FIELD OF THE INVENTION

The present invention relates to an isolated non-immunostimulatory antibody, wherein said antibody lacks or with very low Fc-gamma receptor binding activities, antibody-dependent cell-mediated cytotoxicity, and complement-mediated cytotoxicity. In some embodiments, the antibody retains a substantial portion of an immunoglobulin G2 (lgG2) or G4(lgG4)Fc region. BACKGROUND OF THE INVENTION

The constant domains of antibodies are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and in humans several of these are further divided into subclasses (isotypes), e.g., IgG l, lgG2, lgG3, and lgG4; IgAl and lgA2. The human IgG isotypes, IgG l, lgG2, lgG3, and lgG4 elicit differential responses due to their sequence differences, which result in differential binding the two Fc- gamma receptors (Daeron (1997) Annu. Rev. Immunol. 15:203-234) and/or the initial complement component, Clq (Cooper (1985) Adv. Immunol. 37: 151). Of the various human immunoglobulin classes, human IgGl, lgG3 and IgM are known to activate complement dependent cytotoxicity (CDC) more effectively than lgG2 and lgG4; and human IgGl and lgG3 mediate antibody dependent cellular cytotoxicity (ADCC) more effectively than lgG2 and lgG4.

It is sometimes desirable that antibodies used in therapies do not provoke an immune reaction toward cells harboring the target antigen. Therefore, there is a need in the art for the generation of therapeutic human or humanized monoclonal antibodies that do not possess any or have much reduced effector functionality, yet retain the typical pharmacokinetics of an IgG. The present invention meets this need in the art.

Fc structure is frequently used to make Fc fusion proteins as therapeutics. Fc fusion protein provides long half-life and convenience in purification by protein A chromatography. It is also sometimes desirable that Fc containing molecules used in therapies do not provoke an immune reaction toward cells harboring the target antigen. Therefore, there is a need in the art for the generation of the mentioned therapeutics that do not possess any or have much reduced effector functionality. The present invention meets this need in the art.

Published data and reports indicate that the members of the ELRCXC subfamily of CXCL chemokines are elevated in a number of diseases. There are a total of 16 CXCL family members. The chemokines are reported to be up-regulated in a number of inflammatory diseases, including COPD, in which the level of CXCL 1-3, 5 and 8, also known as Gro-alpha, -beta, -gamma (Haskill, S., et al. Proc. Nat. Acad. Sci. 1990: 87, 7732-7736), ENA-78 (Wang, D. and Richmond, A., Cytokine Reference. Oppenheim, J.J. and Feldman, M. ed., Academic Press, London, 1023-1027; Power, C.A. et al. Gene, 1994: 151, 333-334), and I L-8 (Lizasa, H. and Matsushima , K., Cytokine

Reference. Oppenheim, J.J. and Feldman, M. ed., Academic Press, London, 1061-1067;

Matsushima, K. et al., J. Exp. Med. 1988: 167, 1883-1893) respectively, are elevated (Am. J.

Respir. Crit. Care Med., 163: 349-355, 2001, Am. J. Respir. Crit. Care Med., 168: 968-975, 2003, Thorax, 57: 590-595, 2002). It has been postulated that prolonged and elevated expression of these chemokines could be involved in the development of diseases such as particularly ulcerative colitis, Crohn's disease, COPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease, psoriasis, transplant rejection, gout, cancer, acute lung injury, acute lung disease, sepsis, ARDS, peripheral artery disease, systemic sclerosis, neonatal respiratory distress syndrome, respiratory syncytial virus, flu, Behcets disease, uveitis, periodontal disease particularly gingivitis, exacerbation of asthma and COPD, cystic fibrosis, acne, Bronchiolitis obliterans syndrome, diffuse panbronchiolitis, deep vein thrombosis, preeclampsia, vasculitis, familial Mediterranean fever, reperfusion injury, pain and/or endometriosis. These CXC chemokines are known to stimulate neutrophil chemotaxis by engaging and activating the CXCRl and CXCR2 receptors. Thus, the inhibition of these chemokines could prevent inflammatory cells from infiltrating the lung tissue and thus preventing tissue damage.

