completely that of an oligonucleotide. This can include random substitutions of any of the 20 amino acids at a desired position or any specific subset of these. Alternatively, this process can be carried out to achieve a desired location of a particular amino acid within an amino acid chain, such as the novel CDR sequences according to the invention. In summary, the appropriate nucleotide sequence to express any desired amino acid sequence can be easily achieved using such procedures.

Novel CDR of the present invention.

This results in the ability to synthesize polypeptides, such as antibodies, with any of the desired amino acid sequences.

For example, it is now possible to determine the amino acid sequences of any of the desired domains of an antibody of choice and, optionally, to prepare homologous chains with one or more amino acids substituted by other desired amino acids, to give a range of substituted analogs. .

In applying such methods, it is appreciated that due to the degeneracy of the genetic code, such methods such as random oligonucleotide synthesis and partial degenerate oligonucleotide synthesis will incorporate redundancies for codons that specify a particular amino acid residue at a particular position, although such methods can be used to provide a master set of all possible amino acid sequences and screen these for optional function as antibody structures or for other purposes. In Cwirla et al., Proc. Natl. Acad. Sci.

ES 2 349 348 T3

87: 6378-6382 (1990) an Devlin et al., Science 249: 404-406 (1990) such methods are described.

In accordance with the invention described herein, augmented antibody variants can be generated by combining into a single polypeptide framework one, two or more novel CDR sequences in accordance with the invention, each shown to independently give as a result increased binding activity.

In this way, various novel amino acid sequences within an antibody can be combined, on the same or different CDRs, to produce high affinity antibodies within the present invention.

For example, one can employ such novel CDR sequences and investigate the resulting antibodies for affinity for a particular antigenic structure, such as the F or RSV antigen. Thus, the complete result would be an interactive process of combining various simple amino acid substitutions and investigating the resulting antibodies for antigenic affinity in a step-by-step manner. Such methods were used to prepare some of the plasma antibodies within the present invention. Such methods also represent a convenient, if tedious, way of optimizing the antibody sequences of the present invention, especially the sequences of the CDR regions of such antibodies.

Using the novel sequences and methods of the present invention, said application avoids the time and expense of generating and investigating all possible permutations and combinations of the antibody structure in an effort to find the antibody with maximum efficiency. Conversely, the complete randomization of a single 10 amino acid CDR residue will generate above 10

ES 2 349 348 T3 trillion variants, a virtually impossible number to investigate.

This interactive method can be used to generate double and triple amino acid substitutions in a stepwise process to expand the investigation of antibodies that have higher affinity.

Conversely, it should be recognized that not all locations within the sequences of the different domains of the antibody may be the same. Substitutions of any kind at a particular location can be helpful or detrimental. Furthermore, the substitutions of certain types of amino acids at certain locations may also be more or less how they affect affinity for the particular antigen. For example, it may not be necessary to treat all possible hydrophobic amino acids at a given position. It may be that any hydrophobic amino acid will also be made. Alternatively, an acidic or basic amino acid at a given location can provide large changes in measured affinity. Therefore, it is also necessary to learn the "rules of making such substitutions but determining such" rules may not require studying all possible combinations and substitutions — trends may become apparent after examining less than the maximum number of substitutions.

In accordance with what has already been said, the antibodies of the present invention are ultra-high affinity neutralizing antibodies, with specificity for the same RSV epitope as the antibody of US Patent No. 5,824,307.

Furthermore, the affinities of the ultra-high affinity antibodies of the invention are generally at least 10¹⁰ M^—1. Because these high affinities are not

ES 2 349 348 T3 easily measurable, except for the procedures described herein, said value can commonly be considered as part of a range and can, for example, be within 2 times 10¹⁰ M^-1 or be older than 10¹⁰ M^-1 or it can even be numerically equal to 10¹⁰ M^-1. In such cases, the affinity is indicated by an affinity constant, which is in the nature of a binding constant to give units of reciprocal molarity. As such, the affinity of the antibody for the antigen is proportional to the value of this constant (ie, the higher the constant, the higher the affinity). This constant is easily calculated from the index constants for association-dissociation reactions such as those measured by standard kinetic methodology for antibody reactions.

In a specific embodiment, the high affinity neutralizing antibodies of the present invention have affinity constants for their respective antigens of at least 10¹¹ M^-1, in some cases being in excess of this value, an interval in the region much higher than the measurement capacity.

High affinity neutralizing antibodies and fragments thereof can advantageously be directed to any of the desired antigenic determinants, such as epitopes present on any type of macromolecule, especially peptide epitopes present as part of the three-dimensional structure of protein and polypeptide molecules. Said peptide epitopes can commonly be present on surfaces, or otherwise be part of the structure of, microorganisms and cells, such as cancer cells. Microorganisms that express such peptide epitopes include bacteria and viruses. In the latter case,

ES 2 349 348 T3 proteins and polypeptides displaying peptide epitopes can be molecules expressed on the surfaces of viruses or they can be expressed by cells infected with a virus. For the purpose of evaluating the efficacy of the rules and methods described in accordance with the present invention, a virus was chosen as an available antigenic source to develop antibodies within the present invention. The virus chosen for further analysis was respiratory syncytial virus (RSV). The latter virus was chosen because it is well characterized with respect to its replicative cycle as well as with respect to the antigenic determinants found on its surface. Furthermore, the antigens known to be expressed by cells infected with this virus are well characterized. Thus, the virus presents both a surface G antigen and a surface F antigen, both proteins. The G antigen facilitates the binding of the virus to cell surfaces and the F proteins facilitate the fusion of the virus with cells. Cells thus infected also express the F antigen on their surfaces and the latter result induces fusion of the cells to form a syncytium; from now on the name of the virus. Furthermore, the virus is a convenient subject for analysis in the sense that those cell lines easily infected by this virus are well known and well characterized, thereby making a virus easy to grow and cultivate in vitro. Furthermore, available antibodies for the treatment of this virus are known and commercially available (for example, the antibodies described in US Patent No. 5,824,307). For these reasons, the antibody described in said patent contains the amino acid sequences of the reference antibody described in Figure 1 and whose

ES 2 349 348 T3 CDR sequences are summarized in Table 1. Therefore, the availability of a commercially successful antibody product as well as the well characterized properties of RSV made this an ideal combination to use in assay and optimization of the antibodies of the present invention and the rules described herein for producing high affinity CDRs for use in the construction of such antibodies. The methods described herein for preparing such antibodies are generally easily and advantageously applied in the field of antibody technology. Furthermore, even the specific embodiments provided by the present disclosure and which are antibodies directed specifically to antigenic determinants expressed by RSV, and RSV-infected cells, can have high affinity for other epitopes, especially those present on related viruses. Therefore, as described herein RSV, and the antibody with the variable sequences as shown in Figure 1, are simply a convenient model system used as a benchmark for the development and application of the methods. of the antibody technology taught herein.

A high affinity neutralizing antibody according to the present invention, including active fragments thereof, comprises at least one high affinity complementarity determining region (CDR) wherein said CDR has an amino acid sequence selected to result in a antibody that has an affinity constant (k_to) of at least 10¹⁰ M^—1.

In preferred embodiments, said antibody, or active fragment, comprises at least 2 high affinity CDRs, or at least 3 high affinity CDRs or even at least 4 high affinity CDRs

ES 2 349 348 T3 high affinity. In highly preferred embodiments, said antibodies or fragments thereof comprise 3 or 4 high affinity CDRs. In a preferred embodiment, said active fragment is an Fab fragment.

The high affinity antibodies of the present invention commonly comprise a mammalian constant region, preferably human, and a variable region, said variable region comprising framework regions of the heavy and light chain and CDRs of the heavy and light chain, in the that the heavy and light chain framework regions are derived from a mammalian antibody, preferably a human antibody, and where the CDRs are derived from an antibody of some species other than human, preferably mouse. Where the framework amino acids are also derived from non-human, the latter is preferably mouse.

Furthermore, the high-affinity antibodies of the invention bind to the same epitope as the antibody from which the CDRs are derived, and where at least one of the CDRs of said ultra-high-affinity antibody contains amino acid substitutions, and where said substitutions comprise the substitution of one or more amino acids in the CDR regions by non-identical amino acids, preferably the amino acids of the correspondingly aligned positions of the CDR regions of the human antibody that contribute to the framework and constant domains.

High affinity CDRs can be produced by amino acid substitutions in non-high affinity CDRs to produce said high affinity CDRs or said high affinity CDRs can be directly synthesized to form said high affinity CDRs. Therefore, the ultra-high affinity neutralizing antibodies herein

ES 2 349 348 T3 invention may have amino acid substitutions in only one of the CDR regions, preferably more than one CDR region and most preferably 3 or even 4 of said regions, with possibly as many as 5 or even all 6 of the CDRs containing at least one substituted amino acid.

In applying the methods described herein to produce antibodies of the present invention, the method of preparing the antibodies is not a limiting factor. Therefore, The high affinity neutralizing antibodies of the present invention can be prepared by generating polynucleotide sequences encoding the polypeptides of the antibodies using vectors to insert said polynucleotide sequences into permissive cells capable of not only expressing said polypeptides but also of assemble them within the characteristic tetrameric structures of antibody that are then recovered from the cells or cell cultures, possibly being secreted into the medium by said cells. The technologies for such production processes are already known and patented and are not essential to the practice of the present invention. [see: Morrison et al. US Patent No. 5,807,715]. Furthermore, the polypeptide chains of the antibodies of the present invention can be chemically synthesized, with or without the addition of enzymes, and then chemically joined to form tetrameric structures of the usual H2L2 configuration. Thus, any method of preparing the antibodies described herein can be used.

