Antibodies binding to the F protein of a respiratory syncytial virus (RSV)

p00001High potency recombinant antibodies and method for their production.

FIELD OF THE INVENTION

p00002The present invention relates to high potency antibodies, methods of increasing the potency of antibodies and use of such antibodies for disease prevention and treatment.

BACKGROUND OF THE INVENTION

p00003Antibodies for the prevention and treatment of various diseases, especially those caused by infectious microorganisms, such as viruses, have been developed, and currently are.

p00004One approach has been the development of antibodies, especially neutralizing monoclonal antibodies, some of them with high specific neutralization activity. A drawback of this approach has been the need to produce human antibodies instead of mouse or rat and thereby minimize the development of human anti-mouse or anti-rat antibody responses, potentially resulting in additional immune pathology. .

p00005An alternative approach has been the production of human-murine chimeric antibodies in which the genes encoding the variable regions of the mouse heavy and light chains have been coupled to the genes for human heavy and light chain constant regions in order to produce chimeric antibodies, or hybrids. For example, a humanized anti-RSV antibody has been prepared and is currently being marketed. [See: Johnson, US Patent No. 5,824,307].

p00006In some cases, mouse complementarity determining regions (CDRs) have been grafted into human constant and framework regions in which some of the mouse framework amino acids (amino acids in the antibody's variable region but outside the antibody regions) have been inserted. CDRs) replacing the correspondingly positioned amino acids of a human antibody of the same specificity in order to provide an antibody called "humanized". [See, for example, Queen, US Patent No. 5,693,761 and 5,693,762]. However, such antibodies contain intact mouse CDR regions and have reached uneven efficacy, often exhibiting affinities no greater than 107 to 108 M-1.

p00007WO 9,833,919 describes the humanized antibody Vitaxin® and related grafted antibodies based on mouse monoclonal LM609.

p00008The production of high potency antibodies (i.e. antibodies with high biological activity, such as antigen neutralizing activity), including antibodies with ultra-high affinity for the target antigen, would be desirable from the point of view of both the neutralizing ability of a antibody of this type as from the most practical aspects of requiring less amount of antibody in order to achieve a desirable degree of clinical efficacy, thereby reducing utilization costs.

p00009The affinity of the antibodies is measured by the antibody binding constant to a particular antigen, and said binding constant is often calculated by the rate of the rate constant for the formation of the antibody-antigen complex (at which it is made reference as the "kon" value) to the velocity constant for the dissociation of said complex (the "koff" value). In accordance with the present invention, it has been determined that the potency of an antibody is a function of the kon value, regardless of specificity. The present invention thus provides a solution to the problems of achieving high antibody potency in the sense that the higher the kon value, the higher the antibody potency, thereby providing high potency antibodies and a method for producing the same.

BRIEF SUMMARY OF THE INVENTION

p00010In accordance with one aspect of the present invention, high potency antibodies according to claim 1 are useful in the treatment and / or prevention in a disease. In another aspect, the potency of an antibody increases with increasing the rate constant for the formation of the antigen-antibody complex (the "kon" value).

p00011In one aspect, the present invention relates to high potency antibodies, other than Vitaxin, which include immunologically active portions, fragments, or segments thereof, which have a kon of at least 2.5 x 105 M-1 s- 1, with prevalence at least about 5 x 105 M-1 s-1, and most preferably at least about 7.5 x 105 M-1 s-1. Such antibodies may also have high affinity (at least about 109 M-1).

p00012In another aspect, the present invention relates to high potency neutralizing antibodies, which include portions, fragments, or immunologically active segments thereof having a kon of at least 2.5 x 105 M-1 s-1, preferably at least about 5 x 105 M-1 s-1, and most preferably at least about 7.5 x 105 M-1 s-1. Such antibodies may also have high affinity (at least about 109 M-1).

p00013Methods for increasing the potency of neutralizing antibodies by increasing the kon value with respect to a given antigen without changing the epitope to which the antibody is fixed are also described.

p00014An antibody screening medium is also described for properties that ensure high potency with respect to a desired antigen, said potency being at least 2 to 10 times greater than that of known antibodies.

p00015More specifically, it is an object of the present invention to produce antibodies having kon values ​​at least as high as 2.5 x 105 M-1 s-1, preferably at least 5 x 105 M-1 s-1, and most preferably at less as high as 7.5 x 105 M-1 s-1.

p00016It is also an object of the present invention to provide high affinity and high potency antibodies that have high specificity for the F protein of a respiratory syncytial virus (RSV) causing respiratory system infection.

p00017In one embodiment, the present invention provides antibodies that have substantially the variable chain framework (FR) regions of the antibody described in Figure 1 (with the same specificity as this antibody) but in which the polypeptide structures contain one or more differences of amino acid in a

p00018or more of the CDRs (or complementarity determining regions) thereof. In a preferred embodiment, the antibodies of the present invention will differ from the antibody of Figures 1 or 2 (hereinafter, the "basic structure" or "reference structure") only in the sequences of one or more of the CDRs, which they include L1, L2, L3, H1, H2, and H3. A preferred sequence is shown in Figure 3.

p00019It is another object of the present invention to provide compositions comprising the antibodies described herein wherein said antibodies are suspended in a pharmacologically acceptable carrier, diluent or excipient.

p00020It is a further object of the present invention to provide compositions and medicaments for use in the prevention and / or treatment of diseases, such as those caused by viruses, especially respiratory syncytial virus, wherein the compositions and medicaments comprise a therapeutically effective amount of a composition containing an antibody as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

p00021Figure 1 shows the amino acid sequence of the light and heavy chain variable regions of a high affinity monoclonal antibody whose potency can be increased by the methods of the present invention. For reference purposes, this antibody is the MEDI-493 antibody sequence described in Johnson et al., J. Infect. Dis., 176: 1215-1224 (1997). In this case, the CDR regions are underlined, while the non-underlined residues form the framework regions of the variable regions of each polypeptide structure. In this structure, the CDRs are derived from a mouse antibody, while the framework regions are derived from a human antibody. Constant regions (not shown) are also derived from a human antibody. Figure 1A shows the variable region of the light chain (SEQ ID NO: 1) and Figure 1B shows the variable region of the heavy chain (SEQ ID NO: 2) of the light and heavy chains, respectively.

p00022Figure 2 shows the variable regions of the heavy and light chain for a different basic or reference polypeptide sequence. Again, the CDR regions are underlined. This sequence differs from Figure 1 in the first four residues of the CDR L1 of the light chain, the residue 103 of the light chain and the residue 112 of the heavy chain. All of the high power neutralizing Fab structures of the present invention (CDR structures depicted in Table 2) use the framing sequences of this reference structure.

p00023or basic Fig. 2A shows the variable regions of the light chain (SEQ ID NO: 3) and Fig. 2B shows the variable regions of the heavy chain (SEQ ID NO: 4).

p00024Figure 3 shows the variable regions of the heavy chain (SEQ ID NO: 36) and the light chain (SEQ ID NO: 35) of a preferred embodiment of the present invention. This preferred antibody has several high-kon CDRs (or high-potency CDRs) present, which result in association rate constants (i.e., kon) greater than the basic or reference antibody of Figure 2, and therefore higher power. This preferred antibody has the same framework amino acid sequences as the sequence of Figure 2 and, for the purposes of the present disclosure, is designated as "clone 15" in Tables 2 and 3, below. These sequences are easily generated by methods described herein, all of which are readily known to those skilled in the art. The kinetic constants were measured according to the procedure of Example 1 and the potency was determined as described in Example 2.

p00025Figure 4 shows a schematic diagram of the use of phage M13 for generating Fab fragments in accordance with the present invention and the use of a histidine marker sequence (6 histidine residues) in order to facilitate purification.

p00026Figure 5 shows a schematic diagram for the screening procedure used for the antibodies of the present invention. "SPE" refers to a single point ELISA. "H3-3F4" is a designation for clone 4 of Tables 2 and 3.

DETAILED SUMMARY OF THE INVENTION

p00027In accordance with one aspect of the present invention, high potency antibodies according to claim 1 useful in the treatment and / or prevention of diseases are provided. In another aspect, the potency of the antibody is increased by increasing the rate constant for the formation of the antigen-antibody complex, referred to as the "kon" value, by replacing CDR sequences of said antibody with high CDR sequences. power in place.

p00028In one aspect, the present invention relates to high potency antibodies, other than vitaxin, which include immunologically active portions, fragments, or segments of said high potency antibodies, which have a kon of at least 2.5 x 105 M- 1 s-1, preferably at least about 5 x 105 M-1 s-1, and most preferably at least about 7.5 x 105 M-1 s-1. Such antibodies may also have a high affinity (at least about 109 M-1).

p00029In one aspect, the present invention relates to high potency neutralizing antibodies, including immunologically active portions, fragments or segments thereof, which have a kon of at least 2.5 x 105 M-1 s-1, preferably at least about 5 x 105 M-1 s-1, and most preferably at least about 7.5 x 105 M-1 s-1. Such antibodies may also have a high affinity (at least about 109 M-1).

p00030Methods of producing antibodies, neutralizing or non-neutralizing, having high potency, or biological activity, preferably having an affinity of at least about 109 M1, and having a kon of at least about 2.5 x 105 M-1 are also described. s-1, preferably at least about 5 x 105

M-1-1

p00032s-1, and most preferably at least about 7.5 x 105 M-1 s.

p00033With the emergence of molecular biology methods and recombinant DNA technology, it is now possible to produce antibodies, including active fragments thereof, by recombinant means and thereby generate gene sequences encoding the specific amino acid sequences found in the polypeptide structure of antibodies. This has allowed easy production of antibodies that have characteristic sequences of neutralizing antibodies from different chemical species and sources.

p00034Regardless of their mode of construction, the antibodies have a similar overall three-dimensional structure usually indicated as L2H2 where the molecule commonly comprises 2 light chains (L) of amino acids and 2 heavy chains (H) of amino acids. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions that interact with the target are referred to as "variable" or "V" regions, and are characterized by differences in the amino acid sequence of the different antigen specificity antibodies.