Our published patent application WO2008/130969 teaches that antibody having heavy chain and light chain (SEQ I D NO: 1 and 2, respectively, referred to Pan ELR) decreases the neutrophil chemotaxis through inhibition of CXCRl and CXCR2 receptor activation by neutralizing human I L-8, Gro-alpha, Gro-beta, Gro-gamma, gcp-2 and ENA-78.

Pan ELR is a humanized, IgGl wild type antibody. It specifically suppresses the infiltration of neutrophils in a number of animal models including the LPS inhaled model of lung

inflammation and the cantharidin skin blister model: for example, at 10 mg/Kg i.v. suppresses over 90% of neutrophil, but not monocyte, infiltration demonstrating the specificity of this chemokine pathway. In two GLP toxicology studies, run at doses of 3, 30, 100 and 300 mg/Kg with once weekly administration, the majority of animals in the high dose group (100 - 300 mg/Kg), and a minority of animals in the low (therapeutic) dose groups (3 - 10 mg/Kg), developed one to two skin lesions per animal. These lesions were marked by a monocytic or eosinophilic cell infiltration. To understand if the Fc-region of the wild type IgGl antibody was playing a role in the inflammatory response, an IgGl FcyR and compliment-disabled antibody (PanELR Fc disabled, L234A/H235A (EU numbering), SEQ ID NO:2 and SEQ ID NO: 3 for light and heavy chains, respectively) was made and dosed at a single concentration of 300 mg/Kg once per week. These animals also developed skin lesions at approximately the same incidence rate as the animals dosed with the IgGl wild type antibody, suggesting that Fc effector function disablement of IgGl wt antibodies may not be complete.

In order to understand the role of the Fc portion of PanELR and its interaction with FcyR more fully, as described in WO2013/166099, a stabilized and two aglycosylated forms of lgG4 were made: PanELR lgG4 (PE) antibody (having heavy and light chains with SEQ ID NO:4 and SEQ ID NO: 2 sequences, respectively); PanELR lgG4(PE)N297A antibody (having heavy and light chains with SEQ ID NO: 5 and SEQ ID NO: 2 sequences, respectively; and PanELR lgG4(PE)N297S antibody (having heavy and light chains with SEQ ID NO: 6 and SEQ ID NO:2 sequences, respectively ) in which the the variable regions of the heavy and the light chain from PanELR were transposed onto an lgG4 backbone. Characterization of the lgG4 variants confirmed that they had the same binding and neutralizing characteristics as the IgGl isotype as expected.

In order to characterize the interactions of Pan-ELR IgG variants with complement, a Clq BiaCore binding assay was established that reveled greater than 65% reduction in lgG4(PE) variant of Pan-ELR binding to Clq. This demonstrated that lgG4(PE) had less binding and thus potential, to activate complement than either IgGl wild type or Fc disabled antibodies. While panELR lgG4(PE) may have significantly reduced effector function capability, whether or not this antibody can sufficiently address the issue of monkey skin toxicity awaits further testing. In order to make pan-ELR antibody a safe human therapeutic product , it is may be prudent to completely eliminate the effector function potential inherent to the Fc. We have now discovered Fc variant sequences with close to complete elimination of Fc gamma receptor binding activities.

SUMMARY OF THE INVENTION In one embodiment, the present invention relates to a Fc-containing molecule having decreased affinity for at least one Fc gamma receptor as compared to wild type Fc, comprising a Fc domain with a mutated lgG2 constant region with amino acid residues 233, 234, 235, 237, 238, 268, 309, 330, and 331 (using EU numbering) having amino acids of PAAASAVSS (SEQ ID NO:15), PAAAPALSS (SEQ ID NO:16) , or PAAASQLSS (SEQ ID NO:17) (SEQ ID NO: 7, 8, or 9, respectively, for the entire Fc portions)

In another embodiment, the present invention relates to Fc-containing molecule having decreased affinity for at least one Fc gamma receptor as compared to wild type Fc, comprising a Fc domain with a mutated lgG4 constant region with amino acid residues 233, 234, 235, and 237 having amino acids of PAAA, and furthermore having deleted glycine at position 236 (all in EU numbering) (SEQ ID NO:10 for the entire Fc portion).