The present invention is also directed to the formation of high affinity neutralizing antibodies.

ES 2 349 348 T3 with the properties already listed, which have high affinity as a result predominantly of having sequences of

High affinity CDR. In accordance with the present invention, the CDR sequences of the antibodies described herein have been optimized to confer on the antibody molecule the ultra-high affinity characteristic of the antibodies of the present invention.

Said sequences of

CDR, along with the framework sequences described herein, especially those taught by the sequences of Figures 1

3,

4, 5, 6 and

7, and more specifically when used with constant region sequences characteristic of the antibodies of the organism that acts as a recipient of the antibodies of the present invention when used therapeutically, invention in its produce the antibodies of the present more specific embodiments . However, the methods of the present invention are more specifically directed at the amino acid sequences of CDRs.

To produce immunoglobulins, said antibodies and / or fragments thereof, within the present invention, for example high affinity neutralizing antibodies, the rules taught by the present invention are advantageously used to produce antibody molecules whose structures incorporate the sequences of the high affinity CDR sequences described in accordance with the present invention. Thus, the high affinity neutralizing antibodies of the present invention are, in essence, not truly "monoclonal" antibodies as that term is commonly used, since they do not have to be produced by cloning either. As already mentioned, the sequences of the antibodies of the invention can be synthesized

ES 2 349 348 T3 directly and therefore cannot be identical to any of the antibody sequences, especially not to any of the CDR sequences, currently known. The sequences themselves can be completely novel ab initio and not exactly represented in any antibody produced by any living organism. Thus, the high affinity CDR sequences described herein are found, or achieved, by optimization, as described herein, and then, once said high affinity CDR sequences are known, they are they can be used to fully synthesize functional antibody molecules, both dimeric and tetrameric, bifunctional or monofunctional, by any and all means known to science.

In accordance with what is already known, and to better describe the sequences described according to the invention, including their optimization, the sequence of the variable regions of the heavy and light chain of a reference antibody (here, the anti -RSV of Patent Document No. 5, 5,824,307) are shown in Figure 1A (light chain variable region- SEQ ID NO:

1) and in Figure 1B (SEC heavy chain variable region. ID NO: 2). Also in accordance with the invention, the novel sequences were produced with amino acid differences only in the CDR regions relative to the reference antibody. One means used to effect this result was to introduce mutations in the CDR regions of the so-called starter or reference chains and then to test the resulting recombinational clones for antigen affinity (RSV F protein).

ES 2 349 348 T3

In accordance with what is already known, changes were made first in a CDR sequence to optimize that sequence and determine the "critical residue, or residues, that said positions were optimized through a series of amino acid substitutions limited to that position alone. . Each of the 6 CDRs of the antibody clone were studied in turn until the "critical CDRs (where" critical CDRs means CDRs that have a substantial effect on antibody binding, such as beneficial or high affinity CDRs of Table 3). Not all CDRs were found to be critical. For the antibody used in this particular study, only the H1, H3, L2 and L3 CDRs were found to be critical but the results may be different for a different antibody. Once a "high affinity CDR (ie, a beneficial" CDR) was determined then combinations of the CDRs were studied to optimize the combination of CDR sequences resulting in a high affinity neutralizing antibody of the invention.

As a very specific embodiment, the invention described herein relates to a high affinity neutralizing antibody against respiratory syncytial virus (RSV) having an affinity constant of at least 10¹⁰ M^-1, where said affinity constant could be within at least 2 times this value due to the variability of said determinations and the variability of the affinity of the different cloned antibodies for the antigen (here, the RSV F antigen). Some of the resulting optimized structures within this embodiment had Ka greater than 10¹¹ M^-1 (eg, clones numbered 22 and 23 in Table 4).

ES 2 349 348 T3

This high affinity neutralizing antibody is also an antibody that binds to the same epitope on RSV as the antibody whose light chain variable region has the sequence of SEQ. ID. N °: 1 (Figure 1A) and whose variable region of the heavy chain has the sequence of SEQ. ID. N °: 2 (Figure 1B).

In general, the application used to identify antibodies of the invention, based on the specific example of anti-RSV already described, was to generate nucleotide sequences for the genes expressing the desired antibody chains and insert these into vectors used below. to transform Escherichia coli cells using standard protocols. Cells were grown in wells and the supernatant was sampled and measured for antigen binding using lift and capture ELISA techniques. [See: Watkins et al., (1997) Anal. Biochem. 253, 37-45; Watkins et al. (1998) Anal. Biochem. 256, 169-177. These polynucleotides were designed to provide single amino acid substitutions in the CDRs that could then be screened for increased affinity, with beneficial substitutions (which produce increased affinity) selectively combining for increased affinity. These were then investigated for binding affinity for RSV F antigen against the basic or reference antibody.

Using this protocol, the ELISA data indicated that no single amino acid substitutions in the L1 or H2 CDRs did not result in any increase in antibody clone affinity for the epitope used as antigen (herein, the RSV F antigen). . Therefore, the antibodies of the present invention all contain CDR sequences that differ from the reference antibody.

ES 2 349 348 T3 only on CDRs L2, L3, H1 and H3 (herein, the reference antibody with sequences in Figure 1 was merely a useful reference against which methods for optimizing the affinity of antibody by increasing K_to and any other system could be used just as well).

Antibodies so described herein with respect to RSV also commonly have framework regions derived from a human antibody but, where not so derived, preferably mouse.

For the reference antibody CDRs, the amino acid sequence of each CDR is shown in Table 1 (as given in the sequences of Figure 1). The amino acid residue locations within the CDRs of the basic or reference antibody, which, if substituted by amino acids as taught by the present invention, followed by optimization, produced high affinity CDR sequences (resulting in neutralizing antibodies very high affinity) and thereby a beneficial result (increased affinity), they are indicated in bold and underlined in Table 1 (giving said sequences increased affinity over the reference antibody which is indicated as "beneficial CDRs or" high affinity CDRs). The CDRs of the basic or reference antibody (Figure 1) are referred to herein as "basic or reference CDRs". Thus, Table 1 represents the CDR sequences depicted in Figure 1 (ie, for the anti-RSV reference antibody used herein for control optimization).

Table 1. Sequences of Basic or Reference CDRs

ES 2 349 348 T3

<td>CDR</td><td>Length</td><td>Sequence</td><td>SEC.ID.N2</td>

<td>L1</td><td> 10</td><td>SASSSVGY Μ H</td><td> 3</td>

<td>L2</td><td> 7</td><td>DTSKLAS</td><td> 4</td>

<td>L3</td><td> 9</td><td>FQGSGYPFT</td><td> 5</td>

<td>H1</td><td> 7</td><td>TSGM.SVG</td><td> 6</td>

<td>H2</td><td> 16</td><td>DIWWDDKKDYNPSLKS</td><td> 7</td>

<td>H3</td><td> 10</td><td>SMI TNWYFDV</td><td> 8</td>

With respect to the sequences described herein, the CDR regions as defined for the purposes of the present invention are those segments that correspond to residues 24-33 (CDR L1), 49-55 (CDR L2) and 88 -95 (CDR L3) of the variable regions of the light chain and residues 31-37 (CDR H1), 52-67 (CDR H2) and 100109 (CDR H3) of the variable regions of the heavy chain of the antibodies described In the present memory.

In the production of the antibodies of the present invention, both by generation of clones and by cloning of the polypeptide chains that make up said antibodies, or by direct synthesis of the polypeptide sequences, with or without the use of the polynucleotide sequences that encode them, or by any method that the user may choose, since no method of production of said antibodies results in a limitation of the teaching of the present invention, the basic or reference antibody can be used (variable regions of the heavy and light chain (CDRs plus

Framework) shown in the

Figure 1) as “template to generate the sequences of

Novel CDRs of the antibodies of the present invention and of comparing binding constants, purpose etc. Standard applications were used to characterize and synthesize the six single mutation CDR libraries (see Wu

ES 2 349 348 T3 et al. Proc. Natl. Acad. Sci. 95: 6037-6042 (1998)). The target CDR was first deleted for each of the libraries prior to annealing the nucleotides. For the syntheses of the libraries, the CDRs were defined as in Table 1. Codon-based mutagenesis for oligonucleotide synthesis was employed to produce the CDR sequences of the invention (as described below).

Libraries were initially screened by capture lift to identify the highest affinity variants. Subsequently, these clones were further characterized using capture ELISA and by titration on immobilized antigen.

DNA was sequenced from the highest affinity variants to determine the nature of the beneficial or high affinity substitutions. After investigation, it was determined that eight beneficial or high affinity substitutions had been observed, occurring in only four of the CDRs. These are summarized as the CDR sequences in Table 3 with differences from the reference or basic CDRs in Table 1 which are in bold and underlined. Thus, the CDR sequences in Table 3 can be considered a CDR library of cassettes available for use in the production of a high affinity neutralizing antibody of the present invention where the specificity is directed towards the RSV F antigen.

Data analysis indicated that amino acid substitution at selected locations had greatly increased epitope binding, especially where the nature of the substitution was the insertion of an amino acid selected from the group of phenylalanine, alanine, proline, tryptophan. and tyrosine, most especially phenylalanine, again all

ES 2 349 348 T3 these beneficial and high affinity substitutions at selected positions.

