NZ626716B2 - Antibodies useful in passive influenza immunization - Google Patents

Abstract

Disclosed is a monoclonal antibody including a bi-specific antibody or immunoreactive fragment thereof, that binds to the same epitope of influenza A virus as does MAB579, MAB383, MAB699, MAB700, MAB708, MAB710, MAB711 or MAB723 as defined herein and that neutralises infection by H3 and H7, and which is human or humanised. Also disclosed is the use of the above-described antibody in the manufacture of a medicament for healing or preventing influenza infection in a subject. h is human or humanised. Also disclosed is the use of the above-described antibody in the manufacture of a medicament for healing or preventing influenza infection in a subject.

Description

ANTIBODIES USEFUL IN PASSIVE INFLUENZA IMMUNIZATION d Application This ation claims benefit of US. application Serial Number 61/5 67,046 filed December 2011 which is incorporated herein by reference in its entirety.

Technical Field The invention relates to the field of passive immunization against influenza. More particularly, ic antibodies that bind near to the HA0 maturation cleavage site consensus sequence of influenza hemagglutinin A, including antibodies secreted by human cells are described.

Background Art The hemagglutinin protein (HA) of influenza virus has a globular head domain which is highly heterogeneous among flu strains and a stalk region containing a fusion site which is needed for entry into the cells. HA is present as a trimer on the viral envelope. The uncleaved form of hemagglutinin protein (HA0) is ted by ge by trypsin into HA1 and HA2 portions to permit the fusion site to effect virulence. The two cleaved portions remain coupled using disulfide bonds but undergo a conformational change in the low pH environment of the host cell mal compartment which leads to fusion of the viral and host cell membranes.

The cleavage site contains a consensus sequence that is shared both by influenza A and influenza B and by the various strains of influenza A and B. The uncleaved hemagglutinin protein trimer (HA0) is referred to as the inactivated form, whereas when cleaved into HA1 and HA2 portions, the hemagglutinin protein is referred to as being in the activated form.

Bianchi, E., et al., J. Virol. (2005) 79:7380-7388 describe a “universal” influenza B vaccine based on the consensus sequence of this ge site wherein a peptide comprising this site was able to raise dies in mice when conjugated to the outer membrane protein complex of Neisseria meningitidis. Monoclonal dies which appear to bind to the consensus sequence were also described. In addition, successful passive transfer of antiserum was observed in mice. Other prior art es, such as those bed in W02004/080403 comprising peptides derived from the M2 and/or HA proteins of influenza induce dies that are either of weak efficacy or are not effective across strains. ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] m None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Antibodies described in the art which bind the HA stalk region involve those developed by Crucell, CR6261 and CR8020 described in Throsby, M., et al., PLoS One (2008) 3:e3942, Ekiert, D. C., et al., Science (2011) 333:843-850, and Sui, J., et al., Nat. Struct. M01.

Biol. (2009) 16:265-273. An MAB has also been developed against the conserved M2E antigen as described by a, A. G., et al., PNAS USA (2010) 107:12658-12663. M2E is on the surface of infected cells and is also the target of amantadine and adine. Drug resistance has ed against these antibiotics which suggests that this target does not serve an essential function.

An additional antibody has been described by the ecchia Group: Corti, D., et al., Science (2011) 0-856 which binds and neutralizes both Group 1 and Group 2 strains of influenza A, but the potency is not as high as those described herein as shown in the examples below. In addition, an MAB that is immunoreactive against both influenza A and B as described in Dreyfus, C., et al., Science (2012) 337: 1343-1348 has less potency than those described below.

PCT ation publication No. W0201 1/160083, incorporated herein by reference, describes monoclonal antibodies that are derived from human cells and useful in passive vaccines. The antibodies show high affinities of binding to influenza viral clade H1, which is in Group 1, and some of the antibodies also show high affinities to H9, also in Group 1 and/or to H7 in Group 2 and/or H2 in Group 1. Some of the antibodies disclosed bind only the inactivated trimer form, presumably at the consensus cleavage region, while others are able to bind activated hemagglutinin protein which has already been cleaved.

There remains a need for antibodies that bind additional clades and show enhanced affinity thereto.

Disclosure of the Invention The invention provides monoclonal dies that bind s representative of either or both Group 1 and Group 2 of influenza A with enhanced affinity. Such antibodies are able to confer passive immunity in the event of a pandemic caused, for example, by a previously unidentified influenza strain or a strain against which protection is not conferred by the seasonal vaccines currently available. As at least some of the antibodies bind across many strains, tive of targeting an essential site, they are likely to bind even previously untered strains. Such antibodies are also useful to ameliorate or prevent infection in subjects for whom vaccination failed to produce a fully protective response or who are at high risk due to a weak [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam immune system (e. g., the very young, the elderly, transplant patients, cancer or HIV herapy treated patients).

Thus, in one aspect, the invention is directed to binding moieties, notably monoclonal antibodies or immunoreactive fragments thereof that are y crossreactive with influenza A virus of Group 1 including H1, H2, H5, H6, H8, H9, H11, H13, H16 or Group 2 including H3 and H7 as type specimens, or that show cross-Group reactivity. Some of the antibodies illustrated below bind to an epitope contained in the HA0 protein ically and recognize the native trimeric form of HA, as well as the activated form.

Particularly important are bispecific antibodies and fragments thereof which are able to enhance the range of viral clades that can be bound ically.

As is well understood in the art, non-immunoglobulin based proteins may have similar epitope recognition properties as antibodies and can also provide suitable embodiments, including binding agents based on fibronectin, transferrin or lipocalin. c acid based moieties, such as rs also have these binding properties.

In other aspects, the invention is directed to methods to use the binding moieties of the invention for passively inhibiting viral infection in subjects that are already exposed to the virus or that are already infected. The invention is also directed to recombinant materials and methods to produce antibodies or fragments.

Brief ption of the gs Figure 1 shows the art-known classification of influenza virus into groups of significant clades.

Figure 2 shows the results of binding by MAB579 in vitro to various H7 and H3 strains, both representing Group 2.

Figures 3A and 3B show two lead mAbs, MAB53 (Group 1) and MAB579 (Group 2) have sub-nM affinity across clades ng the respective Groups.

Figures 4A and 4B st the lizing activity of MAB486 and MAB579. As shown in Figure 4A, MAB486, as well as a polyclonal preparation from rabbit immunization are ive in neutralizing HlNl only in the absence of trypsin. In contrast, Figure 4B shows that MAB579 is effective both in the presence and absence of trypsin.

Figure 5 is a m of the bispecific antibody that comprises the variable regions of MAB579 and MAB53.

Figures 6A-6E show the in vivo efficacy of MAB53, MAB579, es of these, and the bispecific antibody shown in Figure 5.

[Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam ation] amandam Unmarked set by amandam [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Modes of Carrying Out the Invention The present invention provides useful binding moieties, including antibodies and fragments thereof as well as effective means to identify cells that secrete such antibodies so that the relevant coding ces can be retrieved and stored for subsequent and facile inant tion of such antibodies.

The antibodies or analogous binding moieties of the invention are useful for both prophylaxis and therapy. Thus, they may be used to protect a subject against challenge by the virus as well as for ent of subjects that are already exposed or infected with influenza.

The subjects of most ultimate interest are human subjects and for use in human subjects, human forms or humanized forms of the binding moieties which are traditional l antibodies or immunoreactive nts thereof are preferred. However, the antibodies containing appropriate binding characteristics as dictated by the CDR regions when used in studies in laboratory animals may retain man characteristics. The antibodies employed in the studies of the examples below, although done in mice, nevertheless contain both variable and constant regions which are human.