p00035The variable regions of the H or L chains contain the amino acid sequence capable of specifically binding antigen targets. Within these sequences there are smaller sequences designated "hypervariable" due to their extreme variability between antibodies of different specificity. Such hypervariable regions are called "complementarity determining regions" or "CDR" regions. These CDR regions account for the basic specificity of the antibody for a particular antigen determining structure.

p00036The CDRs represent non-contiguous stretches of amino acids within the variable regions, but it has been found that the positional locations of these critical amino acid sequences within the heavy and light chain variable regions have similar locations within the amino acid sequences of the structure. of immunoglobulins. The variable heavy and light chains of all antibodies each have 3 CDR regions, none of them being contiguous to the others (designated L1, L2, L3, H1, H2, H3) for the respective light and heavy chains. The accepted CDR regions have been described by Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977). The numbering scheme is shown in Figures 1-3, where the CDRs are underlined and the numbers follow the Kabat scheme.

p00037In all mammalian species, the antibody polypeptides contain constant (ie, highly conserved) and variable regions comprising both CDRs and so-called "framework regions", the latter consisting of amino acid sequences within the variable region but outside the CDRs.

p00038Among the properties commonly used to characterize an antibody, or fragment thereof, are the specificity and affinity of the antibody. Specificity refers to the particular ligand, or antigenic structure, to which the antibody is strongly, or very strongly, bound. Affinity refers to a quantitative measure of the binding strength of the antibody to a particular ligand and is given in terms of an "affinity constant." Such affinity constants can be determined as association or dissociation constants and represent the ratio of equilibrium concentrations of the free ligand and the free antibody to the antibody-ligand complex. As used herein, affinity will be given as an association constant.

p00039Such constants are commonly measured by the kinetics of the formation of the antigen-antibody complex, the association rate constant being designated to form the complex as kon, and the dissociation rate constant being designated as koff. The measurement of such constants is fully within the skill of those skilled in the art. The antibody and the respective antigen combine to form a complex as follows:

p00040Here, the affinity constant is given as an association constant and therefore represents:

p00041where Ka = the association constant (or affinity), while the square brackets indicate molar concentration of the species enclosed between them. For a given series of conditions such as temperature, pressure and ionic strength, the ratio of the concentration of the complex to the product of the concentrations of the reactant species is constant. As long as saturation conditions for the antibody or antigen (ligand) are not reached, a

p00042fifteen Change in the concentration of any of the fixation species will alter the concentration of the complex (Ab-Ag) in an amount dictated by the above equation (since Ka is constant). This interaction operates in accordance with the law of action of the masses.

p00043Additionally, this interrelation depends on the concentrations and not on the absolute amount of the chemical species present, so the overall volume is also relevant for any affinity measurement. So, if the

p00044twenty The reaction takes place in half the volume, a twice as large amount of complex will be formed because each reacting species (Ab and Ag) is now present at a double concentration, and therefore a four times larger amount of complex will be formed. Conversely, dilution can significantly reduce the concentration of the Ab-Ag complex. In general, the kinetics of the antigen-antibody interaction are well known to those skilled in the art.

p0004525 Said antibody-antigen reaction can be described kinetically as a dynamic equilibrium in which the affinity constant can be measured as a ratio of the individual velocity constants for complex formation and dissociation:

p00046Thus, the kon value is the rate constant, or specific reaction rate, of the direct reaction, or of

p0004730 complex formation, measured in units: M-1 s-1. The koff value is the rate constant, or specific reaction rate, for the dissociation of the Ab-Ag complex and is measured in units of s-1.

p00048The kon values ​​for the antibodies, and active fragments thereof, of the present invention were measured using the BIAcore protocol and equipment as described in the Examples.

p00049In accordance with the foregoing, the present invention relates to high potency neutralizing antibodies, with

p0005035 inclusion of portions, fragments and / or immunologically active segments thereof, which have a kon of at least 2.5 x 105 M-1 s-1, preferably having at least about 5 x 105 M-1 s-1, and most preferably at least about 7.5 x 105 M-1 s-1.

p00051As used herein, the terms "portion," "segment," and "fragment," when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, a sequence that forms a subset of a larger sequence. For example, if a polypeptide were subjected to

p00052treatment with any of the common endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, etc., the oligopeptides resulting from said treatment would represent portions, segments or fragments of the starting polypeptide. Such proteinases are commonly used to generate antibody fragments, such as those described herein, although fragments of this type can now be more easily generated by cloning or direct synthesis of the particular polypeptide that is desired to be produced.

p00053The antibodies of the present invention are high potency antibodies, which generally exhibit high kon values. For the purposes of the present disclosure, the term "high power" refers to a power reflected by an EC50 (or effective concentration that results in at least a 50% reduction in the DO450 value in the microneutralization test described below. ) less than about 3 nM (nanomolar or 10-9 molar). Antibodies according to the present invention can be neutralizing (causing destruction of the target species, such as a virus, and thereby reducing viral load). A non-neutralizing antibody for one use may be neutralizing for a different use.

p00054High potency antibodies may have specificity for antigenic determinants found in the microbes and are capable of neutralizing said microbes by binding to them. In accordance with the present invention, such microbes are in most cases viruses, bacteria or fungi, especially organisms that cause respiratory disease and most preferably viruses. A specific example, used in the examples herein, is respiratory syncytial virus (RSV); Another example is the parainfluenza virus (PIV).

p00055High potency antibodies may also have specificity for antigens exhibited on cancer cell surfaces (but generally will not include antibodies, such as vitaxin, that are non-neutralizing. (See: Wu et al., Proc. Natl. Acad. Sci. 95: 6037-6042 (1998)).

p00056High potency antibodies may also have specificity for chemical substances such as toxic substances, or toxins, or for toxin products, which include, but are not limited to, products produced by the metabolism of an organism of such or such toxins. For example, the high potency antibodies of the present invention may be useful in nullifying, or otherwise improving, the effects of addictive drugs, such as cocaine.

p00057The high potency antibodies of the present invention may also have high affinity for the RSV F antigen, and, where such high affinity is exhibited, the affinity constant (Ka) of such antibodies is at least about 109 M-1, with preferably at least about 1010 M-1, and most preferably at least about 1011 M-1.

p00058The antibodies of the present invention exhibit high potency when measured in the microneutralization assay described in Example 2. In said assay, high potency is measured by the EC50 value and commonly has an EC50 value of less than about 3.0 nM ( nanomolar or 10-9 M), and most preferably less than about 1.0 nM. In general, the lower the EC50 value, the greater the potency, or biological activity.

p00059The high potency antibodies of the present invention exhibit such high potency due to their high kon values, which is determined by the amino acid sequences that constitute the framework regions (FR) and complementarity determining regions (CDRs). These antibodies, or active fragments thereof, have high potency determining regions (CDRs) within their amino acid sequences. The high potency neutralizing antibodies of the present invention may comprise at least 2 high potency CDRs, or 3 high potency CDRs, or even 4 high potency CDRs or 5 high potency CDRs, and can comprise even 6 high potency CDRs . Of course, in the latter case, all of the 6 CDRs of the antibody, or active fragments thereof, are high potency CDRs. Accordingly, such high potency neutralizing antibodies of the present invention have high potency CDRs that are constituted by one of each of the light chain CDRs L1 (CDR L1), L2 (CDR L2), and L3 (CDR L3) and heavy chain CDRs H1 (CDR H1), H2 (CDR H2) and H3 (CDR H3).

p00060In specific embodiments of such high potency antibodies, said high potency CDRs have amino acid sequences selected from the group consisting of SEQ ID NO: 11, 12, 13, 56 or 59 for CDR L1, SEQ ID NO: 14, 15, 16 , 17, 18, 19, 20, 21, 22, 57, 58 DTFRLAS and DTFYLSS for CDR L2, SEQ ID NO: 23 for CDR L3, SEQ ID NO: 24 and 25 for CDR H1, SEQ ID NO: 26, 27 , 28, 29, 30, 55 and DIWWDGKKSYNPSLKS for CDR H2, SEQ ID NO: 31, 32, 33 and 34 for CDR H3.

p00061In preferred embodiments, the high potency neutralizing antibodies of the present invention comprise heavy and light variable chains with amino acid sequences selected from the group consisting of SEQ ID NO: 35 and 36.

p00062A process for producing a high potency antibody may comprise:

<dl><dt>(to)</dt><dd> produce a recombinant antibody, including immunologically active fragments thereof, comprising heavy and light chain constant regions derived from a mammalian antibody and </dd></dl>

heavy and light chain variable regions containing one or more lattice regions and / or complementarity determining regions (CDRs) having preselected amino acid sequences;

<dl><dt>(b)</dt><dd> screening said recombinant antibodies to high kon when said antibody reacts in vitro with a selected antigen; and</dd></dl>

<dl><dt>(c)</dt><dd> select antibodies with said high kon. </dd></dl>

p00063Antibodies produced according to the process described above will commonly give high affinity constants and high kon values, producing the latter high biological activity, or potency. In specific embodiments, high potency antibodies produced in accordance with the present invention commonly have a kon of at least about 2.5 x 105 M-1 s-1, preferably at least about 5 x 105 M-1 s-1 , and most preferably at least about 7.5 x 105 M-1 s-1.

p00064In one embodiment, the processes described herein produce a high potency antibody in which the preselected amino acid sequences that produce a high kon (and the resulting high potency) are present, either at the same time in the framework region and at least in two or three CDR regions, perhaps in all 6 CDR regions, of the antibody or are restricted exclusively to CDR regions.

p00065In another embodiment, the preselected amino acid sequences that produce a high kon are present either in the framework region and at least in 3 CDR regions of the antibody or are restricted exclusively to CDR regions.

p00066In a further embodiment, the preselected amino acid sequences that produce a high kon are present either in the framework region and in at least 4 CDR regions of the antibody or are restricted exclusively to CDR regions.