In another embodiment, Fc-containing molecules described above are isolated antibodies.

In another embodiment, the present invention relates to an isolated antibody having light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 11, 12, 13,14 or 15.

In another embodiment, the present invention relates to a method of treating a human with diseases or disorders characterized by elevated or unbalanced level of one or more of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, ENA-78 and GCP-2, particularly ulcerative colitis, Crohn's disease, COPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease, psoriasis, transplant rejection, gout, cancer, acute lung injury, acute lung disease, sepsis, ARDS, peripheral artery disease, systemic sclerosis, neonatal respiratory distress syndrome, respiratory syncytial virus, flu, Behcets disease, uveitis, periodontal disease particularly gingivitis, exacerbation of asthma and COPD, cystic fibrosis, acne, Bronchiolitis obliterans syndrome, diffuse panbronchiolitis, deep vein thrombosis, preeclampsia, vasculitis, familial Mediterranean fever, reperfusion injury, pain and/or endometriosis, comprising the steps of administering an isolated antibody having light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 11, 12, 13, 14 or 15.

In another embodiment, the present invention relates to an isolated antibody having light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 11, 12, 13, 14 or 15 or a pharmaceutical composition comprising thereof in the manufacture of a medicament for the treatment of human diseases or disorders characterized by elevated or unbalanced level of one or more of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, ENA-78 and GCP-2, particularly ulcerative colitis, Crohn's disease, COPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease, psoriasis, transplant rejection, gout, cancer, acute lung injury, acute lung disease, sepsis, ARDS, peripheral artery disease, systemic sclerosis, neonatal respiratory distress syndrome, respiratory syncytial virus, flu, Behcets disease, uveitis, periodontal disease particularly gingivitis, exacerbation of asthma and COPD, cystic fibrosis, acne, Bronchiolitis obliterans syndrome, diffuse panbronchiolitis, deep vein thrombosis, preeclampsia, vasculitis, familial Mediterranean fever, reperfusion injury, pain and/or endometriosis.

In another embodiment, the present invention relates to an isolated antibody having light chain sequence of SEQ I D NO: 2 and a heavy chain sequence of SEQ I D NO: 11, 12, 13, 14 or 15 or a pharmaceutical composition comprising thereof for use in the treatment of human diseases or disorders characterized by elevated or unbalanced level of one or more of human I L- 8, Gro-alpha, Gro-beta, Gro-gamma, ENA-78 and GCP-2, particularly ulcerative colitis, Crohn's disease, COPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease, psoriasis, transplant rejection, gout, cancer, acute lung injury, acute lung disease, sepsis, ARDS, peripheral artery disease, systemic sclerosis, neonatal respiratory distress syndrome, respiratory syncytial virus, flu, Behcets disease, uveitis, periodontal disease particularly gingivitis, exacerbation of asthma and COPD, cystic fibrosis, acne, Bronchiolitis obliterans syndrome, diffuse panbronchiolitis, deep vein thrombosis, preeclampsia, vasculitis, familial Mediterranean fever, reperfusion injury, pain and/or endometriosis.

Other non-limiting aspect of inventions are pharmaceutical compositions, method of making , polynucleotides, host cells for proteins described above.

DESCRIPTION OF THE FIGURES

The Figure 1 shows the schematic diagram of ELISA binding assay format of pan-ELR antibody and Fc gamma receptor.

The Figure 2 shows pan-ELR antibody binding to FcyRl tetramer using the ELISA format described in Figure 1. Pan-ELR antibodies were coated on ELISA 96 well plate and FcyR tetramer in 3-fold serial dilution were added for binding. Bovine serum albumin (BSA) was used as a negtive control for antibody.