The subjects for which the binding moieties including antibodies of the invention are useful in y and laxis include, in addition to humans, any subject that is susceptible to infection by flu. Thus, various mammals, such as bovine, porcine, ovine and other mammalian subjects including horses and old pets will benefit from the prophylactic and therapeutic use of these binding moieties. In on, za is known to infect avian species which will also benefit from compositions containing the antibodies of the invention.

Methods of use for prophylaxis and therapy are conventional and generally well known. The antibodies or other binding moieties are typically provided by injection but oral vaccines are also understood to be effective. Dosage levels and timing of administration are easily optimized and within the skill of the art.

Human cells that secrete useful antibodies can be identified using, in particular, the CellSpotTM method described in U.S. patent 7,413,868, the contents of which are incorporated herein by reference. Briefly, the method is able to screen individual cells obtained from human (or other) subjects in high hput assays taking advantage of labeling with particulate labels and microscopic observation. In one illustrative embodiment, even a single cell can be analyzed for antibodies it secretes by allowing the secreted antibodies to be adsorbed on, or coupled to, a surface and then treating the surface with desired antigens each coupled to a distinctive particulate label. The footprint of a cell can therefore be identified with the aid of a microscope.

Usifihis que, millions of cells can be screened for desirable antibody secretions and [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam even rare dies, such as those herein ble for passive influenza immunization across s can be recovered. Since human subjects have existing antibodies to at least some influenza strains, and since the antibodies obtained by the method of the invention bind a conserved sequence, these antibodies serve the purpose of addressing new strains as well as strains with which human populations have experience.

Methods to obtain suitable dies are not limited to the CellSpotTM technique, nor are they limited to human subjects. Cells that produce suitable antibodies can be identified by various means and the cells may be those of laboratory animals such as mice or other rodents.

The nucleic acid sequences encoding these antibodies can be isolated and a variety of forms of antibodies produced, including chimeric and humanized forms of antibodies produced by non- human cells. In addition, recombinantly produced antibodies or fragments include single-chain antibodies or Fab or Fabz regions of them. Human antibodies may also be ed using hosts such as the XenoMouse® with a humanized immune system. Means for tion of antibodies for screening for suitable binding characteristics are well known in the art.

Similarly, means to construct aptamers with desired binding ns are also known in the art.

As noted above, antibodies or other binding moieties may bind the activated form, the inactivated form or both of the lutinin protein. It is advantageous in some instances that the epitope is at the cleavage site of this protein as it is relatively conserved across strains, but preferably the binding moiety binds both the trimer and the activated form.

The cleavage site for various strains of influenza A and za B is known. For example, the above cited article by Bianchi, et (11., shows in Table l the sequence around the cleavage site of several such strains: Table l Consensus sequence of the solvent-exposed region of the influenza A and B virus maturational cleavage sites VWS/sumypesmmSequence ________________________________________ iqs‘éifngiéw i NIB/HA0 consensus Gib $52) AIHUHAO consensus $52515i253) i (($315 $54) B/HAo Consensusb 33:17ng l AD T.6)1 ‘1 The on of cleavage between HA1 and HA2 is indicated by the arrow.

The consensus is the same for both the Victoria and Yamagata lineages.

[Annotation] m None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam As indicated, strict consensus occurs starting with the arginine residue upstream of the cleavage site and thus preferred consensus ces included in the test peptides of the ion have the sequence RGI/L/F FGAIAGFLE (SEQ ID NO:57). It may be possible to use only a portion of this sequence in the test peptides.

As noted above, once cells that secrete the desired antibodies have been identified, it is straightforward to retrieve the nucleotide sequences encoding them and to produce the desired antibodies on a large scale recombinantly. This also s manipulation of the antibodies so that they can be ed, for example, as single-chain antibodies or in terms of their variable s only.

The retrieved nucleic acids may be physically stored and recovered for later recombinant production and/or the sequence information as to the coding sequence for the antibody may be ved and stored to permit subsequent synthesis of the appropriate nucleic acids. The availability of the information contained in the coding sequences and rapid synthesis and cloning techniques along with known methods of recombinant production permits rapid tion of needed antibodies in the event of a pandemic or other emergency.

For reference, the sequences of human constant regions of both heavy and light chains have been described and are set forth herein as SEQ ID NOS: 1-3. In the above- referenced WOZOl 1/160083, various monoclonal antibodies with variable regions of determined amino acid sequence and nucleotide coding sequences have been recovered that bind with varying degrees of affinity to HA n of various strains of influenza. The structures of variable regions, both light and heavy chains, of those of ular interest herein are set forth for convenience herein as SEQ ID NOS:22-25. These antibodies include MAB8 and MAB53. MAB53 and MAB8 bind with particular ty to H1; further, MAB53 binds tightly to H5, H7 and H9. MAB8 also binds H7 and H2. Neither of these antibodies binds strongly to H3, but MAB579 does bind H3 described herein. H7 and H3 are particularly attractive targets.

In more detail, each of these MABs binds to at least three different clades with reasonable or high affinity. MAB53 binds to HA0 from the H1, H9 and H7 clades and MAB8 binds to HA0 from H1, H7 clades and less strongly to and H3, as demonstrated by ELISA assay against HA0 n. The ties are in the nanomolar range. Reactivity to native trimer of HA from all the Group 1 clades was verified using HA expressed in HEK293 cells with antibody binding measured by flow cytometry.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by m [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam These results were confirmed using an alternative assay system, the biolevel interferometry based binding assay designated ForteBio® biosensor. As measured by this more accurate assay, the affinities are as follows: MAB53 / H1 = 60 pM, H5 = 6 nM, H7 = 70 pM, H9 = 30 pM; MAB8 / H1 = 9 nM, H3 =16 nM, H5 = 0.2 nM.

The additional specific antibodies identified in the present application, MAB383, MAB486, MAB579, MAB699, MAB700, MAB708, MAB710, MAB711 and MAB723 are represented by SEQ ID NOS:4-21 in terms of the amino acid sequences of their variable heavy chain and light chain. These antibodies bind with enhanced affinity to additional clades of influenza s. For example, MAB579 binds with high affinity to both H3 and H7. Thus, these antibodies add to the repertoire of antibodies useful in prophylaxis and ent of influenza.

Multiple technologies now exist for making a single antibody-like molecule that incorporates antigen specificity domains from two separate antibodies (bi-specific antibody).

Thus, a single antibody with very broad strain reactivity can be constructed using the Fab domains of individual antibodies with broad reactivity to Group 1 and Group 2 respectively.

Suitable technologies have been bed by enics (Rockville, MD), Micromet (Bethesda, MD) and Merrimac (Cambridge, MA). (See, e. g., Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, Wittrup KD. A modular IgG-scFv bispecific antibody topology, Protein Eng Des Sel. (2010) 23:221-228; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs. (2011) 1:3(3); Baeuerle PA, Reinhardt C. ific T-cell engaging antibodies for cancer therapy. Cancer Res. (2009) 69:4941-4944.) Thus, it is particularly useful to provide antibodies or other binding es which bind to multiple types of lutinin protein by constructing bispecific antibodies.

Particularly useful combinations are those that combine the binding specificity of MAB53 (H1, H5, H9) with MAB579 (H3, H7).

All of the antibodies of the present ion e at least one of the binding specificities of the newly disclosed antibodies described above. These may be combined with various other antibodies, including those that were described in the above-referenced WO201 83 as well as other members of the new group of antibodies disclosed . All of the possible combinations of such g specificities are within the scope of the present invention.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] amandam Unmarked set by amandam While MAB53 binds with high affinity to HA0, it does not bind HA1 implying binding to the complementary HA2 fragment, which binding was confirmed. As MAB53 does not bind to HA0 when tested by Western blot, it is assumed that the dominant epitope is at least in part mational. It was been found that MAB8 and MAB53 bind to the same or nearby epitopes as demonstrated by their ability to compete with each other for binding to the HA0 protein of the H1 clade.