For the optimization experiment described herein and using the RSV / anti-RSV system, it was found that the most beneficial of the high affinity CDRs resulted from amino acid substitutions in 3 or 4 of the 6 CDRs, and in just 4 amino acid locations in their entirety. Thus, the high affinity neutralizing antibodies of the present invention contain amino acid sequences that differ from that of the base or reference antibody only in complementary determining regions, or in such regions as well as in the surrounding framework regions, as opposed to previously known human-made antibodies. Thus, the antibodies of the present invention are high affinity neutralizing antibodies that contain one or more CDR sequences selected to produce high affinity for the antibody molecule. In the specific embodiment that uses RSV as the target such differences are found only in

L2 (or CDRL2), L3 (CDRL3), H1 (or CDRH1), and H3 (or CDRH3). As indicated, the highest affinities for this antibody occurred only at selected amino acid positions of these CDRs, and in some of the embodiments of the present invention just one amino acid location on each CDR was preferred to give high affinity.

Thus, for CDR H1, it was found that substitution at amino acid 2 of the CDR (counting from the amino terminal end of the particular underlined CDR sequence of Figure 1B), especially by substitution of the serine located at position 2 CDR H1 of the basic or reference antibody with either an alanine or a

ES 2 349 348 T3 proline, was the most beneficial and therefore resulted in higher affinity for the RSV antigen epitope. For CDR H3, it was found that substitution of glycine at position 6 of the CDR sequence, especially either a phenylalanine or tryptophan, most especially by phenylalanine, resulted in increased affinity for the RSV epitope. For CDR L2, substitution of serine at position 3 of the CDR, especially either a phenylalanine or tyrosine, resulted in increased affinity for the F antigen. For CDR L3, substitution of glycine at position 5 of CDR, especially by phenylalanine, tryptophan, or tyrosine, resulted in increased affinity for the RSV epitope.

In accordance with the invention, by combining said amino acid substitutions so that more than one occurred in the same antibody molecule, it was possible to greatly increase the affinity of the antibodies described herein for the RSV F antigen epitope.

Table 2 shows the results of using the novel CDR sequences (for H1,

H3,

L2

L3, respectively) of a number of clones according to the present invention.

As shown in Table 2, a particular antibody may have herein incorporated as many as 1, or 3 novel CDRs of the invention against the basic or reference antibody chains shown in Figures 1A and 1B (for chain light (L2 and L3) and heavy (H1 and H3), respectively). The effects of the novel CDRs are described in terms or Antigen (Ag) titer mark (see below), where the basic or reference antibody is shown at the top and has a mark (“score) of 0.1 . Others

ES 2 349 348 T3 markers are indicated relative to this tag 0.1 of the "basic or reference antibody sequences. The identities of the amino acids that give the highest affinities at the respective locations (i.e., position 2 for H1, position 6 for H3, position 3 for L2, and position 5 for L3) are listed below the amino acids for the antibody. basic or reference

<td>just to</td><td>indicate</td><td>the</td><td>interval</td><td colspan="2">of mutations used</td>

<td>for each CDR.</td><td></td><td></td><td></td><td></td><td></td>

<td>The number</td><td>total</td><td>of</td><td>clones</td><td>examined</td><td>in this</td>

<td>experiment was</td><td>of 37,</td><td>with</td><td>Some</td><td>duplicates</td><td>(indicated</td>

by the value "n in parentheses, for example," n = 4 for clone # 7 indicates that 4 duplicate clones were examined).

Overall, the data showed that there is a correlation between affinity and the number of beneficial or high affinity CDRs, with all higher affinity variants having more than one beneficial or high affinity CDR. Furthermore, all the best clones had an F (Phe) at position 6 within CDR H3. Also, the beneficial or high affinity CDRs were those in which a hydrophobic amino acid, especially an aromatic one, was inserted in place of the residue found in the basic or reference antibody.

The antibody titer assay employed varying concentrations of the antibody using 500 ng of RSV F antigen for each measurement. A graph of comparison data for a number of the combinatorial clones in Table 2 is shown in Figure 2.

In summary, Table 2 shows a number of clones evaluated by the procedures described herein along with the amino acids that occur at key locations (underlined and bold in Tables 1 and 3) of CDRs H1, H3, L2 and L3. The Table also

ES 2 349 348 T3 summarizes the number of differences between the CDRs of these clones versus the corresponding CDRs of the reference antibody (See Table 1 and Figure 1)

The right side of the Table shows a “Mark of

Ag or antigen binding value, which represents an arbitrary qualitative value, which ranges between

0-4 represents a qualitative estimate of the relative binding capacity of the different antibody clones based on their respective titration curves. This value is provided herein for rough qualitative comparisons of different antibodies only and is not intended as a quantitative measure of binding capacity.

Table 2 also shows the number of the novel CDRs for each antibody clone (meaning the number of CDRs in the antibody with at least one amino acid difference from the corresponding CDR of the reference antibody — see Table 1). The number of novel CDRs is also the number of "beneficial" or "high affinity" CDRs present in that antibody molecule. The amino acid differences in the novel CDR would be given in the position in bold and underlined in Table 1 for the reference antibody such that the amino acid in bold and underlined in Table 1 has been replaced by the amino acid indicated for the respective CDR in Table 2 (using standard single letter amino acid designations).

Table 2. Summary of Clone Data

<td>Clone</td><td colspan="4">CDRs</td><td>N ° CDRs Novelty</td><td>Ag brand</td>

<td></td><td>H1</td><td>H3</td><td>L2</td><td>L3</td><td></td><td></td>

ES 2 349 348 T3

<td>Basic</td><td>S</td><td>W</td><td>S</td><td>G</td><td> 0</td><td> 0,1</td>

<td>Simple</td><td>TO</td><td>F</td><td>F</td><td>F</td><td></td><td></td>

<td></td><td>P</td><td></td><td>Y</td><td>W</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>Y</td><td></td><td></td>

<td> 1</td><td>TO</td><td>F</td><td>S</td><td>F</td><td> 3</td><td> 4</td>

<td> 2</td><td>TO</td><td>F</td><td>F</td><td>G</td><td> 3</td><td> 4</td>

<td>3 (n = 3)</td><td>P</td><td>F</td><td>F</td><td>F</td><td> 4</td><td> 4</td>

<td>4 (n = 3)</td><td>P</td><td>F</td><td>F</td><td>Y</td><td> 4</td><td> 3,5</td>

<td>5 (n = 3)</td><td>P</td><td>F</td><td>F</td><td>W</td><td> 4</td><td> 3,5</td>

<td> 6</td><td>P</td><td>F</td><td>Y</td><td>F</td><td> 4</td><td> 3,5</td>

<td>7 (n = 4)</td><td>P</td><td>F</td><td>F</td><td>G</td><td> 3</td><td> 3</td>

<td> 8</td><td>P</td><td>F</td><td>F</td><td></td><td> 3 +</td><td> 3,5</td>

<td>9 (n = 2)</td><td>P</td><td>F</td><td>S</td><td>W</td><td> 3</td><td> 3</td>

<td> 10</td><td>P</td><td>F</td><td>S</td><td>F</td><td> 3</td><td> 3</td>

<td> 11</td><td>P</td><td>W</td><td>F</td><td>W</td><td> 3</td><td> 3</td>

<td>12 (n = 2)</td><td>P</td><td>W</td><td>F</td><td>F</td><td> 3</td><td> 2,5</td>

<td>13 (n = 3)</td><td>S</td><td>F</td><td>F</td><td>F</td><td> 3</td><td> 2,5</td>

<td> 14</td><td>S</td><td>F</td><td>F</td><td>W</td><td> 3</td><td> 2,5</td>

<td>15 (n = 2)</td><td>TO</td><td>F</td><td>S</td><td>G</td><td> 2</td><td> 2,5</td>

<td>16 (n = 2)</td><td>P</td><td>F</td><td>S</td><td>G</td><td> 2</td><td> 2</td>

<td> 17</td><td>P</td><td>W</td><td>S</td><td>W</td><td> 2</td><td> 2</td>

<td>18 (n = 2)</td><td>S</td><td>F</td><td>F</td><td>G</td><td> 2</td><td> 2</td>

<td> 19</td><td>S</td><td>F</td><td>S</td><td>W</td><td> 2</td><td> 2</td>

<td> 20</td><td>S</td><td>F</td><td>S</td><td>F</td><td> 2</td><td> 2</td>

<td> 21</td><td>S</td><td>W</td><td>Y</td><td>F</td><td> 2</td><td> 2</td>

The novel CDRs represented in each of the clones are easily determined by locating the clone in the table, and matching the amino acid indicated for each CDR with the corresponding amino acid for the same CDR close to the basic or reference clone. For convenience, where an amino acid is different in a particular CDR from one of the

ES 2 349 348 T3 clones, the new

Furthermore, for all selected amino acid substitutions the clones are indicated in bold.

shown in the table, are given only in the locations listed below when producing a novel CDR.

relative

Therefore, all the substitutions to the basic or reference antibody of the CDR (meaning amino acid from the end as the

3), in CDR

H1 are in the underline and all positions, again, terminal of the second

CDR H1 in bold in substitutions Figure 1 in CDR H3 are the

Tables in the all all basic or of chose substitutions in CDR L2 substitutions in CDR L3 are in the, again reference.

all with

Should the basic antibody you knew had a very high

RSV. [See clone No. 1, one or of

Johnson et al.,

Thus, for example, reference to the antibody keep in mind that reference because already affinity for the (1997) J.

the table for the beneficial H1 CDR is

Infect.

shows epitopes of

Dis., 176, that for the high affinity o, alanine instead of serine at position 2 of the CDR H1 of the basic or reference antibody, thereby achieving an increased affinity for phenylalanine is given instead of tryptophan in that of the CDR antibody was given in affinity.