The Figure 3 shows pan-ELR antibody binding to FCγR2A( H 131) tetramer using the ELISA method described in Figure 1. Pan-ELR antibodies were coated on ELISA 96 well plate and FCγR2A( H 131) tetramer in 3-fold serial dilution were added for binding; 3A) pan-ELR-2-1 and pan- ELR -3-1; 3B) pan-ELR-2-1 and pan-ELR-3-la; 3C) pan-ELR-2-1 and pan-ELR-3-lb; 3D) pan-ELR-2-1 and pan-ELR-3-lc; and 3E)pan-ELR-2-l and pan-ELR-3-2. Bovine serum albumin (BSA) was used as a negtive control for antibody.

The Figure 4 shows pan-ELR antibody binding to FcyR3A(V158) tetramer using the ELISA method described in Figure 1. Pan-ELR antibodies were coated on ELISA 96 well plate and

FcyR3A(V158) tetramer in 3-fold serial dilution were added for binding; 4A) pan-ELR-2-1 and pan- ELR -3-1; 4B) pan-ELR-2-1 and pan-ELR-3-la; 4C) pan-ELR-2-1 and pan-ELR-3-lb; 4D) pan-ELR-2-1 and pan-ELR-3-lc; and 4E)pan-ELR-2-l and pan-ELR-3-2. Bovine serum albumin (BSA) was used as a negative control for antibody.

Detailed Description

In one aspect of the invention as herein described are antibodies comprising a Fc domain with a mutated lgG2 constant region with amino acid residues 233, 234, 235, 237, 238, 268, 309, 330, and 331 (using EU numbering) having amino acids of PAAASAVSS (SEQ ID NO:15),

PAAAPALSS (SEQ ID NO:16) , or PAAASQLSS (SEQ ID NO:17) (SEQ ID NO: 7, 8, or 9, respectively, for entire Fc portions) have no or very minimal Fc gamma receptors binding. The same was true for antibodies comprising a Fc domain with a mutated lgG4 constant region with amino acid residues 233, 234, 235, and 237 having amino acids of PAAA, and furthermore having deleted glycine at position 236 (all in EU numbering) (SEQ ID NO:10 for entire Fc portion). These Fc mutations are new and could have wide applicabilities to biological active proteins. For example, we have employed them to an isolated antibody having light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 11, 12, 13, or 14, which has no or virtually no effector functions, thus less immunological side effects.

Thus, the present invention relates to a method of treating a human with diseases or disorders characterized by elevated or unbalanced level of one or more of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, ENA-78 and GCP-2, particularly ulcerative colitis, Crohn's disease, COPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease, psoriasis, transplant rejection, gout, cancer, acute lung injury, acute lung disease, sepsis, ARDS, peripheral artery disease, systemic sclerosis, neonatal respiratory distress syndrome, respiratory syncytial virus, flu, Behcets disease, uveitis, periodontal disease particularly gingivitis, exacerbation of asthma and COPD, cystic fibrosis, acne, Bronchiolitis obliterans syndrome, diffuse panbronchiolitis, deep vein thrombosis, preeclampsia, vasculitis, familial Mediterranean fever, reperfusion injury, pain and/or endometriosis, comprising the steps of administering an isolated antibody having light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 11, 12, 13, 14 or 15.