p00067Additionally, the antibodies produced by the processes described herein may be complete tetramer antibodies, having the H2L2 structure, or they may be fragments of such antibody structures, including single chain antibodies or fragments such as Fab or F (ab) fragments. two'.

p00068In accordance with the present invention, the antigen for which antibodies are specific is expressed by respiratory syncytial virus (RSV).

p00069A process for producing a high potency antibody may comprise producing a recombinant antibody comprising the heavy and light chain constant region derived from a mammalian antibody and heavy and light chain variable regions containing framework regions and / or determining regions of Complementarity (CDR) in which at least one CDR is a high (or high potency) CDR that has an amino acid sequence not found in nature and in where the presence of said CDR results in a high kon.

p00070In specific embodiments, the high kon recombinant antibody comprises at least 2 high kon CDRs, possibly 3 high kon CDRs, and even 4 high kon CDRs, and as many as 5 or 6 high kon CDRs. The presence of such CDR sequences results in the antibody, or fragment, exhibiting a high kon, and therefore high potency.

p00071In other embodiments, the high association constant mentioned above of the antibodies produced by the

M-1-1

p00073Methods of the invention is at least about 2.5 x 105 s, preferably at least about 5 x 105 M-1 s-1, and most preferably at least about 7.5 x 105 M-1 s-1.

p00074A process for producing a high potency antibody may comprise:

<dl><dt>(to)</dt><dd> producing a recombinant antibody, including immunologically active fragments thereof, comprising constant heavy and light chain regions derived from a mammalian antibody and heavy and light chain variable regions containing one or more framework regions and / or determining regions of complementarity (CDRs) that have preselected amino acid sequences; </dd></dl>

<dl><dt>(b)</dt><dd> screening said recombinant antibodies for both high affinity and high kon when said antibody reacts in vitro with a selected antigen; and</dd></dl>

<dl><dt>(c)</dt><dd> select antibodies that have both high affinity and high kon. </dd></dl>

p00075The processes described herein produce high potency antibodies that have both high and high kon affinity where the affinity constant is at least 109 M-1 and kon is at least 2.5 x 105 M-1 s-1 , especially where said affinity is at least 1010 M-1 and said kon is at least 2.5 x 105 M-1 s-1, very especially where said affinity constant is at least 1011 M-1 and said kon is at least 2.5 x 105 M-1 s-1, having the most preferred affinity and very high kon embodiments, especially in which said affinity is at least 109 M-1 and said kon is at least 5 x 105 M-1 s-1, and very especially in which the affinity constant is at least 1010 M-1, and kon is at least 2.5 x 105 M-1 s-1, a very particularly preferred embodiment being one that produces a high potency antibody in which the affinity constant is at least 1011 M-1, and the kon is at least 7 , 5 x 105 M-1 s-1. It should also be understood that, when high affinity is also sought, any combination of the aforementioned values ​​of affinity (Ka) and kinetic association (kon) is within the present invention.

p00076These embodiments also include processes in which the preselected amino acid sequence that produces a high kon is present both in the framework region and the CDR regions, or only in the CDR regions, and in which such sequences, selected from SEQ ID NO: 11 to 34 and 55 to 58, are present in 1, 2, 3, 4, 5, or all of the 6 CDR regions, where the individual CDR sequence is selected from the individual sequences described herein. The methods for doing this are fully within the skill of those skilled in the art and will not be discussed in greater detail herein.

p00077The methods described herein are not limited to producing exclusively new high affinity antibodies that are specific for a particular antigen and that have been produced without taking into account existing immunogenic molecules and structures. Thus, the methods described herein provide a means for selected modifications of the structures of known antibody molecules, thereby producing increases in the kon of said antibodies and the concomitant increased biological activity. This is achieved by selective incorporation of the high power CDR sequences described herein.

p00078In separate embodiments of the present invention, the antibody whose potency should be increased will have an initial and / or final affinity constant of at least 109 M-1, preferably at least about 1010 M-1, and most preferably at least about 1011 M-1.

p00079Antibodies produced according to the methods described herein will have higher kon constants after amino acid changes to produce the high potency sequences of the invention and as a result of such amino acid changes, especially in which the kon value after said amino acid changes is at least 2.5 x 105 M-1 s-1, especially at least about 5 x 105 M-1 s-1, and in a very special way at least about 7.5 x 105 M-1 s-1 (regardless of the particular affinity constant) (Ka).

p00080In the application of the methods described herein, it should be understood that the above-mentioned changes in the amino acid sequence used to increase the potency of an antibody, or active fragments thereof,

p00081or the use of selected amino acid sequences to produce high potency antibodies, or high potency active fragments thereof, reach said high potency, or increased potency, by producing high kon values. Since the affinity constant is a numerical ratio of kon to koff, an increased kon can result in increased affinity if the koff value does not change in the same factor. Thus, the high potencies of the antibodies produced according to the present methods are a function of the value of kon and not of the value of Ka (the affinity constant). For example, the use of selected amino acid sequences in the CDRs of an antibody molecule can result in an appreciable increase in both association rate (kon) and dissociation (koff) constants and, if both constants increase according to the same factor , the result is a high power, or greater (due to the greater kon), but without any resulting increase in Ka (since the ratio of kon to koff is the same). Conversely, in cases where the use of such preselected amino acid sequences within the CDRs of a high potency antibody results in a reduced koff and without any increase in kon, the result is an antibody, or active fragment thereof, with greater affinity but with little or no increase in power. Thus, it has been found that little or no change in power occurs in cases where only the koff value changes (since Ka is the ratio of kon to koff) but kon remains constant despite a numerical change in Ka .

p00082According to the present invention, several convenient methods are available for measuring the potency of the antibodies, or active fragments thereof, such as Fab fragments. Such a method uses the cotton rat model, details of which are set forth in the examples provided below. Another is the microneutralization test (see Example 2).

p00083Also in accordance with the present invention, compositions and medicaments for prevention are provided.

p00084or treatment of a disease comprising a therapeutic (or prophylactically) effective amount of a high potency antibody, or active fragment thereof, which has a polypeptide sequence as described herein or produced in accordance with the methods set forth herein. . In a preferred embodiment, the disease is caused by a respiratory syncytial virus and the parainfluenza virus.

p00085The high potency neutralizing antibodies of the present invention are obtained by generating appropriate antibody gene sequences, that is, amino acid sequences, by arranging the appropriate nucleotide sequences and expressing them in a suitable cell line. Any desired nucleotide sequences can be produced using the codon-based mutagenesis method, as described, for example, in US Pat. Nos. 5,264,563 and 5,523,388. Such procedures allow the production of any and all amino acid residue frequencies at any desired codon positions within an oligonucleotide. This may include completely random substitutions of any of the 20 amino acids in a desired position or in any specific subset thereof.

p00086Alternatively, this process can be carried out in such a way that a particular amino acid is achieved at a desired location within an amino acid chain, such as the new CDR sequences according to the invention. In sum, the appropriate nucleotide sequence to express any desired amino acid sequence can be easily achieved and using such procedures the new CDR sequences of the present invention can be reproduced. This results in the possibility of synthesizing polypeptides, such as antibodies, with any desired amino acid sequence. For example, it is now possible to determine the amino acid sequences of any desired domains of an antibody of choice and, optionally, prepare homologous chains that have one or more amino acids replaced by other desired amino acids, in order to obtain a range of substituted analogs.

p00087In the application of such methods, it should be appreciated that due to degeneracy of the genetic code, methods such as random synthesis of oligonucleotides and partial synthesis of degenerate oligonucleotides will incorporate redundancies for codons that specify a particular amino acid residue at a particular position, although said methods can be used to provide a master set of all possible amino acid sequences and screen them for optimal function as antibody structures or for other purposes. Such methods are described in Cwirla et al., Proc. Natl. Acad. Sci. 87: 6378-6382 (1990) and Devlin et al., Science 249: 404406 (1990). Alternatively, such antibody sequences can be chemically synthesized or generated by other routes well known to those skilled in the art.

p00088In accordance with the invention described herein, improved antibody variants can be generated by combining in a single polypeptide structure, one, two or more new CDR sequences such as those described herein (see, for example, SEQ ID NO: 11-34 ), demonstrating that each of them independently results in increased potency or biological activity. In this way, several new amino acid sequences can be combined into a single antibody, in the same or different CDRs, to produce antibodies with desirable levels of biological activity. Such desirable levels will often result from the production of antibodies whose kon values ​​are at least about 2.5 x 105 M-1 s-1.

p00089By way of non-limiting example, 3 new CDR sequences of this type can be used and the resulting antibodies screened for potency, or biological activity, using either the cotton rat protocol or the microneutralization protocol described herein, where said antibody demonstrates high affinity for a particular antigenic structure, such as the RSV F antigen. The overall result would therefore be an iterative process of combining various simple amino acid substitutions and screening of the resulting antibodies for antigenic affinity and potency in a step-by-step mode, thereby ensuring that potency without sacrificing a value is increased. Desirably high, or at least a minimum value, for affinity.

p00090Using the new sequences and methods described herein, such an approach could avoid the time and expense of generating and screening all possible permutations and combinations of antibody structures in an effort to find the antibody with maximum efficiency. . Conversely, the complete randomization of a single CDR of 10 amino acid residues would generate more than 10 trillion variants, a virtually impossible number to screen.

p00091This iterative method can be used to generate double and triple amino acid replacements in a gradual process to narrow the search for antibodies that have higher affinity.

p00092Conversely, it should be appreciated that not all locations within the sequences of the different antibody domains can be the same. Substitutions of any kind at a particular location may be favorable or harmful. Additionally, the substitutions of certain classes of amino acids in certain locations may similarly represent a point in favor or an unfavorable point with regard to affinity. For example, it may not be necessary to test all possible hydrophobic amino acids in a given position. It is possible that any hydrophobic amino acid behaves equally well. Conversely, an acidic or basic amino acid at a given location can provide large fluctuations in the measured affinity. Therefore it is also necessary to learn the "rules" of making such substitutions, but the determination of said "rules" does not require the study of all possible combinations and substitutions. Trends may become apparent after examining a number of substitutions less than the maximum.