All of the dies disclosed herein, including those previously disclosed in the above-referenced WO201 1/160083 bind to the native HA trimer expressed on the surface of HA transfected cells. This was verified using an HA-encoding plasmid ed by S. Galloway and D. auer of Emory University. That is, the trimer yed on the cell surface of the clades recognized by the various MAB’s of the invention is recognized by these MAB’s.

It was shown that MAB53 and MAB8 differ in that MAB8 is released from the HA0 protein when the pH is lowered to 6, whereas MAB53 is not. This difference is significant as it appears predictive of neutralizing capability. In tests for the ability to neutralize HlNl viral infection in a plaque reduction assay in MDCK target cells, low doses of MAB53 of l-5 ug/ml neutralized infection by HlNl, by H7N3, H5N1 and H9N2. r, MAB8 does not neutralize ion by these strains. Thus, neutralizing strains may be preferentially selected by washing bound MAB or fragment at pH 6 during the primary screen, thus removing from HA0 MAB’s that are unlikely to remain bound as the antibody-virus complex enters the cell via the endosomal compartment and thus will be expected to have reduced ability to neutralize the virus. For e, in the CellSpot method HA0 may be bound to solid support (fluorescent beads) and captured by the MAB or a mixture of MAB’s, then washed at pH 6.

It was also shown that mice pretreated with graded doses of MAB53 survive challenge with otherwise lethal titers of HlNl and H5Nl viruses with 100% tion against HlNl challenge. The y is comparable to a prior art antibody described by Crucell which does not show activity against Group 2 strains. Throsby, M., (supra) 3:e3942. The Crucell antibodies are heterosubtypic lizing monoclonal antibodies cross-protective against H5N1 and HlNl red from human IgM+ memory B cells. MAB53 also provided full protection at 10 mg/kg; 90% ed at 2 mg/kg and 50% survived at 0.4 mg/kg. Where challenge by H5Nl was substituted for challenge by HlNl, for MAB53, 10 mg/kg gave 80% survival; 2 mg/kg gave 60% survival and 0.4 mg/kg gave 50% survival. ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam MAB53 and antibodies that bind to the same epitope under the same conditions, i.e., then remain bound when the pH is lowered to 6, are effective as passive vaccines suitable for protection of populations against epidemics and pandemics, and for prophylactic or therapeutic use t seasonal influenza for patients with a weakened immune system.

Combinations of the epitope binding region of MAB53 with the high affinity binding epitopes of the antibodies of the present invention are particularly useful in ucting bispecific antibodies. This clearly permits, for example, effective binding of H7, H3 and H1 in the same antibody when MAB579 binding regions are included in the dy. This is shown in Table 2 which provides the IC50’s for various strains of influenza hemagglutinin protein shown by MAB579.

Table 2 MAB579 IC50 values for various flu strains Subtype Strain IC50 (ug/ml) H3A/Perth/16/200902flflflflflflflflflflflflflfl lipines/2/82 X-79 0.9 AfUdorn/307/1976 1.9 A/New York/55/2004* 1.1 A/Wisconsin/67/2005 1.0 A/HongKong/68 2.8 A/SW/MN/02719 3.9 H4 A/Bufflehead 15.5 H7 A/Canada/rv444/04 1.6 A/Netherlands/219/03 0.6 A/Sanderling/A106- 125 >20 A/Redknot/NJ/1523470/06 >20 vii; A/Ruddy one/A106-892 >20 H10 A/Northern Shoveler 0.8 These values were obtained in the MDCK monolayer microneutralization assay. A graphical representation of the affinity of MAB 579 for various strains is also shown in Figure 2.

As shown, while H3 and H7 are y bound, negligible binding affinity is found for H1.

Thus, it is ularly advantageous to combine the binding region of MAB579 with that of an MAB with high binding to H1. In this case, then, both Group 1 and Group 2 are represented.

One embodiment of the invention includes a biospecific antibody that binds both the epitope bound by MAB53 and that bound by MAB579.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] m MigrationNone set by amandam [Annotation] amandam ed set by amandam In addition to bispecific antibodies per se, the invention contemplates the use of the heavy chain only in constructs for neutralization of viral infection; such antibodies may also be bispecific. It is understood in the art that specificity is mostly conferred by the heavy chain variable regions, and in some stances, heavy chains alone have been successful as active ingredients in vaccines. atively, the heavy chain of appropriate specificity may be associated with s forms of light chain to enhance the ty or y to neutralize virus.

It is particularly noted that the CDR3 region of the heavy chains of the antibodies described herein is extended and contains multiple tyrosine es. It is understood that such tyrosine residues may be sulfonated as a posttranslational event. Thus, also part of the invention are es which comprise the CDR3 regions of the heavy chains of MAB579, MAB699, MAB700, MAB708, MAB710, MAB711 or MAB723 wherein one or more of the tyrosine residues in said region is optionally sulfonated. These s with or without sulfonation may also be used alone as passive vaccines. The sulfonation of the CDR3 region is consistent with criteria for sulfonation as described by tti, F., et al., Bioinformatics (2002) 18:769-770.

Other instances where CDR3 regions of heavy chains have been used successfully alone in neutralization of viral infection are described in Pej chal, R., et al., PNAS (2010) 107:11483-11488 and by Liu, L., et al., J. Virol. (2011) 85:8467-8476.

As used herein, the term “antibody” includes immunoreactive fragments of traditional antibodies even if, on occasion, “fragments” are mentioned redundantly. The antibodies, thus, include Fab fragments, FV single-chain dies which contain a substantially only variable regions, bispecific antibodies and their various fragmented forms that still retain immunospecificity and proteins in general that mimic the activity of “natural” antibodies by comprising amino acid sequences or modified amino acid sequences (i.e., pseudopeptides) that approximate the activity of variable regions of more traditional naturally occurring antibodies.

Antibody ures These are presented in the following order: 1. Amino acid ces of the constant region of human IgG1 heavy chain, human constant kappa and human constant lambda; 2. Heavy and light chain amino acid sequences of the variable regions of the heavy and light chains of MAB 383, 486, 579, 699, 700,708, 710, 711 and 723 (The CDR s are underlined in MAB’s 579, 699, 700, 708, 710, 711 and 723.); [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam 3. Heavy and light chain variable region amino acid sequences of MAB8 and MAB53 described in W02011/160083 (The LC sequences shown in ‘083 also contained constant region and this has been deleted); 4. Nucleotide sequences encoding the constant region of human IgG1 heavy chain, human constant kappa and human constant lambda; . Nucleotide sequences encoding heavy and light chain amino acid sequences of the le regions of the heavy and light chains of MAB 383, 486, 579, 699, 8, 710, 711 and 723; 6. Nucleotide ces encoding heavy and light chain variable region amino acid sequences of MAB8 and MAB53.

With respect to the indicated CDR regions, it should be noted that there is more than one system for identifying CDRs. Most frequently used is the Kabat system originally set forth in Wu, T. T., et al., J. Exp. Med. (1970) 132:211-250. Kabat is a widely adopted system which identifies specific positions as associated with CDRs. An additional system, the a ing scheme provides slightly different results. It is bed in Al-Lazikani, B., et al., J. Molec. Biol. (1997) 273:927-948. Depending on which system is used, slightly ent results for CDRs are indicated. For e, in MAB53 the heavy chain CDR according to Kabat is KYAIN whereas the Clothia system designates GG RKYA N. The heavy chain CDR2 region has an additional G at the N-terminus and the CDR3 an additional AR at the N-terminus. For the light chain, the CDR designations are identical in both systems.

Some criticism has been leveled at both systems by various workers; ore, it is tood that the CDR regions as designated herein and in the claims may vary slightly. As long as the resulting variable regions retain their binding ability, the precise location of the CDR regions is not significant, and those regions designated in the claims are to be considered to include CDRs identified by any accepted system.