As used herein "antibodies" include various modified forms. Modifications include glycosylation variants of the antibodies. Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on antibody function, particularly effector functioning such as those described above, see for example, Boyd et al. (1996) Mol. Immunol. 32: 1311-1318. Glycosylation variants of the antibodies or antibody fragments thereof wherein one or more carbohydrate moiety is added, substituted, deleted or modified are contemplated. Introduction of an asparagine-X-serine or asparagine-X-threonine motif creates a potential site for enzymatic attachment of carbohydrate moieties and may therefore be used to manipulate the glycosylation of an antibody. In Raju et al. (2001) Biochemistry 40: 8868-8876 the terminal sialyation of a TNFR-lgG immunoadhesin was increased through a process of regalactosylation and/or resialylation using beta-1, 4-galactosyltransferace and/or alpha, 2,3 sialyltransferase. Increasing the terminal sialylation is believed to increase the half-life of the immunoglobulin. Antibodies, in common with most glycoproteins, are typically produced as a mixture of glycoforms. This mixture is particularly apparent when antibodies are produced in eukaryotic, particularly mammalian cells. A variety of methods have been developed to manufacture defined glycoforms, see Zhang et al. (2004) Science 303: 371: Sears et al. (2001) Science 291: 2344; Wacker et al. (2002) Science 298: 1790; Davis et al. (2002) Chem. Rev. 102: 579; Hang et al. (2001) Acc. Chem. Res 34: 727. The antibodies (for example of the IgG isotype, e.g. IgGl) as herein described may comprise a defined number (e.g. 7 or less, for example 5 or less, such as two or a single) of glycoform(s).

The antibodies of the invention may be coupled to a non-proteinaeous polymer such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene. Conjugation of proteins to PEG is an established technique for increasing half-life of proteins, as well as reducing antigenicity and immunogenicity of proteins. The use of PEGylation with different molecular weights and styles (linear or branched) has been investigated with intact antibodies as well as Fab' fragments, see Koumenis et al. (2000) Int. J. Pharmaceut. 198: 83-95.

The antibodies of the present invention may be produced in transgenic organisms such as goats (see Pollock et al. (1999) J. Immunol. Methods 231: 147-157), chickens (see Morrow (2000) Genet. Eng. News 20: 1-55, mice (see Pollock et al.) or plants (see Doran (2000) Curr. Opinion Biotechnol. 11: 199-204; Ma (1998) Nat. Med. 4: 601-606; Baez et al. (2000) BioPharm 13: 50-54; Stoger et al. (2000) Plant Mol. Biol. 42: 583-590). The antibodies of the present invention may also be produced by chemical synthesis. However, they are typically produced using recombinant cell culturing technology well known to those skilled in the art. A polynucleotide encoding the antibody of the present invention is isolated and inserted into a replicable vector such as a plasmid for further cloning (amplification) or expression. One expression system is a glutamate synthetase system (such as sold by Lonza Biologies), particularly where the host cell is CHO or NSO. Polynucleotide encoding the antibody is readily isolated and sequenced using conventional procedures (e.g. oligonucleotide probes). Vectors that may be used include plasmid, virus, phage, transposons, minichromosomes of which plasmids are typically used. Generally such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter and transcription termination sequences operably linked to the antibody polynucleotide so as to facilitate expression. Polynucleotide encoding the light and heavy chains may be inserted into separate vectors and introduced (for example by transformation, transfection, electroporation or transduction) into the same host cell concurrently or sequentially or, if desired, both the heavy chain and light chain can be inserted into the same vector prior to said introduction.

In one embodiment, the present invention relates to an expression vector comprising polynucleotide which encode heavy chain comprising polypeptide of SEQ ID NO: 11, 12, 13, 14 or 15; or light chain comprising polypeptide of SEQ ID NO:2.

Codon optimization may be used with the intent that the total level of protein produced by the host cell is greater when transfected with the codon-optimized gene in comparison with the level when transfected with the sequence. Several methods have been published (Nakamura et al. (1996) Nucleic Acids Research 24: 214-215; W098/34640; W097/11086). Due to the redundancy of the genetic code, alternative polynucleotides to those disclosed herein

(particularly those codon optimized for expression in a given host cell) may also encode the antibodies described herein. The codon usage of the antibody of this invention therefore can be modified to accommodate codon bias of the host cell such to augment transcript and/or product yield (e.g. Hoekema et al Mol Cell Biol 1987 7(8): 2914-24). The choice of codons may be based upon suitable compatibility with the host cell used for expression.