H3, serine at position 3 of the

RSV, and a 6 position

CDR L2 of the basic or reference was used and a phenylalanine of the

CDR L3 beneficial or high

Therefore, in the

Table summarizes the novel and beneficial CDRs according to the present invention (that is, CDRs of high affinity CDR sequences whose presence in the reference basic antibody instead of the corresponding reference basic CDR served to greatly increase the affinity of said reference.

ES 2 349 348 T3 antibody to the same RSV epitope) which are present in the antibody structures produced in the supernatants tested for the clones of Table 2. In each case, in bold indicates as the novel and beneficial CDRs, or of the invention differ from the corresponding CDRs of the basic or reference anti-RSV antibody.

Table 3. Sequences for High Affinity CDRs

<td>CDR</td><td>Sequence</td><td>SEC.ID.N2</td>

<td>H1</td><td>TAGMSVG</td><td> 9</td>

<td>H1</td><td>TPGMSVG</td><td> 10</td>

<td></td><td></td><td></td>

<td>H3</td><td>SMJTNFYFDV</td><td> 11</td>

<td></td><td></td><td></td>

<td>L2</td><td>DTFKLAS</td><td> 12</td>

<td>L2</td><td>DTYKLAS</td><td> 13</td>

<td></td><td></td><td></td>

<td>L3</td><td>FQGSFYPFT</td><td> 14</td>

<td>L3</td><td>fqgsyypft</td><td> 15</td>

<td>L3</td><td>FQGSWYPFT</td><td> 16</td>

While the CDR sequences in Table 3 represent the sequences for the high affinity CDRs described according to the invention, it is understood that one or more of these CDRs may be present in the same antibody and the sequences in the table indicate the whole from which appropriate sequences can be selected for each of the high affinity CDRs. Therefore, as shown in Table 3, when a high affinity H1 CDR is present in a high affinity neutralizing antibody of the invention described in

ES 2 349 348 T3 herein has a sequence corresponding to the sequence of SEQ.

ID.

N °:

or 10. If a neutralizing antibody of the claimed invention contains a CDR

H3 of high affinity, said CDR has the sequence of SEQ.

ID. NO: 11. If a high affinity neutralizing antibody of the invention contains a high affinity L2 CDR, said high affinity L2 CDR has an amino acid sequence selected from the group consisting of the sequences of SEQ. ID. N °: 12 and 13. Finally, if a high affinity neutralizing antibody of the present invention contains a high affinity L3 CDR, said CDR has an amino acid sequence selected from the group consisting of the sequences of SEQ. ID. N °: 14, 15 and 16.

As already discussed, in one embodiment, high affinity neutralizing antibodies are antibodies that include a human constant region.

Therefore, in a preferred embodiment, the high affinity neutralizing antibody of the invention, with an affinity of at least 10¹⁰ M^-1, or even at least 10¹¹ M^-1, is a grafted antibody that includes a human constant region and a framework for heavy and light chains in which at least a portion of the framework is derived from a human antibody (or from a consensus sequence of a human antibody framework) .

In another embodiment, all frameworks are derived from a human antibody (or a human consensus sequence).

Thus, a neutralizing antibody to RSV, with an affinity of at least 10¹⁰ M^-1, is a grafted antibody that has a human constant region, one or more CDRs that are derived from a non-human antibody in which at least one of the amino acids in at least one of said CDRs is changed and in which all or a portion of the framework

ES 2 349 348 T3 is derived from a human antibody (or a consensus sequence of a human antibody framework).

Due to the combination of CDR sequences of an antibody with non-CDR regions of another antibody resulting in a form of "grafting of the CDRs onto the rest of the molecule, these have been called" CDR grafted "antibodies. Today, the same product can be formed using genetic engineering techniques without isolating any of the sequences from current antibodies. Provided that the desired CDR sequences and the framework and constant sequence are known, the genes with the desired sequences can be assembled and, using a variety of vectors, grafted onto appropriate cells for expression of the functional tetrameric antibody molecules. Coupling this with the methodology already described allows the assembly of single mutation libraries in which the antibodies have the same sequences as the grafted antibodies.

<td>corresponding and,</td><td>by</td><td>therefore, the</td><td>same</td><td colspan="2">structure and</td>

<td>union affinities.</td><td></td><td></td><td></td><td></td><td></td>

<td>Antibodies</td><td>of</td><td>high affinity</td><td>of the</td><td>invention</td><td>I know</td>

<td>can present in</td><td colspan="2">relatively shape</td><td>pure</td><td>or isolated</td><td>So</td>

as in a supernatant extracted from cells grown in wells or plates. Said supernatants were used to generate the data in Table 1. Therefore the antibodies of the invention can also be present in the form of a composition comprising the antibody of the invention and in which said antibody is suspended in a pharmacologically acceptable carrier. , or excipient. The antibodies of the invention may be present in said composition in a concentration, or in an amount, sufficient to be of therapeutic or pharmacological value in the treatment of diseases, such as

ES 2 349 348 T3

RSV. Such antibodies can also be present in a composition in a more dilute form.

Accordingly, the invention is also directed to provide compositions and medicaments for preventing and / or treating respiratory syncytial virus infections where the composition / medicament is intended to be administered to a patient at risk of, or afflicted with, the same.

Said composition medicament comprises the antibody composition described herein.

A preferred embodiment of the high affinity antibodies of the present invention is the antibody whose light regions have sequences

Heavy chain CDR and

<td>such</td><td>as follow</td><td>: CDR H1 has</td><td>the sequence of</td><td>SEC.</td><td>ID</td><td>. N °:</td>

<td> 9,</td><td>CDR H3 has</td><td>sequence</td><td>SEC. ID. N °: 11,</td><td>CDR</td><td>L2</td><td>has</td>

<td>the</td><td>sequence of</td><td>SEC. ID. N ° 4</td><td>(no change since</td><td>CDR</td><td>L2</td><td>of the</td>

reference sequence of Figure 1A) and CDR L3 has the sequence of SEQ.

ID.

No. 14 (see Table 3).

In this preferred embodiment, the affinity constant is approximately 6.99 X 10¹⁰ (or about 14.3 pM as a dissociation constant) as shown in Table 4 (clone 1). The heavy and light chain variable regions of an antibody comprising this embodiment are shown in Figure 3, along with the framework sequences.

Another preferred embodiment of the high affinity antibodies of the present invention is the antibody whose heavy and light chain CDR regions have the following sequences: CDR H1 has the sequence SEQ. ID. NO: 9, CDR H3 has the sequence of SEQ. ID. NO: 11, CDR L2 has the sequence of SEQ. ID. NO: 12, and CDR L3 has the sequence of SEQ. ID. N °: 5 (see Table 2, clone 2-no difference from CDR L3 reference sequence

ES 2 349 348 T3 of Figure 1A). In this preferred embodiment, the affinity constant is about 7.30 X 10¹⁰ (or about 13.7 pM as a dissociation constant) as shown in Table 4 (clone 2). The heavy and light chain variable regions of an antibody comprising this embodiment are shown in Figure 4, along with the framework sequences.

A further preferred embodiment of the high affinity antibodies of the present invention is the antibody whose heavy and light chain CDR regions have the following sequences: CDR H1 has the sequence of SEQ. ID. NO: 10, CDR H3 has the sequence of SEQ. ID. NO: 11, CDR L2 has the sequence of SEQ. ID. NO: 12 and CDR L3 has the sequence of SEQ. ID. No: 14 (see Table 3 for CDR sequences) whose clone is designated by number 3 in Table 2. In this preferred embodiment, the affinity constant is approximately 8.13 X 10¹⁰ (or about 12.3 pM as a dissociation constant) as shown in Table 4 (clone 3). Figure 5 shows the heavy and light chain variable regions of an antibody comprising this embodiment, along with the framework sequences.

A highly preferred embodiment of the high affinity antibodies of the present invention is the antibody whose heavy and light chain CDR regions have the following sequences: CDR H1 has the sequence of SEQ. ID. NO: 9, CDR H3 has the sequence of SEQ. ID. NO: 11, CDR L2 has the sequence of SEQ. ID. NO: 12 and CDR L3 has the sequence of SEQ. ID. N °: 14 (see Table 3). In this preferred embodiment, the affinity constant is approximately 3.6 X 10¹¹ (or about 2.8 pM as a dissociation constant) as shown in Table 4 (clone 22). Figure 6 shows the regions

ES 2 349 348 T3 variables of the heavy and light chain of an antibody comprising this embodiment, together with the framework sequences.

Another highly preferred embodiment of the present invention is the antibody whose heavy and light chain CDR regions include the following sequences: CDR H1 has the sequence of SEQ. ID. NO: 9, CDR H3 has the sequence of SEQ. ID. NO: 11, CDR L2 has the sequence of SEQ. ID. NO: 12 and CDR L3 has the sequence of SEQ. ID. N °: 15 (see Table 3). In this latter preferred embodiment, the affinity constant is approximately 4 X 10¹¹(approximately 2.5 pM as dissociation constant) as shown in Table 4 (clone 23). The heavy and light chain variable regions of an antibody comprising this embodiment are shown in Figure 7, along with the framework sequences.

In particularly preferred embodiments, the antibodies of the present invention will have the framework regions of the sequences depicted for the framework regions in Figures 1, 3, 4, 5, 6, and 7 (each contains the same framework regions and differ only in CDR sequences). These highly preferred embodiments include the neutralizing antibody in which the variable region of the light chain has the amino acid sequence of SEQ.