p00093In accordance with the present invention, such rules determine the amino acid changes that must be made in the CDR regions of the antibodies, or the amino acid sequences that have to be prepared in completely new and synthetic antibody polypeptides, in order to achieve high affinities. However, it has now been discovered that, although high affinity is often a property of antibodies useful in therapeutic applications, such antibodies do not always have sufficient potency to provide practical utility in such uses.

p00094As already described, affinity is measured by the ratio of the constants kon and koff. For example, a 105 M-1 s-1 kon and a 105 M-1 s-1 koff would be combined to give an affinity constant of 1010 M-1 (see the values ​​in Table 3). However, antibodies that exhibit such high affinity may nevertheless lack the potency required to make them useful therapeutic agents. In accordance with the present invention, the potency of the antibodies depends on the value of the rate kon for the antibody binding reaction. Thus, an antibody, with indifference of the affinity for the respective antigen, will exhibit an increase in potency (such as neutralization activity) in the event that said antibody has a higher kon value, with indifference of Ka or koff.

p00095According to the methods described herein, the increased potency of an existing antibody, regardless of its affinity for the antigen, is achieved by selective changes in one or more of the amino acids present in one or more of the CDR regions of said antibody, whereby said amino acid changes have the effect of producing an increase in the kon value for said antibody, preferably with an increase in the affinity of the antibody. A higher power can be achieved with a higher kon value even if the affinity remains the same or decreases slightly. Such an antibody is very advantageously produced by synthesis of the required polypeptide chains via synthesis in conveniently engineered cells that have the appropriate nucleotide sequences encoding the required polypeptide chains containing the segments incorporated therein. CDR altered. Also according to the methods described herein, a new antibody having a desirable level of potency, or biological activity, it can be prepared de novo by incorporation of selected amino acids at selected locations within the CDR regions of said antibody polypeptide chains using genetically engineered cells as described herein or entirely by chemical synthesis of the polypeptide chains required with subsequent formation of the necessary disulfide bonds.

p00096In this regard, it should be clearly taken into account that antibodies produced in accordance with the methods described herein may be antibodies that possess tetrameric, dimeric or monomeric structures. Thus, the term "antibody" as used herein includes whole tetramer antibody molecules, as commonly found in nature, as well as portions and fragments thereof, including L2H2, LH, Fab, F (ab ' ) 2, and other fragments, being the only requirement of such structures that they retain biological activity as measured by the assays and protocols described herein.

p00097In accordance with the foregoing, the antibodies of the present invention are high affinity monoclonal antibodies. Such antibodies, however, are monoclonal only in the sense that they can be derived from a clone of a single cell type. However, this should not be understood as limiting them to a particular origin. Such antibodies can be easily produced in cells that commonly do not produce antibodies, such as CHO or COS cells. Additionally, such antibodies can be produced in other types of cells, especially mammalian cells or even plant cells, by genetic engineering of said cells to express and assemble the light and heavy chains of the polypeptide that form the antibody product. Additionally, such chains can be chemically synthesized but, since they should be specific for a given antigenic determinant, they would still constitute "monoclonal" antibodies in the sense in which said term is used. Thus, as used herein, it should be understood that the term "monoclonal antibody" denotes more the specificity and purity of the antibody molecules produced by the methods described herein than the mere mechanism used to produce said antibodies.

p00098Also, as used herein, it should be understood that the term "potency" describes the dependence of the effect of the antibody, when used for its proposed purpose, on the concentration of said antibody. Thus, potency means biological activity with respect to a given antigen. By way of non-limiting example, the power,

p00099or biological activity, or biological effect, is measured for an anti-RSV antibody, by the cotton rat procedure or the microneutralization procedure, as described in the Methods section.

p00100Conversely, the affinity of an antibody to the antigen is simply a mathematical measure of the ratio of kon to koff.

p00101Additionally, the affinities (Ka) of the antibodies produced according to the method described herein will typically be within the 1010 M-1 environment. This environment may, for example, be of the order of 10 times, greater or lesser, of 1010 M-1 or be more than 10 times greater than 1010 M-1, or it may even be numerically equal to 1010 M-1. The affinity of the antibody to the antigen is proportional to the value of this constant (that is, the higher the constant, the higher the affinity due to the higher concentration of the complex - see equation for the affinity constant). Said constant is measured by standard kinetic methodology for antibody reactions (as described in Example 1).

p00102In one embodiment, the antibodies produced according to the methods described herein (other than the case in which the term "antibody" means an active portion, fragment or segment, all of which, for the purposes of the present disclosure , are considered to be included within the meaning of the term antibody) will commonly comprise a mammalian constant region, preferably human, and a variable region comprising said variable region heavy and light chain framework regions and heavy and light chain CDRs, wherein the heavy and light chain framework regions have characteristic sequences of a mammalian antibody, preferably a human antibody, and wherein the CDR sequences are similar to those of an antibody of any species other than a human, preferably a mouse. In the case where the framework amino acid sequences are characteristic of those of a non-human animal, the latter is preferably a mouse.

p00103In another embodiment, the antibody is a human antibody in which the antibody has a kon value as described herein to provide enhanced potency.

p00104Additionally, antibodies produced in accordance with the present invention will commonly fix the same epitope as before applying the methods described herein to increase the kon value. Thus, after applying the methods described herein, the antibody will have CDR sequences similar, but not identical, to the CDR sequences prior to the application of the methods set forth herein, in the sense that at least one of the CDRs of said antibody will contain a high potency amino acid sequence, such as one selected from SEQ ID NO: 11-34, 55-59, DIWWDGKHSYNPSLKS, DTFRLAS and DTFYLSS if the antibody is to be used to neutralize a virus such as RSV.

p00105In line with the foregoing, and in order to better describe the sequences set forth in accordance with the invention with respect to a humanized antibody against RSV, a basic sequence or starting sequence of light and heavy chain variable regions of an antibody, or fragment thereof, whose power must be increased, are shown in Figure 1A (variable light chain region - SEQ ID NO: 1) and Figure 1B (heavy chain variable region - SEQ ID NO: 2) or a Fab fragment of such an antibody (for example, the sequences of Figure 2A (variable light chain region -SEQ ID NO: 3) and Figure 2B (heavy chain variable region - SEQ ID NO: 4) . Also according to the invention, specific amino acids different from those of these starter sequences were generated by recombinant methods starting from prepared nucleotide sequences designed to generate said amino acid sequences when expressed in recombinant cells. The products of said cells are the monoclonal antibodies of the present invention. Alternatively, such antibodies can be produced without the use of a genetically engineered or recombinant cell by means of synthesis well known in the art.

p00106In one embodiment of the present invention, the potency is increased using a neutralizing antibody against respiratory syncytial virus (RV) having an affinity constant of at least 109 M-1, and preferably at least 1010 M-1 (for the antigen F thereof) by increasing the kon value up to at least 2.5 x 105 M-1 s-1. The amino acids present in the CDRs of such a Fab fragment are shown in Table 3 (for example, clone 5).

p00107In general, the method used to determine the affinity and kinetic constants of the antibodies before and after the application of the methods of the invention to increase the kon value, consisted of generating nucleotide sequences for the genes that express the antibody chains. desired (according to the present invention) and insert these into vectors that were then used to transform COS-1 cells by standard protocols. The cells were grown in wells and the supernatant was sampled and measured for antigen binding using standard ELISA techniques. These polynucleotides were designed in such a way as to provide one or more amino acid replacements in the CDRs that could then be screened for increased kon values, selectively combining favorable replacements (those that produced increased kon values) for increased affinity. These are subsequently screened for binding affinity for the respective antigen, such as the RSV F antigen versus the basic or reference structure, thereby determining that there was no significant change in affinity as a result of the increase in the values ​​kon.

p00108In specific embodiments, the present invention relates to an isolated antibody comprising an affinity constant of at least 109 M-1, preferably at least 1010 M-1 and most preferably at least 1011 M-1, and in

<dl><dt>-</dt><dd /></dl>

p00109where kon is at least about 2.5 x 105 M-1 s-1, preferably at least about 5 x 105 M-1 s 1, and most preferably at least 7.5 x 105 M-1 s-1 (with inclusion of all combinations thereof).

p00110Also in accordance with the present invention, an isolated antibody of this type can be any kind of antibody already known or newly synthesized and new. Thus, antibodies produced in accordance with the methods described herein will include an antibody selected from the group consisting of naturally existing mammalian antibodies, naturally existing human antibodies, naturally occurring mouse antibodies, single chain antibodies, chimeric antibodies (which have constant regions of an antibody of one species and variable regions of an antibody of a different species), CDR-grafted antibodies (which have the CDR regions of an antibody of one species and the constant regions and, possibly, framework regions of an antibody of a different species), humanized antibodies (in which selected amino acids, of the variable regions of framework and / or CDR regions have been altered so that they are similar to a human antibody although such sequences are derived largely from a different species, such as a mouse), preferably humanized mouse antibodies, altered mammalian antibodies, preferably mouse, most preferably human) in which the amino acids selected from an existing antibody have been altered at some point in the polypeptide chain, commonly by genetic engineering techniques, in order to provide antibody structures similar to the antibody structures on which they are based), and new, fully synthetic antibodies, the latter not existing previously in nature.

p00111Methods of increasing the potency of one of the aforementioned antibody types (described above) are also described which comprise selectively changing the amino acids within the variable regions of the antibody in order to increase the measured kon value of said antibody with respect to a particular antigen Of course, the kon value may be different for the same antibody after the same amino acid changes in which the reaction is measured using a different antigen or antigenic determinant. However, in such cases, affinities will also probably change as the identity of the antigenic determinant changes.

p00112Also according to the methods described herein, the amino acid changes introduced in the polypeptide sequences of such antibodies are preferably limited to the CDR portions of the variable regions of the antibodies although these could also involve changes in the framework regions. .