Antibodies may be produced as a fusion protein with a heterologous signal sequence having a specific cleavage site at the N-terminus of the mature protein. The signal sequence should be recognized and processed by the host cell. For prokaryotic host cells, the signal sequence may be for example an alkaline phosphatase, penicillinase, or heat stable enterotoxin II leaders. For yeast secretion the signal sequences may be for example a yeast invertase leader, a factor leader or acid phosphatase leaders see e.g. WO90/13646. In mammalian cell systems, viral secretory leaders such as herpes simplex gD signal and a native immunoglobulin signal sequence may be suitable. Typically the signal sequence is ligated in reading frame to DNA encoding the antibody.

Origin of replications are well known in the art with pBR322 suitable for most gram- negative bacteria, 2μ plasmid for most yeast and various viral origins such as SV40, polyoma, adenovirus, VSV or BPV for most mammalian cells. Generally the origin of replication component is not needed for mammalian expression vectors but the SV40 may be used since it contains the early promoter.

Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins e.g. ampicillin, neomycin, methotrexate or tetracycline or (b) complement auxiotrophic deficiencies or supply nutrients not available in the complex media or (c) combinations of both. The selection scheme may involve arresting growth of the host cell. Cells, which have been successfully transformed with the genes encoding the antibody of the invention, survive due to e.g. drug resistance conferred by the co-delivered selection marker. One example is the DHFR selection marker wherein transformants are cultured in the presence of methotrexate. Cells can be cultured in the presence of increasing amounts of methotrexate to amplify the copy number of the exogenous gene of interest. CHO cells are a particularly useful cell line for the DHFR selection. A further example is the glutamate synthetase expression system (Lonza Biologies). An example of a selection gene for use in yeast is the trpl gene, see Stinchcomb et al. (1979) Nature 282: 38.

Suitable promoters for expressing antibody of the present invention are operably linked to DNA/polynucleotide encoding the antibody. Promoters for prokaryotic hosts include phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan and hybrid promoters such as Tac. Promoters suitable for expression in yeast cells include 3- phosphoglycerate kinase or other glycolytic enzymes e.g. enolase, glyceralderhyde 3 phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6 phosphate isomerase, 3-phosphoglycerate mutase and glucokinase. Inducible yeast promoters include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein and enzymes responsible for nitrogen metabolism or maltose/galactose utilization.

Promoters for expression in mammalian cell systems include viral promoters such as polyoma, fowlpox and adenoviruses (e.g. adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (in particular the immediate early gene promoter), retrovirus, hepatitis B virus, actin, rous sarcoma virus (RSV) promoter and the early or late Simian virus 40. Of course the choice of promoter is based upon suitable compatibility with the host cell used for expression. A first plasmid may comprise a RSV and/or SV40 and/or CMV promoter, DNA encoding light chain variable region (VL), KC region together with neomycin and ampicillin resistance selection markers and a second plasmid comprising a RSV or SV40 promoter, DNA encoding the heavy chain variable region (VH), DNA encoding the yl constant region, DHFR and ampicillin resistance markers.

Where appropriate, e.g. for expression in higher eukaryotes, an enhancer element operably linked to the promoter element in a vector may be used. Mammalian enhancer sequences include enhancer elements from globin, elastase, albumin, fetoprotein and insulin. Alternatively, one may use an enhancer element from a eukaroytic cell virus such as SV40 enhancer (at bplOO-270), cytomegalovirus early promoter enhancer, polyma enhancer, baculoviral enhancer or murine lgG2a locus (see WO04/009823). The enhancer may be located on the vector at a site upstream to the promoter. Alternatively, the enhancer may be located elsewhere, for example within the untranslated region or downstream of the polyadenylation signal. The choice and positioning of enhancer may be based upon suitable compatibility with the host cell used for expression.