Human IgGl HC amino acid sequence of constant region {SEQ ID NO:1) ASTKGPSVFPZJVPSSKSTSGGTAALGCLVKDYFPEPVTVS"\TSGALTSGVHTFPAVL QSSGZJYSLSSVVTVPSSSLGTQTYICWVN {KPSWTKVDKKVEPKSCDKTHTCPPCPA 1’ '.T .GGPSVE'ZJE'PL’KPKDL .M S {11’ * '.V1 S -l 4.31) * '.VK1:'\TWYVDGVEV {NAK 1K1’ {*2 fiQYNS 1Y<VVSV7 QDWT.\TGK7.YKCKVS\TKAT.1’AP * K'.1 SKAKGQB {j PQVYTZJPPSRD7.7 .TKNQVS JTCLVKGb'YES) AV*'.W*'.S\TGQ1’_L'N\TYK1 11’1’VL DSDG SFFZJYSKLTVDKSRWQQGNVFSCSVM-l7A .{NHYTQKSoSLSPGK [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam ation] amandam ed set by m [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Human LC amino acid seguence of constant kappa region {SEQ ID N02) {JJVAAPSVJ:'-J:-'BBS3.L'QLKSGTASVVCLLNWFYPRILAKVQWKV3NALQSGNSQESVT LiJ Q3SK3STYSLSSTLTLSKA3YILKHKVYACILVTHQGLSSPVJKSb'N{GL'C Human LC amino acid seguence of constant lambda region (SEQ ID NO:31 GQPKAABSVJ Lb'b’b’ S S LT .QANKAT‘ .VCT " S 3FYPGAVTVAWKA3S SPVKAGVETTT.