p00113Although the most advantageous CDR sequences are commonly identified by screening of modified antibody clones whose potency should be increased by the methods described herein, once such high potency clones have been identified, the resulting antibody is very advantageously produced thereafter by synthesis of the appropriate heavy and light polypeptide chains in suitable animal or plant cells after introduction into said suitable vector cells containing the appropriate DNA sequences corresponding to the desired amino acid sequences, taking advantage of the advantage of the genetic code to design the required nucleotide sequences. As a consequence of this approach, completely new antibodies with high potency can initially be produced using the amino acid sequence identities suggested by the methods described herein without the need to select existing antibody sequences for modification. Thus, the methods described herein facilitate the production of high potency antibodies with a completely new structure in the sense that their CDR sequences are high potency CDRs as determined by the methods described herein in order to deliberately increase the kon values ​​of such antibodies and without destroying the specificity and affinity of said antibodies for the proposed antigenic target.

p00114In an embodiment of the methods described herein, an existing antibody is modified to increase the potency thereof by increasing the kon value. In a preferred embodiment, the antibody is one with high affinities, eg, at least about 109 M-1 or 1010 M-1. The antibody is synthesized, using genetically modified animal or plant clones or cells, in order to introduce amino acid changes in the heavy and / or light polypeptide chains of said antibody, preferably such that said antibody changes are introduced in the determining regions of the complementarity (CDRs) of said polypeptide chains, to increase the kon value for fixing said antibody to a particular antigen with concomitant increase in the potency of the antibody. Thus, the methods described herein are advantageously used to produce an antibody molecule in which the kon value of said antibody after amino acid changes in its sequence, preferably the variable regions of said sequence, most preferably the CDR portions, is greater than the kon value exhibited by said antibody before said amino acid changes when the kon values ​​are measured with respect to the same antigen.

p00115In general, in the case where the methods described herein are applied to known antibodies, the kon of said antibodies will be increased at least 2 times, preferably at least 5 times, and most preferably at least 10 times. More specifically, the kon value of said antibody is increased to at least

M-1 M-1-1

p00117about 2.5 x 105 s-1, preferably increasing to at least 5 x 105 s, and most preferably at least 7.5 x 105 M-1 s-1.

p00118Since the methods described herein are equally effective for the design of new high potency recombinant antibodies previously unknown, a method of producing an antibody having a kon value of at least 2.5 x 105 is also disclosed. M-1 s-1, which comprises preparing an antibody whose polypeptide sequences contain selected amino acids at selected locations, especially within the CDR sequences, followed by screening of said antibodies looking for those having a kon value of at least 2.5 x 105 M-1 s-1, or a kon value of at least 5 x 105 M-1 s-1 or even 7.5 x 105 M-1 s-1. Such antibodies will result from the presence of one or more of the high potency CDRs described herein. Such antibodies are easily screened for high kon values.

p00119The methods described herein can be used for the production of antibodies with high potency, or antibodies of increased potency, which have affinity for an antigen characteristic of the respiratory syncytial virus (RSV).

p00120In another embodiment, the present invention relates to compositions and medicaments for use in the prevention or treatment of a disease comprising said therapeutically active amount of the composition or medicament of an antibody prepared by the methods described herein. Thus, such an antibody can be a completely new antibody or a known and clinically useful antibody whose potency has been increased by application of the methods described herein. The diseases that are prevented or treated by the antibodies prepared by the methods described herein can be diseases caused by microorganisms, such as bacteria or viruses, preferably viruses and most preferably RSV.

p00121Antibodies disclosed in this way will also commonly have framework regions derived from a human antibody but, in the case of not being of human origin, they are preferably mouse.

p00122In the generation of the clones, the basic or reference antibody (heavy and light chain variable regions (plus framework CDRs) depicted in Figures 1 and 2) was used as the "template" to generate the new 5 CDR sequences of the antibodies of the present invention, imparting the latest higher kon values. Standard methods were used to characterize and synthesize the 6 CDR libraries of simple mutations (see Wu et al., Proc. Natl. Acad. Sci. 95: 6037-6042 (1998). The target CDR was first deleted for each of the libraries before nucleotide reassociation. For the synthesis of the libraries, the CDRs of a reference antibody (see Figure 2) were defined as in Table 1. Codon-based mutagenesis was used to

p0012310 oligonucleotide synthesis in order to produce the CDR sequences of the invention (as described above).

p00124Libraries were initially screened by capture survey to identify maximum affinity variants. These clones were then further characterized using capture ELISA and by titration on an immobilized antigen. After said screening, the antibodies are then screened for their respective kon values, whose positive effects are then measured by potency determination. Figures 4 and 5 show details

p00125fifteen Additional information about the preparation and screening procedures used herein.

p00126Table 1. Basic CDR sequences as provided in Figure 2.

p00127CDR Residues of Fig. 22 Sequence SEQ ID NO.

L1 24-33 SASSSVGYMH 5

L2 49-55 DTSKLAS6

L3 88-96 FQGSGYPFT7

H1 31-37 TSGMSVG 8

H2 52-67 DIWWDDKKDYNPSLKS 9

H3 100-109 SMITNWYFDV 10

p00134In accordance with the present invention, the DNA of the highest kon variants was sequenced to determine the nature of the beneficial or high potency replacements. After screening, the antibodies are then prepared with the high kon amino acid replacements, either individually or in various

p00135twenty combinations, in order to maximize the effects of such substitutions and thereby produce high affinity antibodies that also exhibit high potency.

p00136As a general rule, it was found that the most beneficial of high-kon CDRs were the result of amino acid replacements in up to 6 CDRs. Thus, the high potency neutralizing antibodies (i.e., high kon) disclosed herein contain amino acid sequences that differ from that of the

p0013725 base or reference antibody (for example, as shown in Figures 1 and 2) only in the complementarity determining regions L1 (or CDRL1), L2 (or CDRL2), L3 (or CDRL3), H1 (or CDRH1) and H3 (or CDRH3).

p00138Table 2. Sequences of CDRs that tend to induce high potency in antibodies

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>1 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>L2 </dt><dd>X 14</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24 </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 31 </dd></dl>

<dl><dt>2 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>L2 </dt><dd>X fifteen</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 32 </dd></dl>

<dl><dt>3 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>L2 </dt><dd>X 16</dd></dl>

<dl><dt> L3 </dt><dd>. FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 32 </dd></dl>

<dl><dt>4 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>L2 </dt><dd>X 17</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 32 </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>5 </dt><dd>L1 - SASSSVGYMH </dd><dt>5 </dt><dd /></dl>

<dl><dt>L2 </dt><dd>X fifteen</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 31 </dd></dl>

<dl><dt>6 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>L2 </dt><dd>X fifteen</dd></dl>

<dl><dt> L3 </dt><dd>X 2. 3</dd></dl>

<dl><dt> H 1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 33 </dd></dl>

<dl><dt>7 </dt><dd>L1 X eleven</dd></dl>

<dl><dt> L2 </dt><dd>X fifteen</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 31 </dd></dl>

<dl><dt>8 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>L2 </dt><dd>X 60</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 31 </dd></dl>

<dl><dt>9 </dt><dd>L1 X 12</dd></dl>

<dl><dt> L2 </dt><dd>X 61</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 25</dd></dl>

<dl><dt> H2 </dt><dd>X 26</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>10</dt><dd> L1 X 12</dd></dl>

<dl><dt> L2 </dt><dd>X 18</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 25</dd></dl>

<dl><dt> H2 </dt><dd>X 27</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>11</dt><dd> L1 X 13</dd></dl>

<dl><dt> L2 </dt><dd>X 19 </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 25</dd></dl>

<dl><dt> H2 </dt><dd>X 27</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>12</dt><dd> L1 X </dd><dt>12</dt><dd /></dl>

<dl><dt> L2 </dt><dd>X twenty</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 27</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>13</dt><dd> L1 X eleven</dd></dl>

<dl><dt> L2 </dt><dd>X twenty</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 28</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>14</dt><dd> L1 X 13</dd></dl>

<dl><dt> L2 </dt><dd>X twenty-one </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 29</dd></dl>

<dl><dt> H3 </dt><dd>X 32 </dd></dl>

<dl><dt>15</dt><dd> L1 X 12</dd></dl>

<dl><dt> L2 </dt><dd>X 22</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 30</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>16</dt><dd> L1 X 59</dd></dl>

<dl><dt> L2 </dt><dd>- DTSKLAS 6 </dd></dl>

<dl><dt>L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>- TSGMSVG 8 </dd></dl>

<dl><dt>H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>- SMITNWYFDV 10 </dd></dl>

<dl><dt>17 </dt><dd>L1 - SASSSVGYMH 5 </dd></dl>

<dl><dt>L2 </dt><dd>X fifteen</dd></dl>

<dl><dt> L3 </dt><dd>X 2. 3 </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>- DIWWDDKKDYNPSLKS 9 </dd></dl>

<dl><dt>H3 </dt><dd>X 33 </dd></dl>

<dl><dt>18</dt><dd> L1 X 56</dd></dl>

<dl><dt> L2 </dt><dd>X 22</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 62</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>19</dt><dd> L1 X eleven</dd></dl>

<dl><dt> L2 </dt><dd>X 57</dd></dl>

<dl><dt> L3 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 26</dd></dl>

<dl><dt> H3 </dt><dd>X 3. 4 </dd></dl>

<dl><dt>20</dt><dd> L1 X 13</dd></dl>

<dl><dt> L2 </dt><dd>X 58 </dd></dl>

<dl><dt>Clone</dt><dd> CDR High Power CDR Sequence SEQ ID NO. </dd></dl>

<dl><dt>L2 </dt><dd>- FQGSGYPFT 7 </dd></dl>

<dl><dt>H1 </dt><dd>X 24</dd></dl>

<dl><dt> H2 </dt><dd>X 55</dd></dl>

<dl><dt> H3 </dt><dd>X 32 </dd></dl>

p00139Thus, for the amino acid sequences of Figure 3, amino acids selected from the sequence of Figure 2 were replaced as a means to increase the potency of the antibody with the heavy and light chain sequences shown in Figure 2.