ID. NO: 17 and the heavy chain variable region has the amino acid sequence of SEQ. ID. No: 18; the neutralizing antibody in which the variable region of the light chain has the amino acid sequence SEQ. ID. NO: 19 and the heavy chain variable region has the amino acid sequence of SEQ. ID. No: 20; the neutralizing antibody in which the variable region of the light chain has the amino acid sequence of SEQ. ID. N °: 21 and the region

ES 2 349 348 T3 heavy chain variable has the amino acid sequence of SEQ. ID. No: 22; the neutralizing antibody in which the variable region of the light chain has the amino acid sequence of SEQ.

ID. NO: 23 and the variable region of the variable chain has the amino acid sequence of SEQ. ID. No: 24; the neutralizing antibody in which the variable region of the light chain has the amino acid sequence of SEQ.

ID. NO: 25 and the heavy chain variable region has the amino acid sequence of SEQ. ID. N °: 26.

It should be kept in mind that while the high affinity neutralizing antibodies of the present invention can be assembled from the regions

CDRs and non-CDRs derived from current neutralizing antibodies by splicing amino acid segments together (and antibodies thus assembled would be within the invention described herein) the antibodies of the present invention are most conveniently prepared by sequences of appropriate genes engineered into vectors that can then be transferred into suitable cell lines for expression eventual of the antibody molecules assembled by engineered cells. In fact, such recombinant procedures were employed to prepare the antibodies described herein. Furthermore, because the sequences of the chains of high affinity antibodies are known from the description herein, such antibodies could also be assembled by direct synthesis of the appropriate chains and then allow self-determination. assembly within tetrameric bivalent antibody structures (H2L2).

ES 2 349 348 T3

The method of preparing the high affinity neutralizing antibodies of the invention involved the creation of a combinatorial library which was used to prepare antibody-producing clones comprising the beneficial CDRs of the invention that could then be screened for affinity. for RSV epitopes (eg Figure 2).

EXAMPLE 1

Kinetic Analysis of Humanized RSV Mabs Using BIAcoreTM

Interaction kinetics between high affinity anti-RSV Mabs and RSV F protein were studied by surface plasmon resonance using a Pharmacia BIAcore ™ biosensor. A recombinant baculovirus expressing a C-terminal truncated F protein provided an abundant source of antigen for kinetic studies. The supernatant, which contained the secreted protein F, was enriched approximately 20-fold by successive chromatography on concanavalin A and Q-sepharose columns. The fractions pooled against 10 mM sodium citrate (pH 5.5) were dialyzed, and concentrated to approximately 0.1 mg / ml. In a normal experiment, an aliquot of protein F (100 ml) was amine coupled to the BIAcore sensor chip. The immobilized amount gave approximately 200 response units (Rmax) signal when saturated with mouse monoclonal antibodies H1129 or H1308F (prepared as in US Patent 5,824,307, the disclosure of which is incorporated herein by reference. ). This indicated that there were an equal number of "A and" C antigenic sites on the F protein preparation followed by the coupling procedure. Two irrelevant Mabs no

Related ES 2 349 348 T3s (RVFV 4D4 and CMV H758) showed no interaction with immobilized F protein. A typical kinetic study involved the injection of 35 ml of Mab at varying concentrations (25-300 nM) in PBS buffer containing 0.05% Tween-20 (PBS / Tween). The flow rate was maintained at 5 ml / min, giving a binding phase of 7 min. Followed by Mab injection, the flow was exchanged with PBS / Tween buffer for 30 min to determine the dissociation index. The sensor chip was regenerated between cycles with a 2 min pulse of 10 mM HCl. The regeneration step caused a minimal loss of binding capacity of the immobilized F protein (4% loss per cycle). This small decrease did not change the calculated values ​​of the index constants for union and dissociation (also called Kon and Koff, respectively).

More specifically, for the Kassoc (or Kon) measurement, protein F was directly immobilized by the EDC / NHS method (EDC = N-ethyl-N^,- [3-diethylamino-propyl] carbodiimide; NHS = N-hydroxysuccinimide) with the F protein that is injected onto the EDC / NHS activated sensor chip. In summary, 4 pg / ml of protein F was prepared in 10 mM NaOAc, pH 4.0 and approximately a 30 µl injection gave approximately 500 RU (Response Units) of protein F immobilized under the conditions referred to above. The blank flow cell (surface immobilized with VnR CM-dextran) was subtracted by kinetic analysis. The column could be regenerated using 100 mM HCl (with 72 seconds of contact time required for complete regeneration). This treatment extracted bound Fab completely without damaging the immobilized antigen and could be used for over 40 regenerations. For Kon measurements, Fab concentrations were 12.5 nM, 25 nM, 50 nM, 100

ES 2 349 348 T3 nM, 200 nM and 400 nM. The dissociation phase was analyzed from 230 seconds (30 seconds after the start of the dissociation phase) to 900 seconds. Kinetics were analyzed using the 1: 1 Langmuir test (global test). Measurements were made in HBS-EP buffer (10 mM HEPES, pH

7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) surfactant P20).

For measurements of combinatorial clones, as described herein, Kon and Koff were measured separately. Kon was measured under conditions that were the same.

<td>same as</td><td colspan="2">those for the clones</td><td>of</td><td colspan="2">single mutation and</td>

<td colspan="2">were analyzed in a way</td><td>Similary.</td><td></td><td></td><td></td>

<td>To</td><td>measure the</td><td>^Kdissoc^(okayoff⁾</td><td>^,</td><td>they used</td><td>the</td>

<td>following</td><td>terms.</td><td>In summary,</td><td>I know</td><td>immobilized</td><td> 4100</td>

RU of protein F (as above) with CM-dextran used as a target. Herein, 3000 RU of Fab was bound (with Fab dissociated high enough to compensate for machine jitter). HBS plus 5 nM F protein (approximately 350-2000 times greater than the Kdissoc or Kdt. Dissociation equilibrium constant) was used as a buffer. The dissociation phase was 6-15 hours at a flow rate of 5 µl / min. Under the conditions used herein, the assembly of the dissociated Fab was minimal. For details, see the manual with the biosensor.

The binding of high-affinity anti-RSV antibodies to protein F, or other epitopic sites on RSV, described herein, was calculated from the ratio of the first-order index constant for dissociation to the constant second-order index for the union or association (K_d= K_diss/ K_assoc). The kassoc value was calculated based on the following index equation: dR / dt ^ kassocfMabJRmax - (k_aces_0C[Mab] + kdiss) R

ES 2 349 348 T3 in which R and R_max are the units of response in infinity time, respectively. A “plot” of dr / dt as a function of R gives a slope of [K_assoc [Mab] + K_dlss). Since these slopes are linearly related to [Mab], the value k can be derived_assoc of an update of the graphical representation (“replot) of the slopes against [Mab]. The slope of the new line is equal to Kassoc. Although the Kdiss value can be extrapolated from the y-intercept, a more accurate value was determined by direct Kdiss measurement. Following the Mab injection phase, the PBS / Tween buffer flows through the sensor chip. From this point on, [Mab] = 0. Therefore, the equation indicated above for dR / dt reduces to:

dr / dt = ko dR / R = k_oiss dt

Then the integration of this equation gives:

^In (^R0/^Rt) - ^kdiss^t in which R₀/ R₁ are the units of response at time 0 (start from the dissociation phase) and t, respectively. Finally, the plotting of In (R0 / Rt) as a function of t gives a slope of Kdiss.

In the preferred embodiment herein, the numerical values ​​from said antibody variants were as follows:

Table 4. Summary of Kinetic Constants from

Ultra-High Affinity Antibodies.

<td>Clone ID</td><td>CDRs</td><td>kassoc (M^-1sec^-1)</td><td>kdiss (sec^-1)</td><td>Ka (M^-1)</td>

<td> 1</td><td>AFSF</td><td>1.13 X 10⁵</td><td>1.62 X 10^-6</td><td>6.98 X 10¹⁰</td>

<td> 2</td><td>AFFG</td><td>1.33 X 10⁵</td><td>1.82 X 10^-6</td><td>7.31 X 10¹⁰</td>

<td> 3</td><td>PFFF</td><td>1.10 X 10⁵</td><td>1.35 X 10^-6</td><td>8.15 X 10¹⁰</td>

ES 2 349 348 T3

<td> 22</td><td>AFFF</td><td>1.34 X 10^{3 * 5 * * * *}</td><td>3.7 0 X 10^-7</td><td>3.62 X 10¹⁰</td>

<td> 23</td><td>AFFY</td><td>1.22 X 10⁵</td><td>3.03 X 10^-7</td><td>4.03 X 10¹⁰</td>

Herein, the CDRs represent the amino acids that substitute for the reference amino acids at the key positions (or critical positions) of the CDRs shown in Table 1 (in bold and underlined) for a reference antibody. Thus, for example, clone 22 has an alanine at position 2 of CDR H1 (residue 32 of the heavy chain variable region-SEQ ID NO: 24) instead of the serine shown at position at Table 1 (SEQ ID. NO: 6), a phenylalanine at position 6 of CDR H3 (residue 105 of the variable region of the heavy chain-SEQ. ID. NO: 24) instead of the tryptophan shown at that position in Table 1 ( SEQ ID NO: 8), a phenylalanine at position 3 of CDR L2 (residue 51 of the variable region of the light chain-SEQ ID NO: 23) instead of the serine shown at that position in Table 1 (SEQ ID NO: 4), and a phenylalanine at position 5 of CDR L3 (residue 92 of the variable region of the chain

<td>light-SEC. ID.</td><td>N °:</td><td>23) in</td><td>place</td><td>of</td><td>the wisteria shown in</td>

<td>that position in</td><td>the</td><td>Table 1</td><td>(SEC.</td><td>ID.</td><td>N °: 5).</td>

<td>Agree,</td><td>in</td><td colspan="2">the formation</td><td>of</td><td>said clones, the Table</td>

represents a set of potential CDRs from which the high affinity CDRs of the antibodies of the present invention are extracted. For example, clone 23 of Table 4 uses the same CDRs as clone 22 with the exception of CDR L3, which has a tyrosine at position 5 of CDR L3 (Table 3-SEQ ID NO: 15 ) instead of the glycine shown at that position in Table 1 (SEQ.