P SKQSNNKYAAS SYT . S‘ .TP'TQWKSHRSYSCQVJ H LGS JV L'K JVVPAL'CS MAB383 HC Amino acid seguence of variable domain (SEQ ID NO:4) QVQLVQSGALVKRPGASVKVSCRASGYJJL J SJ:' GJ:' SWVRQAPGQG HZWMGW SAYNG3 TKSPQKLQCRVJMJ J 3i S JNJAYMLTRST. S33JAVYYCARAPPLYYSSWSS3YWGQ GTLLTVSS MAB383 LC Amino acid sequence of le domain (SEQ ID NO:51 3--Q JQSPGJT.ST.SBG:'.RAJ- .SCRASQSVSSNYLAWYQQKHGQAPRP FYGASRRAT 3VP3RFSGSGSGJ3J:'JHJ S{THUR:".3J:AVYYCQQYGSSPRJJ:GQGJK K MAB486 HC Amino acid seguence of variable domain (SEQ ID NO:61 QVQ- GGMVQPGGSRRLSCAASGFSFSTYGMHWVRQAPGKG‘ HZWVAV SY3G:'.K QYYL3SVKG {J:' J-S.-R3NSK3TLYLQMNSLTAL3TAVYYCVK:' SA{{T.T.RYJ:' LW‘ .T.SS PF3\l 'GQGA.LVTVSS MAB486 LC Amino acid seguence of variable domain (SEQ ID NO:7} 3:V TQSP3ST.AVST.G:'.RAJ NCKSSQTVLYTSNKKNYLAWYQQKPGQPPKT.-."YWA STRESGVP3RFSGSGSGT3FTT.T"SST.QAT.3VAVYYCQQYYJSPYJb'GQGJK'.:'. K MAB579 HC Amino acid seguence of variable domain (SEQ ID NO:8) QVQLVQSGALVKKPGASVKVSCK J SGY J J:' JAY J -- HWVRQAPGQ {L LWMGW NAGNG J KYSQRJ:'KG {VJ LT .RST . J S :23 JAT .Yb'CARGRL' J YYY3KTNWLNS -- J R3 J SARJ JYlVl P3ILYFQHWGJGTQVTVSS MAB579 LC Amino acid sequence of le domain (SEQ ID NO:91 3:- QMTQSPSTLSASVG3RVJ -- JC {ASQJ NNY‘ .AWYQQKPGKAPKLL-_YKASSL.L-ti U) G) GSGSGJ : 'J:' JT.J SST.QR33J:'AJYYCQ.L'YNN3SPLJJ:'GGGJKVL K MAB699 HC Amino acid seguence of variable domain (SEQ ID N010) QLQLVQSGA.LVKKPGASVKL Y J J:' J SY J LHWVRQAPGQ J T. LWMGW \lAGNGK J KYBPKJLRG {VJ - J R3 J SAJ JV3MHLS SL J S .L'3 JAVYJ:'CARGRL'SYYY3RS3WLNSH P3ILYFQYWGQGTLV--VSS MAB699 LC Amino acid seguence of variable domain (SEQ ID NO:ll} 3:- STLSASVG3RVJ --AC {ASQS"SSW .AWYQQKPGKAPKT.‘ ."YKASQLIL-lJ U) G) VPSRFSGSGSGJ : '.J:' JT.J NST.QB33J:'AJYYCQLYNVYSPLJb'GGGJ {V3--K [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam ed set by amandam MAB700 HC Amino acid sequence of variable domain (SEQ ID N012) QVQLVLSGADVKKPGASVTVSCKASGYIPRSPIMHWVRQVPGQ {L 4WMGW NAGNGK TKYSQKb'QG {V__VIRDISASIAYM T‘IITITISSTIES IDIAVYYCARGPETYYY_. DSSNWLNSH PD,LYLQYWGQGTPVTVSS MAB700 LC Amino acid sequence of le domain (SEQ ID NO:132 HTQSPSTLSASVGDRVIL IC {ASQST SSWT .AWYQQKPGKAPKLL"YKASILH4 VP SQFSGSGSGI 4PILI SSLQPDDPAIYYCQLYNNNSPLIPGGGIKV2_. K MAB708 HC Amino acid sequence of variable domain (SEQ ID N014) SGADVKRPGASVTVSCKASGYIPRSPIMHWVRQVPGQ {L4WMGW NAGNGK b'QG {V__VIRDISANIAYM T‘IITITISSTIES IDIAVYYCARGPETYYY_. DSSNWLNSH QHWGQGTPVTVSS MAB708 LC Amino acid sequence of variable domain (SEQ ID NO:l5) 3T- QMTQSPSTLPASVGDRVIL IC {ASQST SSWT .AWYQQKPGKAPKLL-YKASSLL-Ei GSGSGI *'EILI SSLQPDDPAIYYCQLYNNNSPLIb'GGGIKV'._. K MAB710 HC Amino acid sequence of variable domain (SEQ ID N016) QVQT.Q 4SGA 4VKKPGASVQVSCKASGYTFTSYSVHWVRQAPGQ {P4WMGW WAGNGK IKYPQKh'KG {VI__ IRDILARIVN HT.SST.TS .DTAVYFCARGP-—I_. DSYYYDRNDWLNSH PD,LYFQHWGQGTVV-_VSS MAB710 LC Amino acid sequence of variable domain (SEQ ID NO: 17) 3T-VMTQSPSTLSASVGDRVIL SC {ASQS TTDSW .AWYQQKPGKAPKT |T|__ YKASNLlJ4 VPSRFSGSGSGI *'EILI SSLQPDDE'AIYYCQ.YNVHL IPGGGI{VTD" K MAB7ll HC Amino acid sequence of variable domain (SEQ ID N018) QVQTV 4SGA 4VKKPGASVK IC 4ASGYIPNIYI" PGQ {L4WMGW NAANG IKYSRKLRS {VinKRDISARISYM 4LSSLGS -—I_..DTAVYYCARGPETYYFDKTNWLNS PD,LYFQiWGQGTLVTVSS MAB7ll LC Amino acid sequence of variable domain (SEQ ID N019) 3T-VMTQSPSTLSASVGDPVIL IC {ASQST STWT .AWYQQKPGKAPKLL-_YKASNLti.4 VPARFSGSGSGI *EILI SSLQPDDPAIYYCQELYNNDSPL__LGGGIIV'._. K MAB723 HC Amino acid sequence of variable domain (SEQ ID N020) QVQLVQSGAAVNKPGASVKVSCKASGYSPISYILHWVRQAPGQ {P4W GW NAGNGK VKYPRKTQGR I IRDVSAIIVHM TTTITIRSTIJ. S _.IDIGLYYCARGPLSYFFDTSNHLNSH P 3,LYFQFWGQGTLVTVSS MAB723 LC Amino acid sequence of variable domain (SEQ ID N021) 3T STLSASVGDRVIL IC {ASQST SSYT .AWYQQKPGKAPKLLTYKASNLlJ4 GSGSGI *E'ILI SSLQPDDPAIYYCQLYNNNSPLIPGAGIKV2_. K [Annotation] amandam None set by amandam ation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam MAB8 HC amino acid seguence of variable domain (SEQ ID N022) LVQTWV*SGGGTVKPGGSLRLSCAASGEIbSIYIMSWVRQAPGQGL*WVSS IRISSN :YYADSV*GREI SRDNAKNSLYLQMHSLRV_LDTAVYYCAR: SGVVGPVPFDYWGQG TLITVSS MAB8 LC amino acid seguence (SEQ ID N023) DIQMTQSPSSLSASVGDRVILICKASQILSKYLNWYQQKPGRAPKLL:YSASSLQSG VPSRFTGSGSGIDEILI DFAIYYCQQSYRBSQ"IFGBGIKVDLK MAB53 HC amino acid sequence of variable domain (SEQ ID NO:24) QVQLVQSGALVRKPGSSVKVSCKVSGG KKYA NWVRQAPGQGLLW GG A FNI ANYAQKFQGKVI SIVYM*TSSLKSLDIALYYCARGMNYYSDYFDYWGQGS LVTVSP MAB53 LC amino acid seguence (SEQ ID N025) L VHIQSPGILSLSPGLRAIUSCRASQSVRSNNLAWYQHKPGQAPRLL__FGASSRAI PDRFSGSGSGIDFIHI DbAVYYCQQYGSSPALIFGGGIKVL K Human IgGl HC nucleotide seguence of constant region (introns are underlined) (SEQ ID N026} GCCTCCACCAAGGGCCCATCAGTCTTCCCCCTGGCACCCTCTACCAAGAGCACCTCT GGGGGCACAACGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCT CAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGCAGCAAGGC AGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGA GAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAA CCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATAT CCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCC TCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGC AGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGC CCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGIGCCCIAGAGIAGCCIGCAICCA GGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTG AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA TGATCTCCCGGACCCCIGAGGICACAIGCGIGGIGGIGGACGTGAGCCACGAAGACC CIGAGGICAAGIICAACIGGIACGIGGACGGCGTGGAGGTGCATAATGCCAAGACAA AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGC GAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCT GTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC [Annotation] amandam None set by m [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAG CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGICIICICAIGCICCGIGAIG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA Human LC nucleotide seguence of constant kappa region {SEQ ID N027) CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA TCTGGAACIGCIAGCGIIGIGIGCCIGCIGAAIAACIICIAICCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Human LC nucleotide seguence of constant lambda region (SEQ ID N028) GGTCAGCCCAAGGCTGCCCCCTCTGTCACTCTGTTCCCGCCCTCTAGCGAGGAGCTT AACAAGGCCACACIGGIGIGICICAIAAGIGACIICIACCCGGGAGCCGTG ACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACA CCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCT GAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACC GTGGAGAAGACAGTGGTCCCTGCAGAATGCTCT MAB383 HC Nucleotide seguence of variable domain (SEQ ID N029) CAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAGGCCTGGGGCCTCAGTGAAG GTCTCCTGCAGGGCTTCTGGTTACACCIIIACIAGCIICGGIIICAGCIGGGIGCGA CAGGCCCCAGGACAAGGGCIIGAGIGGAIGGGGIGGAICAGCGCTTACAATGGTGAC ACAAAGTCTCCACAGAAGCTCCAGGGCAGAGTCACCATGACTACAGACACATCCACG AACACAGCCTACATGGAGCTGAGGAGCCTCATATCTGACGACACGGCCGTGTATTAT TGTGCGAGAGCCCCCCCCCIGIAIIACAGIAGCIGGICCICAGACTACTGGGGCCAG GGAACCCTGCTCACCGTCTCCTCA MAB383 LC tide seguence of variable domain (SEQ ID N030) GATATCCAGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTCAGTAGCAACTACTTAGCCTGGTACCAG CAGAAACATGGCCAGGCTCCCAGGCCCCTCATCTACGGTGCATCCAGAAGGGCCACT CCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGGAACCTGAAGAIIIIGCAGIGIAIIAIIGICAGCAGTATGGTAGTTCA CCTCGAACTTTTGGCCAGGGGACCAAACTGGAAATCAAAC MAB486 HC Nucleotide sequence of variable domain (SEQ ID NO:312 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCATGGTCCAGCCGGGGGGGTCCCGGAGA CTCTCCTGTGCAGCCICIGGAIICAGCIICAGIACCIAIGGCAIGCACIGGGICCGC CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTCATATGATGGAGAAAAG CAAIAIIAICIAGACICCGIGAAGGGACGATTCACCATCTCCAGAGACAATTCCAAG GACACCCTCTATCTGCAAATGAACAGTCTGACAGCTGAGGACACGGCTGTGTATTAC [Annotation] m None set by amandam [Annotation] amandam ionNone set by m [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam AAGGAATCAGCGCGICGAIIAIIACGAIAIIIIGAGIGGIIAIIAAGIICG CCTTTTGACAACTGGGGCCAGGGAGCCCTAGTCACCGTCTCCTCA MAB486 LC Nucleotide ce of variable domain (SEQ ID N032) GATATCGTGATGACCCAGTCTCCAGACICCCIGGCIGIGICIIIGGGCGAGAGGGCC ACCATCAACTGCAAGTCCAGCCAGACTGTTTTATACACCTCCAACAAGAAAAATTAC TTAGCCTGGTACCAACAGAAGCCAGGGCAGCCICCIAAACIGCICAIIIACIGGGCA TCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAG CAAIAIIAIACGICICCCIACACATTTGGCCAGGGGACCAAGCTGGAGATCAAA MAB579 HC