p00140The antibodies selected from high kon (and active fragments thereof) resulting from the methods given to

p001415 known herein are shown in Table 2 (all of which have the framework sequences of Figure 2) where the reference clone is the clone having the heavy and light chain variable region sequences shown in Figure 2 (SEQ ID NO: 3 and 4 for light and heavy sequences, respectively).

p00142Table 2 indicates the amino acid sequences (all sequences in the standard single letter amino acid code) of the high-kon CDRs employed in the high-potency antibodies prepared in accordance with

p0014310 the methods disclosed in this report. In Table 2, the locations of the fundamental amino acid substitutions made in the corresponding CDRs of Table 1 (ie, the locations in which the CDRs differ in amino acids) are indicated by a box around the amino acid (s).

p00144According to the invention, by combining such amino acid substitutions so that more than one occurred in the same antibody molecule, it was possible to significantly increase the potency of the antibodies.

p00145fifteen disclosed in this report.

p00146In general, there is a correlation between kon and antibody potency, with all of the highest kon variants having more than one beneficial or high kon CDR, even having all of the 6 substituted CDRs.

p00147In one embodiment, an antibody prepared in such a manner that has increased kon is an RSV neutralizing antibody, with an affinity of at least 109 M-1 and preferably at least 1010 M-1, which is also a

p00148twenty Humanized antibody that includes a human constant region and a framework region for heavy and light chains wherein at least a portion of the framework is derived from a human antibody (or from a consensus sequence of a human antibody framework).

p00149In another embodiment, the entire framework is derived from a human antibody (or a human consensus sequence).

p0015025 In another embodiment, an antibody produced in accordance with the present invention, with an affinity of at least 109 M-1 and preferably at least 1010 M-1, is a grafted antibody having 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 part of the framework is derived from a human antibody (or a consensus sequence of a human antibody framework).

p0015130 As long as the desired CDR sequences, and the constant and framework sequence are known, genes can be assembled with the desired sequences and, using a variety of vectors, inserted into appropriate cells for expression of the functional tetramer antibody molecules. The coupling of this with the methodology already described, allows the assembly of libraries of simple mutations in which the antibodies have the same sequences as the corresponding grafted antibodies and, consequently, the same

p0015235 fixing structure and affinities.

p00153The combinations of the CDR sequences described in Table 2 may be present in whole tetramer antibody molecules or in active fragments, such as a Fab fragment. The potency data for clones 1 to 5 shown in Table 3 correspond to Fab fragments, while the data for clones 16 and 17 of Table 3 correspond to whole antibody molecules (clone 16 is MEDI-493 with the

p0015440 sequence described in Johnson et al (1997)).

p00155The whole antibody molecules according to the present invention include antibody molecules having heavy chain sequences (most constant variable region) selected from the group consisting of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51 and 53 and with light chain sequences (more constant variable region) selected from the group consisting of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, and 54.

p00156The relatively high kon antibodies of the invention may be present in a relatively pure or isolated form as well as in a supernatant extracted from cultured cells in wells or in plates. The antibodies of the invention may therefore 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 diluent or excipient. The antibodies of the invention may be present in such a composition at a concentration, or in an amount, sufficient to be therapeutically or pharmacologically valuable in the treatment or prevention of diseases (e.g., prevention of RSV, including increased incidence of asthma and breathlessness that often occur after such infections). Such antibodies may also be present in a more dilute composition.

p00157Accordingly, the invention is also directed to provide compositions and medicaments for use in the prevention and / or treatment of diseases, especially viral diseases, most especially respiratory syncytial virus infections, said composition or medicament comprising a therapeutically effective amount of the Antibody composition described herein.

p00158In a particular embodiment, a high potency neutralizing antibody of the present invention has the heavy chain and light chain domain sequences depicted in Figure 3B (SEQ ID NO: 36) and Figure 3A (SEQ ID NO: 35 ), respectively. The CDRs of the heavy and light chains are the same as those given in Table 2 for clone 15.

p00159It should be noted that while the increased kon antibodies of the present invention could be assembled from CDR regions and non-CDR regions derived from real neutralizing antibodies by joint remodeling of amino acid segments (and the antibodies thus assembled would be within the invention described herein), The antibodies of the present invention are more conveniently prepared by genetically engineered gene sequences appropriate to produce vectors that can then be transfected into cell lines suitable for eventual expression of the antibody molecules assembled by the modified cells. In fact, such recombinant procedures were employed to prepare the antibodies described herein. Additionally, since the sequences of the high affinity antibody chains are known by the exposure of this specification, such antibodies could also be assembled by direct synthesis of the appropriate chains then allowing them to self-assemble into tetramer antibody structures.

p00160General Materials and Methods

p00161Monoclonal antibodies. MEDI-493 is a humanized IgG1 (COR) / kappa (K102) MAb (heavy and light chain variable region sequences are depicted in Figure 1) containing the murine MAb 1129 antigen binding determinants [Johnson et al, J. Infect. Dis., 176, 1215-1224 (1997); Beeler and van Wyck Coelingh,

p00162J. Virol., 63, 2941-2950 (1989)].

p00163RSV Fusion Inhibition Assay. The ability of the antibodies to block RSV-induced fusion after viral fixation to the cells was determined in a fusion inhibition assay. This assay was identical to the microneutralization assay, except that the cells were infected with RSV (long) for 4 hours before the antibody was added [Taylor et al., J. Gen. Virol., 73, 2217-2223 (1992) ].

p00164BIAcore analysis. The epitope analysis of the MAbs was performed using a BIAcore biosensor (BIAcore, Piscataway, NJ) [Karlsson et al, J. Immunol. Methods, 145, 229-240 (1991); Johne, Mol. Biotechnol., 9, 65-71 (1998)] with a Microfluidics plasmon resonance system. The antigen used for this assay was a truncated RSV protein (A2) F (amino acids 1-526) expressed in baculovirus. The purified RSV F protein was covalently coupled to a CM5 sensor chip activated with N-hydroxysuccinimide / 1-ethyl-3- [3-dimethylaminopropyl] -carbodiimide according to the manufacturer's protocol, and the unreacted active ester groups were made react with 1M ethanolamine. A primary injection of 1 μm or 10 μM MEDI-493 was followed by a washing step with HBSS, and then by a secondary injection of MEDI-493 or RHSZ19. The sensograms were analyzed using BIA evaluation software.

p00165Calorimetry of Isothermal Titration. The affinity of the solution of each MAb for RSV F protein was determined by isothermal titration calorimetry [Wiseman et al., Anal. Biochem., 179, 131-137 (1989)]. A solution of 1.4 ml of 4.5 μM RSV F protein was titrated with injections of 5.5 μl of 26 μM MEDI-493 or RSHZ19. After each injection of MAb, the amount of heat released was measured, which is proportional to the amount of fixation. The useful antigen was a truncated RSV F protein (A2) (amino acids 25-524) expressed in Drosophila cells. The titrations were carried out at 44º and 55ºC until the optimum signal to noise ratio was reached. The thermal stability of the MAbs and the F protein at these temperatures was demonstrated by deployment experiments by circular dichroism. The affinities were corrected at 37 ° C for comparison with in vivo data using the van't Hoff integrated equation [Doyle and Hensley, Methods Enzymol., 295, 88-99 (1998)]. Van't Hoff's correction is based exclusively on the enthalpy change of F protein binding that was measured directly by calorimetry. Since the fixation enthalpy changes for MEDI-493 and RSHZ19 were found to be very similar, the temperature corrections for their Kds were almost identical.

p00166Prophylaxis of the Cotton Rat. In vivo efficacy is determined using the cotton rat model [Prince et al, J. Virol., 55, 517-520 (1985)]. Cotton rats (Sigmodon hispidus, average weight 100 grams) are anesthetized with methoxyfluorane, bled, and receive 0.1 ml of purified MAb by intramuscular injection (im) at doses of 5, 2.5, 1.25, or 0.625 mg / kg body weight, or bovine serum albumin control (BSA) at 5 mg / kg body weight. 24 hours later, the animals are weighed again, bled to determine the concentration of MAB in the serum, and are faced by intranasal (in) instigation of 105 PFU to strains A (long) or B (18537) of RSV. 4 days later the animals are sacrificed and their lungs are removed. The lungs are homogenized in 10 parts (weight / volume) of Hanks balanced salt solution, and the resulting suspension was used to determine the pulmonary viral titers by bald test. Serum antibody titers at the time of confrontation are determined by an anti-human IgG ELISA.

p00167Example 1

p00168Kinetic Analysis of Humanized RSV Mabs by BIAcore TM

p00169The interaction kinetics between high affinity anti-RSV Mabs and RSV F protein was studied by surface plasmon resonance using a Pharmacia BIAcoreTM biosensor. A recombinant baculovirus expressing a truncated C-terminal F protein provided an abundant source of antigen for kinetic studies. The supernatant, which contained the secreted F protein, was enriched approximately 20 times by successive chromatography on concanavalin A and Q-Sepharose columns. The pooled fractions were dialyzed against 10 mM sodium citrate (pH 5.5), and concentrated to approximately 0.1 mg / ml. In a typical experiment, an aliquot of the F protein (100 ml) was subjected to amine coupling to the BIAcore sensor chip. The immobilized amount gave approximately 2000 response units (Rmax) of signal when saturated with H1129 or H1308F (prepared according to US Patent 5,824,307). This indicated that there was an equal number of "A" and "C" antigenic sites in the preparation of protein F after the coupling procedure. Two irrelevant non-related Mabs (RVFV 4D4 and CMV H758) exhibited 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 fixation phase of 7 min. After Mab injection, the flow was changed with PBS / Tween buffer for 30 min to determine the rate of dissociation. The sensor chip was regenerated between cycles with a 2 min pulse of 10 mM HCl. The regeneration step caused a minimum loss of binding capacity of immobilized F protein (4% loss per cycle). This small decrease did not change the calculated values ​​of the speed constants for fixation and dissociation (also called kon and koff, respectively).