ID. N °: 5). Likewise, Table 2 shows the substitutions in the corresponding critical positions.

ES 2 349 348 T3

EXAMPLE 2

Microneutralization Assay

Neutralization of the antibodies of the present invention was determined by microneutralization assay. This microneutralization assay is a modification of the procedures described by Anderson et al. (1985). Antibody dilutions were made in triplicate using a 96-well plate. 10 TCID50 respiratory syncytial virus (RSV-long strain) were incubated with serial dilutions of the antibody (or Fabs) to be tested for 2 hours at 37 ° C in the wells of a 96-well plate. Then, RSV-susceptible HEp-2 cells (2.5 x 10⁴) to each well and cultured for 5 days at 37 ° C in CO₂ at 5%. After 5 days, the medium was aspirated and the cells were washed and fixed to the plates with 80% methanol and 20% PBS. RSV replication was then determined by expression of protein F. Fixed cells were incubated with a biotin-conjugated anti-protein F monoclonal antibody (pan protein F, C-site-specific Mab 133-1H), washed and avidin was added. conjugated with horseradish peroxidase to cells. The wells were again washed and the turnover of the TMB (thionitrobenzoic acid) substrate was measured at 450 nm. Neutralizing titer was expressed as the antibody concentration that caused at least a 50% reduction in absorbance at 450 nm (the OD450) of the virus-only control cells. The results for various antibodies of the present invention are shown by the graph in Figure 8, while the results using the Fab fragments are represented in the graph of Figure 9.

ES 2 349 348 T3

Background and References Cited:

1. Hall, CB, Douglas, RG, Geiman, JM et al., N.Engl.J.Med. 293: 1343, 1975.

2. Hall, CB, McBride, JT, Walsh, EE et al., N.Engl.J.Med. 308: 1443, 1983.

3. Hall, CB, McBride, JT, Gala, CL et al., JAMA254: 3047,1985.

Four. Wald, ER, et al., J. Pediat. 112: 154, 1988.

5. Kapikian.AZ, Mithcell, RH, Chanock, RM et al., Am.J. Epidemiol. 89: 405, 1969.

6. Prince, GA, Hemming, VG, Horswood, RL et al., Virus Res. 3: 193, 1985.

7. Hemming, VG, Prince, GA, Horswood, RL et al., J.Infecí.Dis. 152: 1083, 1985.

8. Wright, PF, Belshe, RB, et al., Infect Immun. 37: 397, 1982.

9. Conrad, DA, Christenson, JC, et al., Peditr.Infect.Dis.J. 6: 152, 1987.

10. LoBuglio, AF, Wheeler, RL, Trang, J. et al., Proc.Natl.Acad. Sci. 86: 4220, 1989.

eleven. Steplewski, Z., Sun, LK, Shearman, CW et al., Proc.Natl.Acad. Sci. 85: 4852, 1988.

12. Boulianne, GL, Hozumi, N., Shulman, MJ Nature. 312: 643, 1984.

13. Sun, LK, Curtís, P., Rakowicz-Szulczynska, E. et al., Proc.Natl.Acad. Sci. 84: 214, 1987.

14. Liu, AY, Mack, PW, Champion, CI, Robinson, RR, Gene 54: 33, 1987.

fifteen. Morrison, SL, Johnson, MJ, Hersenber, LA, Oi, VT Proc.Natl.Acad. Sci. 81: 6851, 1984.

16. Morrison, SL Science 229: 1202, 1985.

17. Sahagan, BG, Dorai, H., Saltzgaber-Muller, J. et al., J. Immunol. 137: 1066, 1986.

18. Taked, S., Naito, T., Hama, K., Noma, T., Honjo, T., Nature 314: 452, 1985.

19. Carson, DA, Freimark, BD, Adv. Immunol. 38: 275, 1986.

twenty. Beeler, JA, et al., J.Virol. 63: 2941-2950, ​​1989.

twenty-one. Coelingh, et al., Virology, 143: 569-582, 1985.

22. Anderson et al. Microneutralization test for respiratory syncytial virus based or an enzyme immunoassay, J. Clin. Microbiol. (1985) 22: 1050-1052.

2. 3. Johnson et al., Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus, J. Infectious Diseases (1997) 176: 1215-1224.

ES 2 349 348 T3

SEQUENCE LISTING &lt;110&gt; Young, James F.

Koenig, Scott Johnson, Leslie S.

Huse, William D.

Wu, Herren Watkins, Jeffry D.

&lt;120&gt; Ultra High Affinity Neutralizing Antibodies &lt;130&gt; 469201-520 &lt;140&gt;

&lt;141&gt;

&lt;150&gt; 60 / 178,426 &lt;151&gt; 27-01-2000 &lt;160&gt; 26 &lt;170&gt; PatentIn Ver. 2.1 &lt;210&gt; 1 &lt;211&gt; 106 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: Mouse human chimeric antibody light chain variable chain &lt;400&gt; 1

<td>Asp 1</td><td>lie</td><td>Gln</td><td>Met</td><td>Thr 5</td><td>Gln</td><td>To be</td><td>Pro</td><td>To be</td><td>Thr 10</td><td>Leu</td><td>To be</td><td>To</td><td>To be</td><td>Val fifteen</td><td>Gly</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr</td><td>Xle</td><td>Thr</td><td>Cys</td><td>To be</td><td>To</td><td>To be</td><td>To be</td><td>To be</td><td>Val</td><td>Gly</td><td>Tyr</td><td>Met</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>His</td><td>Trp</td><td>Tyr</td><td>Gln</td><td>Gln</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Lys</td><td>To</td><td>Pro</td><td>Lys</td><td>Leu</td><td>Leu</td><td>lie</td><td>Tyr</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Asp</td><td>Thr</td><td>To be</td><td>Lys</td><td>Leu</td><td>To</td><td>To be</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>To be</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be</td><td>To be</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Phe</td><td>To</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Phe</td><td>Gln</td><td>Gly</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Pro</td><td>Phe</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Phe</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>lie</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

100 105 &lt;210&gt; 2

ES 2 349 348 T3 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Mouse human chimeric antibody heavy chain variable chain &lt;400&gt; 2

<td>Gln 1</td><td>Val</td><td>Thr</td><td>Leu</td><td>Arg 5</td><td>Glu</td><td>To be</td><td>Gly</td><td>Pro</td><td>To 10</td><td>Leu</td><td>Val</td><td>Lys</td><td>Pro</td><td>Thr fifteen</td><td>Gln</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Phe</td><td>To be 25</td><td>Gly</td><td>Phe</td><td>To be</td><td>Leu</td><td>To be 30</td><td>Thr</td><td>To be</td>

<td>Gly</td><td>Met</td><td>To be 35</td><td>Val</td><td>Gly</td><td>Trp</td><td>lie</td><td>Arg 40</td><td>Gln</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Lys Four. Five</td><td>To</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>Leu fifty</td><td>To</td><td>Asp</td><td>lie</td><td>Trp</td><td>Trp 55</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Asp 60</td><td>Tyr</td><td>Asn</td><td>Pro</td><td>To be</td>

<td>Leu 65</td><td>Lys</td><td>To be</td><td>Arg</td><td>Leu</td><td>Thr 70</td><td>lie</td><td>To be</td><td>Lys</td><td>Asp</td><td>Thr 75</td><td>To be</td><td>Lys</td><td>Asn</td><td>Gln</td><td>Val 80</td>

<td>Val</td><td>Leu</td><td>Lys</td><td>Val</td><td>Thr 85</td><td>Asn</td><td>Met</td><td>Asp</td><td>Pro</td><td>To 90</td><td>Asp</td><td>Thr</td><td>To</td><td>Thr</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>To</td><td>Arg</td><td>To be 100</td><td>Met</td><td>lie</td><td>Thr</td><td>Asn</td><td>Trp 105</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Val</td><td>Trp 110</td><td>Gly</td><td>Gln</td>

Gly Thr Thr Val Thr Val Ser Ser

115 120 &lt;210&gt; 3 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the complementary determining region L1 of the reference anti-RSV antibody &lt;400&gt; 3

Ser Ala Ser Ser Ser Val Gly Tyr Met His

10 &lt;210&gt; 4 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the L2 complementarity determining region of the reference anti-RSV antibody

ES 2 349 348 T3 &lt;400&gt; 4

Asp Thr Ser Lys Leu Ala Ser

5 &lt;210&gt; 5 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the amino acid sequence of the reference anti-RSV antibody determining region

Artificial: L3 Complementarity Sequence of &lt;400&gt; 5

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

5 &lt;210&gt; 6 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the amino acid sequence of the reference anti-RSV antibody determining region

Artificial: Complementarity Sequence H1 of &lt;400&gt; 6

Thr Ser Gly Met Ser Val Gly

5 &lt;210&gt; 7 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the amino acid sequence of the reference anti-RSV antibody determining region