Nucleotide sequence of variable domain (SEQ ID NO:332 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTTTCCTGCAAGACTTCTGGATACACCTTCACAGCCIAIACIAIACACIGGGIGCGC CAGGCCCCCGGACAAAGGCIIGAGIGGAIGGGAIGGAICAACGCTGGCAATGGTCAC ACGAAATATTCACAGAGGTTCAAGGGCAGAGTCACCATTACCAGGGACACATCCGCG AGGACAACCTACATGGAGCTGCGCAGTCTGACATCTGAGGACACGGCTCTATATTTC TGTGCGAGAGGGCCCGAGACATATTATTATGATAAAACCAAIIGGCIGAACICCCAI CCAGATGAATACTTCCAGCACTGGGGCCACGGCACCCAGGTCACCGTCTCCTCA MAB579 LC Nucleotide seguence of variable domain (SEQ ID NO:34) GATATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCCAGTCAGACIAIIAAIAACIACIIGGCCIGGIAICAGCAG GGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGG GTCCCATCAAGATTCAGTGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGC AGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGAATATAATAATGATTCT ACTTTCGGCGGAGGGACCAAAGTGGAGATCAAA MAB699 HC Nucleotide sequence of variable domain (SEQ ID NO:352 CAGGTGCAGCTGGTGCAGTCCGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG CTTTCCTGCAAGGCTTCTGGGTACACCIICACIICCIAIACICIACAIIGGGIGCGC CAGGCCCCCGGACAGACACIIGAGIGGAIGGGAIGGAICAACGCTGGCAACGGTAAA ACAAAATATCCACCGAAGTTCAGGGGCAGAGTCACCATTACCAGGGACACGTCCGCG ACCACAGTCGACATGCATCTAAGCAGCCTGACATCTGAAGACACGGCTGTGTATTTC TGTGCGAGAGGGCCCGAAAGTTATTACTATGATAGAAGIGAIIGGCIGAACICCCAI CCAGAIGAAIACIICCAGIACIGGGGCCAGGGCACCCTGGTCATCGTCTCCTCA MAB699 LC Nucleotide seguence of variable domain (SEQ ID NO:362 GATATCGTGCTGACGCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGGGACAGAGTC ACCATCGCTTGCCGGGCCAGTCAGAGTATTAGCAGCTGGCTGGCCTGGTATCAGCAG AAACCAGGGAAAGCCCCTAAACTCCTGATCTACAAGGCGTCTCAGTTAGAAAGTGGG GTCCCATCAAGATTCAGCGGCAGCGGATCTGGGACAGAGTTCACTCTCACCATCAAC AGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACIIIAIAAIGIIIAIICI CCGCTCACTTTCGGCGGGGGGACCAGGGTGGACATCAAA [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam ed set by amandam MAB700 HC Nucleotide seguence of variable domain (SEQ ID NO:37} CAGCTGGTGGAGTCTGGGGCTGACGTGAAGAAGCCTGGGGCCTCAGTGACG GTTTCCTGCAAGGCCTCAGGATACACCTTCAGGAGIIIIACIAIGCAIIGGGIGCGC CAGGTCCCCGGACAAAGGCIIGAGIGGAIGGGAIGGAICAACGCTGGCAATGGTAAA ACAAAGTATTCTCAGAAGTTCCAGGGCAGAGTCATCGTTACCAGGGACACATCCGCG GCCTACATGGAGCTGAGCAGCCTAACATCTGAAGACACGGCTGTTTATTAC AGAGGGCCCGAAACAIAIIACIAIGAIAGIAGIAAIIGGCIGAAIICCCAI CCAGAIGAAIAICICCAGIACIGGGGCCAGGGCACCCCGGTCACCGTCTCCTCA MAB700 LC Nucleotide sequence of variable domain (SEQ ID NO:38) GATATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCGTCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCCAGTCAGAGIAIIAGIAGCIGGIIGGCCIGGIAICAGCAG AAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTACTTTAGAAAGTGGG GTCCCATCCAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGC AGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGAGTATAATAATAATTCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA MAB708 HC Nucleotide seguence of variable domain (SEQ ID N039) CAGGTGCAGCTGGTGCAGTCTGGGGCTGACGTGAAGAGGCCTGGGGCCTCAGTGACG TGCAAGGCTTCAGGATACACCTTCAGGAGCIIIACIAIGCAIIGGGIGCGC CAGGTCCCCGGACAAAGGCIGGAGIGGAIGGGAIGGAICAACGCTGGCAATGGTAAA ACAAAATATTCCCAGAAGTTTCAGGGCAGAGTCATCGTTACCAGGGACACATCCGCG AACACGGCCTACATGGAGCTGAGCAGCCTGACATCTGAAGACACGGCTGTTTATTAC TGTGCGAGAGGGCCCGAAACAIAIIAIIAIGAIAGIAGIAAIIGGCIGAACICCCAI CCAGATGAATATTTCCAGCACTGG MAB708 LC Nucleotide seguence of variable domain (SEQ ID NO:40) GATATCCAGATGACCCAGTCTCCTTCCACCCTGCCTGCGTCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCCAGTCAGAGIAIIAGIAGCIGGIIGGCCIGGIAICAGCAG AAACCAGGGAAAGCCCCTAAACTTCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGG GTCCCATCCAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGC AGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGGAGTATAATAATAATTCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA MAB710 HC Nucleotide sequence of variable domain (SEQ ID NO:4l) CAAGTGCAGCTGCAGGAGTCGGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGCAG GTTTCCTGCAAGGCTTCTGGGTACACCIICACGICCIAIAGCGIACAIIGGGIGCGC CAGGCCCCCGGACAAAGGCCIGAGIGGAIGGGAIGGAICAACGCTGGCAACGGAAAG ACAAAATATCCACAGAAGTTCAAGGGCAGAGTCACCATAACCAGAGACACATTAGCG CGCACTGTCAACATACATCTAAGCAGCCTGACATCCGAAGACACGGCTGTGTATTTC TGTGCGAGAGGGCCCGATAGTTATTACTATGATAGAAAIGALIGGCIGAACICCCAI CCAGATGAATACTTCCAGCACTGGGGCCAGGGCACCGTGGTCATCGTCTCCTCA [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by m MAB710 LC Nucleotide seguence of variable domain (SEQ ID N042) GATATCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTGGGAGACAGAGTC ACCATCTCTTGCCGGGCCAGTCAGAGTATTGACAGIIGGIIGGCCIGGIAICAGCAG AAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTAATTTAGAAAGTGGG GTCCCATCAAGATTCAGCGGCAGCGGATCTGGGACAGAATTCACTCTCACCATCAGC AGCCTGCAGCCTGATGATTTTGCGACTTATTACTGCCAACICIAIAAIGIICAIIIG ATCACTTTCGGCGGAGGGACCAGGGTGGACATCAAA MAB711 HC Nucleotide sequence of variable domain (SEQ ID NO:432 CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG ATCACCTGCGAGGCIICIGGAIACACIIICAAIACCIAIACIAIACAIIGGCIGCGC CAGGCCCCCGGACAAAGACIIGAGIGGAIGGGGIGGAICAACGCTGCCAATGGTCAT ACAAAATATTCACGGAAGCTCAGGTCCAGAGTCACCATTAAGAGGGACACATCCGCG AGGACAAGTTACATGGAGCTGAGCAGCCTGGGATCTGAAGACACGGCTGTCTATTAC TGTGCGAGAGGGCCCGAAACATATTACTTTGATAAGACGAAIIGGCIGAACICCCAI CCAGATGAATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA MAB711 LC Nucleotide seguence of variable domain (SEQ ID NO:44) GATATCGTGATGACGCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCCAGTCAGAGIAIIICIACCIGGIIGGCCIGGIAICAGCAG AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCCAATTTAGAAAGTGGG GTCCCAGCAAGATTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGC AGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGAATATAATAATGATTCT CCGCTGATTTTAGGCGGAGGGACCACGGTGGAGATCAAA MAB723 HC Nucleotide ce of variable domain (SEQ ID NO:45) CAGGTGCAGCTGGTGCAGTCTGGGGCTGCGGTGAACAAGCCTGGGGCCTCAGTGAAG GTTTCCTGCAAGGCTTCTGGATACAGCIICACIAGIIACACIIIGCAIIGGGIGCGC CAGGCCCCCGGACAAAGGCCIGAGIGGAIAGGGIGGAICAACGCTGGCAATGGTAAA GTAAAATATCCACGGAAGTTGCAGGGCAGAATCACCATAACCAGGGACGTATCCGCT ACGACAGTTCACATGGAACTGAGGAGCCTGACATCTGAGGACACGGGTCTATATTAC TGTGCGAGAGGGCCCGAAAGIIACIICIIIGAIACIICIAAICAICIGAACICCCAI CCAGAIGAAIACIICCAGIICIGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA MAB723 LC Nucleotide ce of le domain (SEQ ID NO:46) GATATCGTGCTGACGCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCCAGTCAGAGIAIIAGIAGIIACIIGGCCIGGIAICAACAG AAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTAATTTAGAAAGTGGG GTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGC AGCCTGCAGCCTGATGATTTTGCAACTTATTATTGCCAAGAATATAATAATAACTCT CCGCTCACTTTCGGCGCAGGGACCAAGGTGGAGATCAAA [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam MAB8 HC variable domain nucleotide seguence (SEQ ID NO:47} CAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCICIGGIIICACIIICAGIACCIAIACIAIGAGIIGGGICCGC CAGGCTCCAGGGCAGGGGCIAGAGIGGGICICGICCAIIACIAGGACTAGTAGTAAT ATATACTACGCAGACTCAGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG AACTCACTGTATCTGCAGATGCATAGCCTGAGAGTCGAAGACACGGCTGTGTATTAC TGTGCGAGAATCAGCGGGGTAGTGGGACCIGICCCCIIIGACIACIGGGGCCAGGGA ACCCTGATCACCGTCTCCTCT MAB8 LC variable domain nucleotide sequence (SEQ ID NO:48) GACATCCAGATGACCCAGICICCAICIICCCIGICIGCAICIGIAGGAGACAGAGTC ACCATCACTTGCCGGGCAAGTCAGACCATTAGCAAGTATTTAAATTGGTATCAGCAG GGGAGAGCCCCIAAACICCIGAICIACICIGCGICCAGIIIGCAAAGTGGG GTCCCATCAAGGTTCACTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACC AGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGACCCTCC CAGATCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA MAB53 HC variable domain tide seguence (SEQ ID N049) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCGGGGTCCTCGGTGAAG GTCTCCTGCAAGGTTTCTGGAGGCATCATTAGGAAAIAIGCIAICAACIGGGIGCGA CAGGCCCCCGGACAAGGGCTTGAGTGGATGGGAGGGAICAICGCIAICIIIAAIACA GCAAACTATGCACAGAAATTCCAGGGCAGAGTCACGATTACCGCGGACGAGTCCACG AGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCCTTTATTAC TGTGCGAGAGGAAIGAAIIACIACAGIGACIACIIIGACIACIGGGGCCAGGGAAGC CTTGTCACCGTCTCCCCA MAB53 LC variable domain nucleotide seguence (SEQ ID N050) GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGAAGCAACAACTTAGCCTGGTACCAG CACAAACCTGGCCAGGCTCCCAGGCICCICAICIIIGGIGCAICCAGCAGGGCCACT GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGGAGCCTGAAGAIIIIGCAGIAIAIIACIGICAGCAGTATGGTAGCTCA CCTGCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA The following examples are offered to illustrate but not to limit the invention.