p00170More specifically, for the measurement of Kassoc (or kon), the F protein was immobilized directly by the EDC / NHS method (EDC = N-ethyl-N '- [3-diethylaminopropyl) -carbodiimide]). In summary, 4 μg / ml of protein F were prepared in 10 mM NaOAc, pH 4.0 and an injection of approximately 30 μl gives approximately 500 RU (response units) of immobilized F protein under the conditions referred to above. The blank flow cell (immobilized VnR-CM dextran surface) was subtracted for kinetic analysis. The column could be regenerated using 100 mM HCl (requiring 72 seconds of contact time for full regeneration). This treatment completely eliminated the fixed Fab without damaging the immobilized antigen, and could be used for more than 40 regenerations. For kon measurements, Fab concentrations were 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. The dissociation phase was analyzed from 230 seconds (30 seconds after the beginning of the dissociation phase) to 900 seconds. Kinetics were performed by Langmuir's 1: 1 adjustment (global adjustment). The measurements were made in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) of P20 Surfactant.

p00171For measurements of combinatorial clones, as set forth herein, the kon and koff values ​​were measured separately. The kon value was measured under conditions that were the same as those used for single mutation clones and performed analogously.

p00172For the measurement of koff (or kdissoc) the following conditions were used. In summary, 4100 RU of F protein (as previously) were immobilized with CM-dextran used as the target. In this case, 3000 RU of Fab was set (with dissociated Fab high enough to counteract the fluctuation of the machine). As a buffer, HBS plus 5 nM of F protein was used (approximately 350-2000 times greater than kdissoc or kd - the equilibrium dissociation constant). The dissociation phase was 6 to 15 hours at a flow rate of 5 μl / min. Under the conditions used herein, the resetting of the dissociated Fab was minimal. For more details, see the manual with the biosensor.

p00173The binding of high affinity anti-RSV antibodies to the F protein, or other epitopic sites in RSV, described herein was calculated from the ratio of the first order rate constant for dissociation of the rate constant. second order for fixation or association (kd = kdiss / kassoc). The value for kassoc was calculated based on the following speed equation:

p00174where R and Rmax are the response units in time t and infinity, respectively. A graph of dr / dt in

p001755 R function gives a slope of (kassoc [Mab] + kdiss). Since these slopes are linearly related to the [Mab] value, the Kassoc value can be derived from a repetition of the slope graph against [Mab]. The slope of the new line is equal to Kassoc. Although the value of kdiss can be extrapolated from the intersection with the Y axis, a more exact value was determined by direct measurement of kdiss. After the injection phase of the Mab, the PBS / Tween buffer is allowed to flow through the sensor chip. From this point, [Mab] = 0. The equation exposed

p0017610 previously for dR / dt it is therefore reduced to:

p00177The integration of this equation then gives:

p00178where R0 / Rt are the response units for time 0 (beginning of the dissociation phase) and t,

p00179fifteen respectively. Finally, the graphical representation of ln (R0 / Rt) as a function of t gives a slope of kdiss. In the preferred embodiment of this specification, the numerical values ​​of such antibody variants are shown in Table 3.

p00180For the clones of Tables 2 and 3, the reference clone is the Fab fragment with the sequences shown in Figure 2 and the CDRs shown in Table 1. Clones 1-15 are Fab fragments having the

p00181twenty lattice sequences of Figure 2 and the indicated CDR combinations of clones 1-15 of Table 2 (where "X" indicates a high power CDR (ie, a CDR whose presence against the reference sequence gives as a result high power and a power greater than the reference Fab)). Where there is no "X" next to the CDR in Table 2, the sequence is just the corresponding sequence of the reference Fab (from Table 1 and Figure 2).

p0018225 Table 3. Summary of Kinetic Constants for High Power Antibodies

p00183Clone No. Kon x 105 (M-1s-1) Koff x 10-4 (s-1) EC50 (nM)

p00184Ref. 1.85 6.5 3.52 1 3.65 3.26 2.26 2 5.31 4.22 5.05 3 6.05 4.22 4.70 4 7.57 4.62 3, 55 5 4.16 3.06 2.61 6 1.85 3.20 2.88 7 3.70 2.51 1.59 8 3.75 2.73 2.67 9 6.63 2.82 0, 29 10 5.27 2.99 1.06 11 5.71 7.17 20.9 12 7.9 4.53 3.24 13 7.43 2.30 0.81 14 7.35 2.50 2, 23 15 7.81 2.80 0.56 16 2.04 7.35 6.12 17 1.09 2.49 2.7

p00185Table 4. RSV Microneutralization Titles at the End Point of IgGs and Mutant Speed ​​Fabs on “High”

<dl><dt>Type </dt><dd>Clone No. Average IC50 g / ml IC50 standard Difference times Mean IC50 (Control) g / ml Standard (IC50 Control) Difference, times (IC50 Control) Number of Trial Repetitions </dd></dl>

<dl><dt>IgG </dt><dd> 16 0,4527 0,208 - 0,5351 0,238 - 8 </dd></dl>

<dl><dt>" </dt><dd> 24 0,0625 0,0268 7 0,0645 0,0223 8 3 </dd></dl>

<dl><dt>" </dt><dd> 18 0,0342 0,022 13 0,0354 0,0187 15 4 </dd></dl>

<dl><dt>" </dt><dd> 23 0,0217 0,0331 21 0,0289 0,0110 19 5 </dd></dl>

<dl><dt>" </dt><dd> 21 0,0231 0,0141 20 0,0223 0,0083 24 6 </dd></dl>

<dl><dt>" </dt><dd> 20 0,0337 0,0309 13 0,0383 0,0283 14 5 </dd></dl>

<dl><dt>" </dt><dd> 25 0,0357 0,0316 13 0,0354 0,0261 15 7 </dd></dl>

<dl><dt>" </dt><dd> 22 0,0242 0,0163 19 0,0235 0,0076 23 7 </dd></dl>

<dl><dt>" </dt><dd> 26 0,0376 0,0268 12 0,0375 0,0213 14 6 </dd></dl>

<dl><dt>" </dt><dd> 19 0,0171 0,0018 27 0,0154 0,00417 35 2 </dd></dl>

<dl><dt>Fab </dt><dd> 12 0,157 - 3 0,125 - 4 1 </dd></dl>

<dl><dt>" </dt><dd> 27 0,0179 - 25 0,0171 - 31 1 </dd></dl>

<dl><dt>" </dt><dd> 11 >1,00 - - >1,00 - - 1 </dd></dl>

<dl><dt>" </dt><dd> 9 0,0407 0,0112 11 0,0326 0,009 16 2 </dd></dl>

<dl><dt>" </dt><dd> 28 0,177 - 3 0,157 - 34 1 </dd></dl>

<dl><dt>" </dt><dd> 13 0,0287 0,00417 16 0,0310 0,00982 17 2 </dd></dl>

<dl><dt>" </dt><dd> 10 0,0464 0,00791 10 0,0351 0,0126 15 2 </dd></dl>

<dl><dt>" </dt><dd> 15 0,0264 0,00141 17 0,0258 0,00071 21 2 </dd></dl>

<dl><dt>" </dt><dd> 29 0,0414 - 11 0,0411 - 13 1 </dd></dl>

<dl><dt>" </dt><dd> 14 0,120 0,0222 4 0,1022 0,0260 5 2 </dd></dl>

<dl><dt>" </dt><dd> 30 0,194 0,462 2 0,176 0,0625 3 2 </dd></dl>

p00186The results in Table 4 compare Fab fragments and whole tetramer antibody molecules related by IC50 values ​​(or the concentration in μg / ml that gives 50% inhibition against controls similar to those in Table 3). Clone 16 of the Table is the reference antibody with the CDRs described in Table 2.

p001875 Clones 16 and 17 of Table 3 are real monoclonal antibodies with the framework sequences of Figure 1 and constant regions such as those described in Johnson et al (1997). The framework sequences of these antibodies may differ slightly from those of the Fab fragments.

p00188Clones 18 to 26 of Table 4 are tetrameric antibody molecules similar to clones 16 and 17 but have high potency CDR sequences. Clone 21 of the antibody has the same CDR sequences as clone 9 10 of Fab, clone 22 of the antibody has the same CDR sequences as clone 10 of Fab, clone 23 of the antibody

p00189it has the same CDR sequences as Fab clone 11, clone 24 of the antibody has the same CDR sequences as clone 12 of Fab, clone 25 of the antibody has the same CDR sequences as clone 13 of Fab, and clone 26 of the antibody has the same CDR sequences as Fab clone 15. Clones 18, 19 and 20 of the antibody in Table 3 are full length tetramer antibodies with the CDR combinations given in Table 2. The framework sequences of these antibodies may differ slightly from those of the Fab fragments.

p00190The underlined amino acids of the CDR sequences in Table 2 represent the amino acid residues located at the key locations within the high potency CDRs of the high potency antibodies produced by the methods of the present invention. For example, to increase the potency of an antibody by producing a higher kon value, the amino acids located in the key positions set forth herein by the residues in bold and underlined in Table 1 for the reference antibody would be replaced by amino acids. listed under CDRs in Table 2 (also with bold and underlined). Thus, these single letter codes represent the amino acids that replace the reference amino acids at the key positions (or critical positions) of the CDRs shown in Figure 2 (bold residues in the sequences in Table 2) for a reference antibody whose potency should be increased.