Artificial: Complementarity sequence H2 of del &lt;400&gt; 7

Asp lie Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser

10 15 &lt;210&gt; 8 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

ES 2 349 348 T3 &lt;223&gt; Description of the amino acid sequence of the reference anti-RSV antibody determining region

Artificial: Complementarity sequence H3 of del

Ser Met lie Thr Asn Trp Tyr

5 &lt;400&gt; 8

Phe Asp Val &lt;210&gt; 9 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the amino acid present

Artificial Sequence: Sequence in determining regions complementarity of high invention affinity of the &lt;400&gt; 9 antibodies

Thr Ala Gly Met Ser Val Gly 1 5 &lt;210&gt; 10 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of present amino acid complementarity of the invention Artificial Sequence: Sequence in the determining regions high affinity of the &lt;400&gt; 10 antibodies

Thr Pro Gly Met Ser Val Gly

5 &lt;210&gt; 11 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of present amino acid complementarity of the invention Artificial Sequence: Sequence in the determining regions high affinity of the &lt;400&gt; 11 antibodies

<img file="ES2349348T3_D0001.tif" />

ES 2 349 348 T3 &lt;210&gt; 12 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence present in the high affinity complementarity determining regions of the antibodies of the invention &lt;400&gt; 12

Asp Thr Phe Lys Leu Ala Ser

5 &lt;210&gt; 13 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence present in the high affinity complementarity determining regions of the antibodies of the invention &lt;400&gt; 13

Asp Thr Tyr Lys Leu Ala Ser

5 &lt;210&gt; 14 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence present in the high affinity complementarity determining regions of the antibodies of the invention &lt;400&gt; 14

Phe Gln Gly Ser Phe Tyr Pro Phe Thr

5 &lt;210&gt; 15 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence present in the determining regions of

ES 2 349 348 T3 high affinity complementarity of the antibodies of the invention &lt;400&gt; 15

Phe Gln Gly Ser Tyr Tyr Pro Phe Thr

5 &lt;210&gt; 16 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence present in the high affinity complementarity determining regions of the antibodies of the invention &lt;400&gt; 16

Phe Gln Gly Ser Trp Tyr Pro Phe Thr

5 &lt;210&gt; 17 &lt;211&gt; 106 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the light chain of clone 1 of Figure 3A

<td colspan="11"> &lt;400&gt;17</td><td rowspan="2">To be</td><td rowspan="2">To</td><td rowspan="2">To be</td><td rowspan="2">Val fifteen</td><td rowspan="2">Gly</td>

<td>Asp 1</td><td>lie</td><td>Gln</td><td>Met</td><td>Thr 5</td><td>Gln</td><td>To be</td><td colspan="2">Pro Ser</td><td>Thr 10</td><td>Leu</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr twenty</td><td>lie</td><td>Thr</td><td>Cys</td><td>To be</td><td>To 25</td><td>To be</td><td>To be</td><td>To be</td><td>Val</td><td>Gly 30</td><td>Tyr</td><td>Met</td>

<td>His</td><td>Trp</td><td>Tyr 35</td><td>Gln</td><td>Gln</td><td>Lys</td><td>Pro</td><td>Gly 40</td><td>Lys</td><td>To</td><td>Pro</td><td>Lys</td><td>Leu Four. Five</td><td>Leu</td><td>lie</td><td>Tyr</td>

<td>Asp</td><td>Thr fifty</td><td>To be</td><td>Lys</td><td>Leu</td><td>To</td><td>To be 55</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg 60</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td>

<td>Gly 65</td><td>To be</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Phe 70</td><td>Thr</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be 75</td><td>To be</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp 80</td>

<td>Asp</td><td>Phe</td><td>To</td><td>Thr</td><td>Tyr 85</td><td>Tyr</td><td>Cys</td><td>Phe</td><td>Gln</td><td>Gly 90</td><td>To be</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Phe 95</td><td>Thr</td>

<td>Phe</td><td>Gly</td><td>Gly</td><td>Gly 100</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>lie 105</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 18 &lt;211&gt; 120

ES 2 349 348 T3 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the heavy chain of clone 1 of Figure 3B &lt;400&gt; 18

<td colspan="2" rowspan="2">Gln Val 1</td><td colspan="10">Thr Leu Arg Glu Ser Gly Pro Ala Leu Val</td><td rowspan="2">Lys</td><td rowspan="2">Pro</td><td rowspan="2">Thr fifteen</td><td rowspan="2">Gln</td>

<td colspan="3"> 5</td><td colspan="7"> 10</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Phe</td><td>To be</td><td>Gly</td><td>Phe</td><td>To be</td><td>Leu</td><td>To be</td><td>Thr</td><td>To</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Met</td><td>To be</td><td>Val</td><td>Gly</td><td>Trp</td><td>lie</td><td>Arg</td><td>Gln</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Lys</td><td>To</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>Leu</td><td>To</td><td>Asp</td><td>lie</td><td>Trp</td><td>Trp</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Asp</td><td>Tyr</td><td>Asn</td><td>Pro</td><td>To be</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Lys</td><td>To be</td><td>Arg</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be</td><td>Lys</td><td>Asp</td><td>Thr</td><td>To be</td><td>Lys</td><td>Asn</td><td>Gln</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Leu</td><td>Lys</td><td>Val</td><td>Thr</td><td>Asn</td><td>Met</td><td>Asp</td><td>Pro</td><td>To</td><td>Asp</td><td>Thr</td><td>To</td><td>Thr</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>To</td><td>Arg</td><td>To be</td><td>Met</td><td>lie</td><td>Thr</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Val</td><td>Trp</td><td>Gly</td><td>Gln</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Thr</td><td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 19 &lt;211&gt; 106 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the light chain of clone 2 of Figure 4A &lt;400&gt; 19

ES 2 349 348 T3

<td>Asp 1</td><td>lie</td><td>Gln</td><td>Met</td><td>Thr 5</td><td>Gln</td><td>To be</td><td>Pro</td><td>To be</td><td>Thr 10</td><td>Leu</td><td>To be</td><td>To</td><td>To be</td><td>Val fifteen</td><td>Gly</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr twenty</td><td>lie</td><td>Thr</td><td>Cys</td><td>To be</td><td>To 25</td><td>To be</td><td>To be</td><td>To be</td><td>Val</td><td>Gly 30</td><td>Tyr</td><td>Met</td>

<td>His</td><td>Trp</td><td>Tyr 35</td><td>Gln</td><td>Gln</td><td>Lys</td><td>Pro</td><td>Gly 40</td><td>Lys</td><td>To</td><td>Pro</td><td>Lys</td><td>Leu Four. Five</td><td>Leu</td><td>lie</td><td>Tyr</td>

<td>Asp</td><td>Thr fifty</td><td>Phe</td><td>Lys</td><td>Leu</td><td>To</td><td>To be 55</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg 60</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td>

<td>Gly 65</td><td>To be</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Phe 70</td><td>Thr</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be 75</td><td>To be</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp 80</td>

<td>Asp</td><td>Phe</td><td>To</td><td>Thr</td><td>Tyr 85</td><td>Tyr</td><td>Cys</td><td>Phe</td><td>Gln</td><td>Gly 90</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Pro</td><td>Phe 95</td><td>Thr</td>

<td>Phe</td><td>Gly</td><td>Gly</td><td>Gly 100</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>lie 105</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 20 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the heavy chain of clone 2 of Figure 4B &lt;400&gt; 20

<td>Gln 1</td><td>Val</td><td>Thr</td><td>Leu</td><td>Arg 5</td><td>Glu</td><td>To be</td><td>Gly</td><td>Pro</td><td>To 10</td><td>Leu</td><td>Val</td><td>Lys</td><td>Pro</td><td>Thr fifteen</td><td>Gln</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>Leu twenty</td><td>Thr</td><td>Cyn</td><td>Thr</td><td>Phe</td><td>To be 25</td><td>Gly</td><td>Phe</td><td>To be</td><td>Leu</td><td>To be 30</td><td>Thr</td><td>To</td>

<td>Gly</td><td>Met</td><td>To be 35</td><td>Val</td><td>Gly</td><td>Trp</td><td>lie</td><td>Arg 40</td><td>Gln</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Lys Four. Five</td><td>To</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>Leu fifty</td><td>To</td><td>Asp</td><td>lie</td><td>Trp</td><td>Trp 55</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Asp 60</td><td>Tyr</td><td>Asn</td><td>Pro</td><td>To be</td>

<td>Leu 65</td><td>Lys</td><td>To be</td><td>Arg</td><td>Leu</td><td>Thr 70</td><td>lie</td><td>To be</td><td>Lys</td><td>Asp</td><td>Thr 75</td><td>To be</td><td>Lys</td><td>Asn</td><td>Gln</td><td>Val 80</td>

<td>Val</td><td>Leu</td><td>Lys</td><td>Val</td><td>Thr 85</td><td>Asn</td><td>Met</td><td>Asp</td><td>Pro</td><td>To 90</td><td>Asp</td><td>Thr</td><td>To</td><td>Thr</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>To</td><td>Arg</td><td>To be 100</td><td>Met</td><td>lie</td><td>Thr</td><td>Asn</td><td>Phe 105</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Val</td><td>Trp 110</td><td>Gly</td><td>Gln</td>

Gly Thr Thr Val Thr Val Ser Ser 115 120 &lt;210&gt; 21 &lt;211&gt; 106 &lt;212&gt; PRT

ES 2 349 348 T3 &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the light chain of clone 3 of Figure 5A &lt;400&gt; 21