Example 1 Affinity of MAB53 and MAB579 The affinity of MAB53 was ed in the above cited PCT publication. This dy binds HA strongly from clades H5, H7, H1 and H9, with less ty for H2 and H3.

MAB579 binds HA with high affinity with respect to H7 and H3. Figures 3A and 3B show typical results using the standard ForteBioTM assay for each antibody.

[Annotation] amandam None set by amandam [Annotation] amandam ionNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam ionNone set by m [Annotation] amandam Unmarked set by m Neutralization of Infection by MAB486 and MAB579 MAB’s 486 and 579 were tested for inhibition of infection by H1N1 and H3N2 (A/Perth/16/2009) and plaque formation in MDCK cell monolayers in the presence or absence of trypsin in the initial infection phase. MAB486 and pAb XCP (a rabbit polyclonal raised t the cleavage site consensus sequence) neutralize H1N1 (A/California/04/2009) only in the absence of trypsin as shown in Figure 4A, and are unable to inhibit infection and plaque formation if the virus is first activated with trypsin. This shows that antibodies directed to the fusion region with epitopes relying on intact fusion peptide (i.e., protease tible) are not as effective in controlling viral infection. As shown in Figure 4B, MAB579 inhibits infection in both the presence and absence of trypsin.

The ability of MAB53 to neutralize infection was previously reported, but a comparison of the affinities and EC50 for in vitro neutralization are compared to those for the Crucell monoclonal antibodies CR6261 in Table 3 below.

Table 3 Potency in vitro of Trellis mAbs vs. mAbs cloned from Crucell patents MAB53 MAB53 1” Strain Potency KD (nM) EC50 (ug/mL) EC50 (ug/mL) Difference ’fii’iiiiWX/Eiiiéilé’é””””””””””””””””””””””””6Ii”””””””””””””0144030x”””””” H5N1 ANN/1204 0.5 0.10 3.7 40X H2N2 A/Mallard/MN/2008 nd 1.20 nd H9N2 Mallard/MN/98 nd 0.10 nd MAB579 MAB579 “CR8020” Strain Potency KD (nM) EC50 ) EC50 (ug/mL) Difference """A"Ev};365557655663“”35""""""""""""""""i6""""""""""""""""""""3T5"""""""""""""""""3;"""""" H3l\2 H3N2 A/Perth/16/2009 0.8 0.05 2.0 40X H3N2 A/New 5/2004 0.2 2.0 10.0 5X H3l\'2 A/Hong Kong/8/68 0.2 2.0 7.6 3X H7l\'7 A/Netherlands/219/03 0.4 0.7 13.1 20X H7l\'3 A/Canada/rv444/04 0.6 0.5 nd H4l\'4 A/Bufflehead nd 15.0 >40 3x H10N7 A/Northern Shoveler 0.8 The values for EC50 were obtained as described above.

[Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by m [Annotation] amandam Unmarked set by amandam Example 3 Determination of Epitopes Pepscan M Technology was used to map the binding sites of MAB53 and MAB579. About 6,000 unique peptides of varying lengths and with varying length connecters to constrain the ends of each peptide to mimic native structure were synthesized for H1 and for H3. Binding to the stalk region by MAB53 and MAB579 was confirmed using rabbit sera to globular head or stalk as competitors and by direct binding to peptides from the stalk region. As noted above, MAB486 binds both Group 1 and Group 2 but only in the preactivated state before protease cleavage of HA0 to disulfide linked HA1 and HA2. It was concluded that the epitope for cross-clade binding is a discontinuous epitope spanning two rs of the native ic HA0.

Example 4 In Vivo Potency [MAB53] and Pharmacokinetics [MAB53 and MAB579) The strains used in these experiments were: HlNl: A/CA/04/09; H5Nl: A/Vietnam/l203/04/HPAI; H3N2: A/Perth/16/09; H7N3: A/Red Knot/NJ/1523470/06.

To test prophylaxis, MAB53 was provided to mice as a single intraperitoneal dose of mg/kg at Day -1 which was followed at Day 0 by a dose of virus 10 times the LD50 delivered intranasally. The potency of MAB53 was determined to exhibit EC50 at 0.4 mg/kg as ed to the l antibody CR626l which is reported to exhibit an EC50 of 1-1.5 mg/kg (Koudstaal, W., et al., J. Infect. Dis. (2009) 200:1870-1873).

To test therapeutic iveness, MAB was given as a single eritoneal dose of mg/kg at Day +3 for most strains or at Day +1 for H7N3. MAB53 was fully ive with respect to HlNl and H5Nl whereas essentially all control mice were dead by Day 10.

MAB579 was essentially fully effective against H3N2 and H7N3 whereas virtually all control mice were dead before Day 10.

Weight loss was also measured and declines were no worse than 20% in the d mice.

In comparison to treatment with Tamiflu® (oseltamivir phosphate), mice (10 per group) were anesthetized and infected intranasally with 10 times the LD50 dose of virus (HlNl Influenza A/Ca/04/09). MAB53 (or control e-matched human IgG) was given i.p. at Day +l nfection. Tamiflu® was given by oral gavage twice daily for 4 days starting on [Annotation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam [Annotation] amandam None set by amandam ation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam Day +1 post-infection. Both mortality and morbidity (assessed by weight loss) were far more severe for the Tamiflu® cohort compared to the MAB53 cohort.