In eukaryotic systems, polyadenylation signals are operably linked to DNA/polynucleotide encoding the antibody. Such signals are typically placed 3' of the open reading frame. In mammalian systems, non-limiting examples include signals derived from growth hormones, elongation factor-1 alpha and viral (e.g. SV40) genes or retroviral long terminal repeats. In yeast systems non-limiting examples of polydenylation/termination signals include those derived from the phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, polyadenylation signals are typically not required and it is instead usual to employ shorter and more defined terminator sequences. The choice of polyadenylation/termination sequences may be based upon suitable compatibility with the host cell used for expression.

In addition to the above, other features that can be employed to enhance yields include chromatin remodelling elements, introns and host-cell specific codon modification.

Suitable host cells for cloning or expressing vectors encoding the antibody are prokaroytic, yeast or higher eukaryotic cells. Suitable prokaryotic cells include eubacteria e.g. enterobacteriaceae such as Escherichia e.g. E. coli (for example ATCC 31,446; 31,537; 27,325), Enterobacter, Erwinia, Klebsiella Proteus, Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratia marcescans and Shigella as well as Bacilli such as B. subtilis and B. licheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa and Streptomyces. Of the yeast host cells, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces (e.g. ATCC 16,045; 12,424; 24178; 56,500), yarrowia (EP402, 226), Pichia pastoris (EP 183 070, see also Peng et al. (2004) J. Biotechnol. 108: 185-192), Candida, Trichoderma reesia (EP 244 234), Penicillin, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger are also contemplated.

Higher eukaryotic host cells include mammalian cells such as COS-1 (ATCC No.CRL 1650) COS-7 (ATCC CRL 1651), human embryonic kidney line 293, baby hamster kidney cells (BH K) (ATCC CRL.1632), BH K570 (ATCC NO: CRL 10314), 293 (ATCC NO.CRL 1573), Chinese hamster ovary cells CHO (e.g. CHO-K1, ATCC NO: CCL 61, DH FR-CHO cell line such as DG44 (see Uriaub et al. (1986) Somatic Cell Mol. Genet.12: 555-556), particularly those CHO cell lines adapted for suspension culture, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells (ATCC CRL-1587), H ELA cells, canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), Hep G2 and myeloma or lymphoma cells e.g. NS0 (see US 5,807,715), Sp2/0, Y0.

Such host cells may also be further engineered or adapted to modify quality, function and/or yield of an antibody. Non-limiting examples include expression of specific modifying (e.g. glycosylation) enzymes and protein folding chaperones.

Host cells transformed with vectors encoding an antibody may be cultured by any method known to those skilled in the art. Host cells may be cultured in spinner flasks, roller bottles or hollow fibre systems but for large scale production that stirred tank reactors are used particularly for suspension cultures. The stirred tankers may be adapted for aeration using e.g. spargers, baffles or low shear impellers. For bubble columns and airlift reactors direct aeration with air or oxygen bubbles maybe used. Where the host cells are cultured in a serum free culture media, the media is supplemented with a cell protective agent such as pluronic F-68 to help prevent cell damage as a result of the aeration process. Depending on the host cell characteristics, either microcarriers may be used as growth substrates for anchorage dependent cell lines or the cells may be adapted to suspension culture (which is typical). The culturing of host cells, particularly invertebrate host cells may utilise a variety of operational modes such as fed-batch, repeated batch processing (see Drapeau et al. (1994) Cytotechnology 15: 103-109), extended batch process or perfusion culture. Although recombinantly transformed mammalian host cells may be cultured in serum-containing media such as fetal calf serum (FCS), such host cells may be cultured in synthetic serum-free media such as disclosed in Keen et al. (1995) Cytotechnology 17: 153-163, or commercially available media such as ProCHO-CDM or UltraCHO™ (Cambrex NJ, USA), supplemented where necessary with an energy source such as glucose and synthetic growth factors such as recombinant insulin. The serum-free culturing of host cells may require that those cells are adapted to grow in serum free conditions. One adaptation approach is to culture such host cells in serum containing media and repeatedly exchange 80% of the culture medium for the serum-free media so that the host cells learn to adapt in serum free conditions (see e.g. Scharfenberg et al. (1995) in Animal Cell Technology: Developments towards the 21st century (Beuvery et al. eds, 619-623, Kluwer Academic publishers).