p00191For the clones of Table 4, clone 18 has the full length sequences given by SEQ ID NO: 41 (heavy chain) and 42 (light chain); clone 19 has the full length sequences given by SEQ ID NO: 45 (heavy chain) and 46 (light chain), clone 20 has the full length sequences given by SEQ ID NO: 47 (heavy chain) and 48 ( light chain), clone 21 has the full length sequences given by SEQ ID NO: 51 (heavy chain) and 52 (light chain), clone 22 has the full length sequences given by SEQ ID NO: 53 (heavy chain) and 54 (light chain), clone 23 has the full length sequences given by SEQ ID NO: 49 (heavy chain) and 50 (light chain), clone 24 has full length sequences given by SEQ ID NO: 43 (heavy chain) and 44 (light chain), clone 25 has the full length sequences given by SEQ ID NO: 37 (heavy chain) and 38 (light chain), and clone 26 has the sequences of total length given by SEQ ID NO: 39 (heavy chain) and 40 (light chain).

p00192In this case, clone 18 (IgG) and clone 27 (Fab) have the same CDRs, clone 19 (IgG) and clone 29 (Fab) have the same CDRs, clone 20 (IgG) and clone 28 (Fab) have the same CDRs, clone 21 (IgG) and clone 9 (Fab) have the same CDRs, clone 22 (IgG) and clone 10 (Fab) have the same CDRs, clone 23 (IgG) and clone 11 (Fab) have the same CDRs, clone 24 (IgG) and clone 12 (Fab) have the same CDRs, clone 25 (IgG) and clone 13 (Fab) have the same CDRs, clone 26 (IgG) and clone 15 (Fab) have the same CDRs. Thus, the data in Table 4 correlate the activity of Fab fragments with that of a complete antibody molecule.

p00193Thus, the present invention includes high power whole tetramer neutralizing antibodies in which said antibody has a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 37, 39, 41, 45, 47, 49, 43, 51 and 53, and a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52 and 54, preferably where said antibodies are the antibodies of clones 18 -26.

p00194Example 2

p00195Microneutralization test

p00196The 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 ["Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay, J. Clin. Microbiol. 22, 1050-1052 (1985)]. The procedure used here is described in Johnson et al [J. Infectious Diseases, 180, 35-40 (1999)]. Dilutions of the antibodies were made in triplicate using a 96-well plate. 10 TCID50 respiratory syncytial virus (RSV - long chain) were incubated with serial dilutions of the antibody (or Fabs) for testing for 2 hours at 37 ° in the wells of a 96-well plate. RSV-sensitive HEp-2 cells (2.5 x 104) were then added to each well and cultured for 5 days at 37 ° C in 5% CO2. 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. The fixed cells were incubated with a biotin-conjugated anti-protein monoclonal antibody F (pan F protein, Mab 133-1H site C specific) were washed and added to Avidin wells conjugated with horseradish peroxidase. The wells were washed again and the TMB (thionitrobenzoic acid) substrate renewal was measured at 450 nm. The neutralization titer was expressed as the concentration of antibody that caused at least 50% reduction in absorbance at 450 nm (OD450) from control cells containing only the virus.

SEQUENCE LIST

p00197<110> Young, James F. Koenig Scott

p001985 Johnson, Leslie S. Huse, William D. Wu, Herren Watkins, Jeffry D.

p00199<120> High potency recombinant antibodies and methods to produce them

10 <130> 469201-525

p00200<140>

p00201<141>

p00202<150> US 60 / 186,252

p00203<151>

p0020415 <160> 59

p00205<170> PatentIn Ver. 2.1

p00206<210> 1

p00207<211> 106

p00208<212> PRT 20 <213> Artificial Sequence

p00209<220>

p00210<223> Description of the Artificial Sequence: Variable region sequence of the light chain of humanized antibody Medi-493.

p00211<400> 1

p00212<210> 2

p00213<211> 120

p00214<212> PRT

p00215<213> Artificial Sequence

p00216<220>

p00217<223> Description of the Artificial Sequence: Variable region sequence of the heavy chain of humanized antibody Medi-493

p00218<400> 2

p00219<210> 3

p00220<211> 106

p00221<212> PRT

p00222<213> Artificial Sequence

10 <220>

p00223<223> Description of the Artificial Sequence: Variable region sequence of the light chain of a humanized antibody.

p00224<400> 3

p00225<210> 4

p00226<211> 120

p00227<212> PRT

p00228<213> Artificial Sequence

p00229<220>

p00230<223> Description of the Artificial Sequence: Variable region sequence of the heavy chain of a humanized antibody.

p00231<400> 4

p00232<210> 5

p00233<211> 10

p00234<212> PRT

p00235<213> Artificial Sequence

15 <220>

p00236<223> Description of the Artificial Sequence: Light chain CDR reference sequence.

p00237<400> 5

<dl><dt>20 </dt><dd><210><211><212> <213> 6 7 PRT Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

p00238<223> Description of the Artificial Sequence: Light chain CDR reference sequence.

p00239<400> 6

<dl><dt><210></dt><dd> 7 </dd></dl>

<dl><dt><211></dt><dd> 9 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: Light chain CDR reference sequence. </dd></dl>

<dl><dt><400></dt><dd> 7 </dd></dl>

<dl><dt><210></dt><dd> 8 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: CDR heavy chain reference sequence. </dd></dl>

<dl><dt><400></dt><dd> 8 </dd></dl>

<dl><dt><210></dt><dd> 9 </dd></dl>

<dl><dt><211></dt><dd> 16 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: CDR heavy chain reference sequence. </dd></dl>

<dl><dt><400></dt><dd> 9 </dd></dl>

<dl><dt><210></dt><dd> 10 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: CDR heavy chain reference sequence. </dd></dl>

<dl><dt><400></dt><dd> 10 </dd></dl>

<dl><dt><210></dt><dd> 11 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

p00240<223> Description of the Artificial Sequence: High power CDR sequence.

p00241<400> 11

<dl><dt><210></dt><dd> 12 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 12 </dd></dl>

<dl><dt><210></dt><dd> 13 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 13 </dd></dl>

<dl><dt><210></dt><dd> 14 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 14 </dd></dl>

<dl><dt><210></dt><dd> 15 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 15 </dd></dl>

<dl><dt><210></dt><dd> 16 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 16 </dd></dl>

<dl><dt><210></dt><dd> 17 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 17 </dd></dl>

<dl><dt><210></dt><dd> 18 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 18 </dd></dl>

<dl><dt><210></dt><dd> 19 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 19 </dd></dl>

<dl><dt><210></dt><dd> 20 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 20 </dd></dl>

<dl><dt><210></dt><dd> 21 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 21 </dd></dl>

<dl><dt><210></dt><dd> 22 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 22 </dd></dl>

<dl><dt><210></dt><dd> 23 </dd></dl>

<dl><dt><211></dt><dd> 9 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 23 </dd></dl>

<dl><dt><210></dt><dd> 24 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 24 </dd></dl>

<dl><dt><210></dt><dd> 25 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 25 </dd></dl>

p00242<210> 26

p00243<211> 16 <210> 31

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 26 </dd></dl>

<dl><dt><210></dt><dd> 27 </dd></dl>

<dl><dt><211></dt><dd> 16 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 27 </dd></dl>

<dl><dt><210></dt><dd> 28 </dd></dl>

<dl><dt><211></dt><dd> 16 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 28 </dd></dl>

<dl><dt><210></dt><dd> 29 </dd></dl>

<dl><dt><211></dt><dd> 16 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 29 </dd></dl>

<dl><dt><210></dt><dd> 30 </dd></dl>

<dl><dt><211></dt><dd> 16 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 30 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 31 </dd></dl>

<dl><dt><210></dt><dd> 32 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 32 </dd></dl>

<dl><dt><210></dt><dd> 33 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 33 </dd></dl>

<dl><dt><210></dt><dd> 34 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 34 </dd></dl>

<dl><dt><210></dt><dd> 35 </dd></dl>

<dl><dt><211></dt><dd> 106 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 35 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 36 120 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 36 </dd></dl>

<dl><dt><210><211><212><213></dt><dd> 37 450 PRT Artificial Sequence </dd></dl>

<dl><dt>5 </dt><dd><220> <223> Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 37 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 38 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 38 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 39 450 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 39 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 40 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 40 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 41 450 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 41 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 42 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 42 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 43 450 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 43 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 44 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 44 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 45 450 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 45 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 46 213 PRT Artificial Sequence </dd></dl>

<dl><dt>51 </dt><dd /></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 46 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 47 450 PRT Artificial Sequence </dd></dl>

<dl><dt>10 </dt><dd><220> <223> Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 47 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 48 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 48 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 49 450 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 49 </dd></dl>

<dl><dt><210><211><212><213></dt><dd> 50 213 PRT Artificial Sequence </dd></dl>

<dl><dt>5 </dt><dd><220> <223> Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 50 </dd></dl>

<dl><dt><210></dt><dd> 51 </dd></dl>

<dl><dt>10 </dt><dd> <211> 450 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 51 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 52 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 52 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 53 450 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Heavy chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 53 </dd></dl>

<dl><dt>5 </dt><dd><210><211><212> <213> 54 213 PRT Artificial Sequence </dd></dl>

<dl><dt><220> <223> </dt><dd>Description of the Artificial Sequence: Light chain of high potency antibody. </dd></dl>

<dl><dt><400></dt><dd> 54 </dd></dl>

<dl><dt><210></dt><dd> 55 </dd></dl>

<dl><dt><211></dt><dd> 16 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 55 </dd></dl>

<dl><dt><210></dt><dd> 56 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 56 </dd></dl>

<dl><dt><210></dt><dd> 57 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 57 </dd></dl>

<dl><dt><210></dt><dd> 58 </dd></dl>

<dl><dt><211></dt><dd> 7 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: High power CDR sequence. </dd></dl>

<dl><dt><400></dt><dd> 58 </dd></dl>

<dl><dt><210></dt><dd> 59 </dd></dl>

<dl><dt><211></dt><dd> 10 </dd></dl>

<dl><dt><212></dt><dd> PRT </dd></dl>

<dl><dt><213></dt><dd> Artificial Sequence </dd></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><223> </dt><dd>Description of the Artificial Sequence: Basic CDR Sequence.</dd></dl>

<dl><dt><400></dt><dd> 59 </dd></dl>