For controls, all of the mice were dead by eight days post infection. For those treated with Tamiflu®, all but two mice were dead before eight days post infection; these two mice ed at least to Day 14. In the group d with MAB53, eight of the ten mice survived past Day 8 to Day 14.

With respect to weight loss, the control group declined in weight to 70% of their initial weight after eight days. The declines in weight were reversed at Day 4 for the mice treated with MAB53 and the original weight was exceeded by Day 14. In the Tamiflu® treated mice, weight loss was reversed by Day 6 but only 92% of the original weight was attained by Day 14.

Pharmacokinetics were also examined in mice for MAB53 and MAB 579. These show a half-life in mice of about 7-14 days corresponding to a half-life in humans of 3-4 weeks.

This corresponds to that typical for an IgGl K MAB. The bispecific antibody MAB579/53Bi (see Example 5) shows a similar half-life.

Example 5 Construction of MAB579/53Bi The construction of MAB579/53Bi provides an scFv n of MAB53 coupled to the constant region of MAB579 as shown in Figure 5. Construction of such ific antibodies is well known in the art (Marvin, J. 8., Acta Pharmacologica Sinica (2005) 26:649-658). Thus, MAB579/53Bi provides bivalent binding at both ends of the molecule along with an intact Fc region. Table 4 shows that the bispecific dy retains the affinity of the ndent antibodies as measured by ioTM in nM. The ific antibody further retains the neutralization capability of the individual antibodies of which it is composed and has an EC50 of 3.5 ug/ml against H1N1; 6.0 ug/ml against H5N1, and 2.2 ug/ml t H3N2.

Table 4 Affinity by ioTM (nM) Strain MAB53 MAB579 Bi-Specific H5 Vietnam/1203/2004 0.5 2.5 H3 HongKong/8/1968 0.2 0.2 H3 Perth 16/09 0.7 1.3 H7 Netherlands/219/03 0.4 0.3 [Annotation] amandam None set by amandam [Annotation] m MigrationNone set by amandam [Annotation] amandam ed set by amandam ation] amandam None set by amandam [Annotation] amandam MigrationNone set by amandam [Annotation] amandam Unmarked set by amandam In vivo Potency of MAB579/53Bi In vivo efficacy was measured as described generally in Example 4 with the results as shown in Figures 6A-6E.

As shown in Figure 6A and 6C, mice were infected with A/Ca/04/09 (HlNl) (representing influenza Group 1) on Day 0, and d by IP injection with 10 mg/ml MAB53 alone, 10 mg/ml MAB579 alone or either a mixture of MAB53 and MAB579 (Figure 6A) or the bispecific antibody (Figure 6C) at Day 2, (Figure 6C also shows weight loss curves). Controls received IgG at 20 mg/kg. The mixture of MAB’s was administered at 10 mg/kg each and the bispecific antibody was administered at 10 mg/kg. As shown in Figure 6A, the mixture of MAB53 and MAB579, as well as MAB53 alone, were tive, while MAB579 and control resulted in no survivors after 10 days. As shown in Figure 6C, the bispecific antibody was equally effective as the mixture.

Similar results were obtained in the ous protocol for mice infected with a Group 2 representative Philippines 2/82 (H2N3) as shown in Figures 6B and 6D. (Figure 6D also shows weight loss curves.) As shown in Figure 6B, the mixture was effective against this virus as was MAB579, but MAB53 alone and control resulted in death after 10 days. In Figure 6D, it is demonstrated that the bispecific antibody is equally effective as the mixture.

Figure 6E shows the results of treatment of ion using the analogous protocol where the challenge was with both Group 1 and Group 2 representatives HlNl and H2N3 which both infected the same mouse. A combination of MAB579 and MAB53 each at 3 mg/kg was completely protective and at 1 mg/kg each protected 80% of the mice. This is significant e ection occurs in nature resulting in recombined virus that may cause a pandemic.

[Annotation] AmandaM None set by AmandaM [Annotation] AmandaM MigrationNone set by AmandaM [Annotation] AmandaM Unmarked set by AmandaM

Claims (3)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A monoclonal dy optionally including a bi-specific antibody or immunoreactive fragment f, that either is, or binds to the same epitope of influenza A virus as an antibody sing: i) a heavy chain variable domain sequence of SEQ ID NO:8 and a light chain variable domain sequence of SEQ ID NO:9; ii) a heavy chain variable domain sequence of SEQ ID NO:4 and a light chain variable domain sequence of SEQ ID NO:5; iii) a heavy chain variable domain sequence of SEQ ID NO:10 and a light chain variable domain sequence of SEQ ID NO:11; iv) a heavy chain variable domain ce of SEQ ID NO:12 and a light chain le domain ce of SEQ ID NO:13; v) a heavy chain variable domain sequence of SEQ ID NO:14 and a light chain variable domain sequence of SEQ ID NO:15; vi) a heavy chain variable domain sequence of SEQ ID NO:16 and a light chain variable domain ce of SEQ ID NO:17; vii) a heavy chain variable domain sequence of SEQ ID NO:18 and a light chain variable domain sequence of SEQ ID NO:19; or viii) a heavy chain variable domain sequence of SEQ ID NO:20 and a light chain variable domain sequence of SEQ ID NO:21, and that neutralizes infection by H3 and H7, and which is human or humanized.

2. The antibody or fragment of claim 1 which is bispecific and binds to the same epitope as i) an antibody comprising a heavy chain variable domain sequence of SEQ ID NO:8 and a light chain variable domain sequence of SEQ ID NO:9; and to the same epitope as ii) an antibody comprising a heavy chain variable sequence of SEQ ID NO:24 and a light chain variable domain sequence of SEQ ID NO:25. [Annotation] AmandaM None set by AmandaM [Annotation] AmandaM MigrationNone set by AmandaM [Annotation] AmandaM Unmarked set by AmandaM

3. The antibody or fragment of claim 1 or 2 wherein a) the antibody comprises a heavy chain le region that comprises CDR1 of the sequence AYTIH, and CDR2 of the sequence WINAGNGHTKYSQRFKGR, and CDR3 of the sequence GPETYYYDKTNWLNSHPDEYFQH; and b) the antibody comprises a heavy chain variable region that comprises CDR1 of the sequence SYTLH, and CDR2 of the sequence WINAGNGKTKYPPKFRGR, and CDR3 of the sequence GPESYYYDRSDWLNSHPDEYFQY; and c) the antibody comprises a heavy chain variable region that comprises CDR1 of the sequence SFTMH, and CDR2 of the sequence WINAGNGKTKYSQKFQGR, and CDR3 of the ce GPETYYYDSSNWLNSHPDEYLQY; and d) the antibody comprises a heavy chain variable region that comprises CDR1 of the sequence SFTMH, and CDR2 of the sequence WINAGNGKTKYSQKFQGR, and CDR3 of the sequence YDSSNWLNSHPDEYFQH; and e) the antibody comprises a heavy chain variable region that comprises CDR1 of the ce SYSVH, and CDR2 of the sequence WINAGNGKTKYPQKFKGR, and CDR3 of the sequence GPDSYYYDRNDWLNSHPDEYFQH; and f) the antibody comprises a heavy chain variable region that ses CDR1 of the sequence TYTIH, and CDR2 of the sequence WINAANGHTKYSRKLRSR, and CDR3 of the sequence FDKTNWLNSHPDEYFQH; and g) the antibody comprises a heavy chain variable region that comprises CDR1 of the sequence SYTLH, and CDR2 of the sequence GKVKYPRKLQGR, and CDR3 of the sequence GPESYFFDTSNHLNSHPDEYFQF; and