<td>Asp 1</td><td>lie</td><td colspan="2">Gln Met</td><td colspan="2">Thr Gln 5</td><td>To be</td><td>Pro</td><td>To be</td><td>Thr 10</td><td>Leu</td><td>To be</td><td>To</td><td>To be</td><td>Val fifteen</td><td>Gly</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr</td><td>lie</td><td>Thr</td><td>Cys</td><td>To be</td><td>To</td><td>To be</td><td>To be</td><td>To be</td><td>Val</td><td>Gly</td><td>Tyr</td><td>Met</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>His</td><td>Trp</td><td>Tyr</td><td>Gln</td><td>Gln</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Lys</td><td>To</td><td>Pro</td><td>Lys</td><td>Leu</td><td>Leu</td><td>lie</td><td>Tyr</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Asp</td><td>Thr</td><td>Phe</td><td>Lys</td><td>Leu</td><td>To</td><td>To be</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>To be</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be</td><td>To be</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Phe</td><td>To</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Phe</td><td>Gln</td><td>Gly</td><td>To be</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Phe</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Phe</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>lie</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 22 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the heavy chain of clone 3 of Figure 5B &lt;400&gt; 22

ES 2 349 348 T3

<td>Gln 1</td><td>Val</td><td>Thr</td><td>Leu</td><td>Arg 5</td><td>Glu</td><td>To be</td><td>Gly</td><td colspan="2">Pro Wing 10</td><td>Leu</td><td>Val</td><td>Lys</td><td>Pro</td><td>Thr fifteen</td><td>Gln</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Phe</td><td>To be</td><td>Gly</td><td>Phe</td><td>To be</td><td>Leu</td><td>To be</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Met</td><td>To be</td><td>Val</td><td>Gly</td><td>Trp</td><td>lie</td><td>Arg</td><td>Gln</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Lys</td><td>To</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>Leu</td><td>To</td><td>Asp</td><td>lie</td><td>Trp</td><td>Trp</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Asp</td><td>Tyr</td><td>Asn</td><td>Pro</td><td>To be</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Lys</td><td>To be</td><td>Arg</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be</td><td>Lys</td><td>Asp</td><td>Thr</td><td>To be</td><td>Lys</td><td>Asn</td><td>Gln</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Leu</td><td>Lys</td><td>Val</td><td>Thr</td><td>Asn</td><td>Met</td><td>Asp</td><td>Pro</td><td>To</td><td>Asp</td><td>Thr</td><td>To</td><td>Thr</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>To</td><td>Arg</td><td>To be</td><td>Met</td><td>lie</td><td>Thr</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Val</td><td>Trp</td><td>Gly</td><td>Gln</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Gly Thr Thr Val Thr Val Ser Ser

115 120 &lt;210&gt; 23 &lt;211&gt; 106 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the light chain of clone 22 of Figure 6A &lt;400&gt; 23

<td>Asp 1</td><td>lie</td><td>Gln</td><td>Met</td><td>Thr 5</td><td>Gln</td><td>To be</td><td>Pro</td><td>To be</td><td>Thr 10</td><td>Leu</td><td>To be</td><td>To</td><td>To be</td><td>Val fifteen</td><td>Gly</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr twenty</td><td>lie</td><td>Thr</td><td>Cys</td><td>To be</td><td>To 25</td><td>To be</td><td>To be</td><td>To be</td><td>Val</td><td>Gly 30</td><td>Tyr</td><td>Met</td>

<td>His</td><td>Trp</td><td>Tyr 35</td><td>Gln</td><td>Gln</td><td>Lys</td><td>Pro</td><td>Gly 40</td><td>Lys</td><td>To</td><td>Pro</td><td>Lys</td><td>Leu Four. Five</td><td>Leu</td><td>lie</td><td>Tyr</td>

<td>Asp</td><td>Thr fifty</td><td>Phe</td><td>Lys</td><td>Leu</td><td>To</td><td>To be 55</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg 60</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td>

<td>Gly</td><td>To be</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be</td><td>To be</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Phe</td><td>To</td><td>Thr</td><td>Tyr 85</td><td>Tyr</td><td>Cys</td><td>Phe</td><td>Gln</td><td>Gly 90</td><td>To be</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Phe 95</td><td>Thr</td>

<td>Phe</td><td>Gly</td><td>Gly</td><td>Gly 100</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>lie 105</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 24 &lt;211&gt; 120 &lt;212&gt; PRT

ES 2 349 348 T3 &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the heavy chain of clone 22 of Figure 6B &lt;400&gt; 24

<td>Gln 1</td><td>Val</td><td>Thr</td><td colspan="2">Leu Arg 5</td><td>Glu</td><td>To be</td><td>Gly</td><td colspan="2">Pro Wing 10</td><td colspan="2">Leu Val</td><td>Lys</td><td colspan="2">Pro Thr fifteen</td><td>Gln</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Phe</td><td>To be</td><td>Gly</td><td>Phe</td><td>To be</td><td>Leu</td><td>To be</td><td>Thr</td><td>To</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Met</td><td>To be</td><td>Val</td><td>Gly</td><td>Trp</td><td>lie</td><td>Arg</td><td>Gln</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Lys</td><td>To</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>Leu</td><td>To</td><td>Asp</td><td>lie</td><td>Trp</td><td>Trp</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Asp</td><td>Tyr</td><td>Asn</td><td>Pro</td><td>To be</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Lys</td><td>To be</td><td>Arg</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be</td><td>Lys</td><td>Asp</td><td>Thr</td><td>To be</td><td>Lys</td><td>Asn</td><td>Gln</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Leu</td><td>Lys</td><td>Val</td><td>Thr</td><td>Asn</td><td>Met</td><td>Asp</td><td>Pro</td><td>To</td><td>Asp</td><td>Thr</td><td>To</td><td>Thr</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>To</td><td>Arg</td><td>To be</td><td>Met</td><td>lie</td><td>Thr</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Val</td><td>Trp</td><td>Gly</td><td>Gln</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Gly Thr Thr Val Thr Val Ser Ser 115 120 &lt;210&gt; 25 &lt;211&gt; 106 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the light chain of clone 23 of Figure 7A &lt;400&gt; 25

ES 2 349 348 T3

<td>Asp 1</td><td>lie</td><td>Gln</td><td>Met</td><td>Thr 5</td><td>Gln</td><td>To be</td><td>Pro</td><td>To be</td><td>Thr 10</td><td>Leu</td><td>To be</td><td>To</td><td>To be</td><td>Val fifteen</td><td>Gly</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr twenty</td><td>lie</td><td>Thr</td><td>Cys</td><td>To be</td><td>To 25</td><td>To be</td><td>To be</td><td>To be</td><td>Val</td><td>Gly 30</td><td>Tyr</td><td>Met</td>

<td>His</td><td>Trp</td><td>Tyr 35</td><td>Gln</td><td>Gln</td><td>Lys</td><td>Pro</td><td>Gly 40</td><td>Lys</td><td>To</td><td>Pro</td><td>Lys</td><td>Leu Four. Five</td><td>Leu</td><td>lie</td><td>Tyr</td>

<td>Asp</td><td>Thr fifty</td><td>Phe</td><td>Lys</td><td>Leu</td><td>To</td><td>To be 55</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg 60</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td>

<td>Gly 65</td><td>To be</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Phe 70</td><td>Thr</td><td>Leu</td><td>Thr</td><td>lie</td><td>To be 75</td><td>To be</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp 80</td>

<td>Asp</td><td>Phe</td><td>To</td><td>Thr</td><td>Tyr 85</td><td>Tyr</td><td>Cys</td><td>Phe</td><td>Gln</td><td>Gly 90</td><td>To be</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Phe 95</td><td>Thr</td>

<td>Phe</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td><td>lie</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

100 105 &lt;210&gt; 26 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of the Artificial Sequence: Amino acid sequence of the variable region of the heavy chain of clone 23 of Figure 7B &lt;400&gt; 26

<td>Gln 1</td><td>Val</td><td>Thr</td><td>Leu</td><td>Arg or 5</td><td>Glu</td><td>To be</td><td>Gly</td>

<td>Thr</td><td>Leu</td><td>Thr</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Phe</td>

<td>Gly</td><td>Met</td><td>To be 35</td><td>Val</td><td>Gly</td><td>Trp</td><td>lie</td><td>Arg 40</td>

<td>Trp</td><td>Leu fifty</td><td>To</td><td>Asp</td><td>lie</td><td>Trp</td><td>Trp 55</td><td>Asp</td>

<td>Leu 65</td><td>Lys</td><td>To be</td><td>Arg</td><td>Leu</td><td>Thr 70</td><td>lie</td><td>To be</td>

<td>Val</td><td>Leu</td><td>Lys</td><td>Val</td><td>Thr 85</td><td>Asn</td><td>Met</td><td>Asp</td>

<td>Cys</td><td>To</td><td>Arg</td><td>To be 100</td><td>Met</td><td>lie</td><td>Thr</td><td>Asn</td>

<td>Gly</td><td>Thr</td><td>Thr 115</td><td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be 120</td>

Pro To 10 Leu Val Lys Pro Thr fifteen Gln To be 25 Gly Phe To be Leu To be 30 Thr To Gln Pro Pro Gly Lys Four. Five To Leu Glu Asp Lys Lys Asp 60 Tyr Asn Pro To be Lys Asp Thr 75 To be Lys Asn Gln Val 80 Pro To 90 Asp Thr To Thr Tyr 95 Tyr Phe 105 Tyr Phe Asp Val Trp 110 Gly Gln