The antibody secreted into the media may be recovered and purified using a variety of techniques to provide a degree of purification suitable for the intended use. For example the use of the antibody for the treatment of human patients typically mandates at least 95% purity, more typically 98% or 99% or greater purity (compared to the crude culture medium). Cell debris from the culture media is typically removed using centrifugation followed by a clarification step of the supernatant using e.g. microfiltration, ultrafiltration and/or depth filtration. A variety of other techniques such as dialysis and gel electrophoresis and chromatographic techniques such as hydroxyapatite (HA), affinity chromatography (optionally involving an affinity tagging system such as polyhistidine) and/or hydrophobic interaction chromatography (HIC, see US 5, 429,746) are available. The antibodies, following various clarification steps, can be captured using Protein A or G affinity chromatography. Further chromatography steps can follow such as ion exchange and/or HA chromatography, anion or cation exchange, size exclusion chromatography and ammonium sulphate precipitation. Various virus removal steps may also be employed (e.g. nanofiltration using e.g. a DV-20 filter). Following these various steps, a purified (for example a monoclonal) preparation comprising at least 75mg/ml or greater, or lOOmg/ml or greater, of the antibody is provided. Such preparations are substantially free of aggregated forms of antibodies.

Bacterial systems may be used for the expression of antibodies. Such fragments can be localised intracellularly, within the periplasm or secreted extracellularly. Insoluble proteins can be extracted and refolded to form active proteins according to methods known to those skilled in the art, see Sanchez et al. (1999) J. Biotechnol. 72: 13-20; and Cupit et al. (1999) Lett Appl Microbiol 29: 273-277.

The skilled person will appreciate that, upon production of the antibody, in particular depending on the cell line used and particular amino acid sequence of the antibody, post- translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, deamidation, oxidation, disulfide bond scrambling, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation. The present invention encompasses the use of antibodies which have been subjected to, or have undergone, one or more post-translational modifications.

Deamidation is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid and aspartic acid (D) at approximately 3:1 ratio. To a much lesser degree, deamidation can occur with glutamine residues in a similar manner. Deamidation in a CDR results in a change in charge of the molecule, but typically does not result in a change in antigen binding, nor does it impact on PK/PD.

Oxidation can occur during production and storage (i.e. in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but occasionally can occur at tryptophan and free cysteine residues.

Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling.

Isomerization typically occurs during production, purification, and storage (at acidic pH) and usually occurs when aspartic acid is converted to isoaspartic acid through a chemical process. N-terminal glutamine in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu). Most pGlu formation happens in the production bioreactor, but it can be formed non- enzymatically, depending on pH and temperature of processing and storage conditions. pGlu formation is considered as one of the principal degradation pathways for recombinant mAbs. C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant mAbs. Variants of this process include removal of lysine from one or both heavy chains. Lysine clipping does not appear to impact bioactivity and has no effect on mAb effector function.

Purified preparations of an antibody or fragments thereof of the present invention as described herein may be incorporated into pharmaceutical compositions for use in the treatment of the human diseases, disorders and conditions described herein. The terms diseases, disorders and conditions are used interchangeably. The pharmaceutical preparation may comprise an antibody in combination with a pharmaceutically acceptable carrier. The antibody may be administered alone, or as part of a pharmaceutical composition.

Typically such compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice, see e.g. Remingtons Pharmaceutical Sciences, 16th edition (1980) Mack Publishing Co. Examples of such carriers include sterilized carriers such as saline, Ringers solution or dextrose solution, optionally buffered with suitable buffers to a pH within a range of 5 to 8.

Pharmaceutical compositions may be administered by injection or continuous infusion (e.g. intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular or intraportal). Such compositions are suitably free of visible particulate matter. Pharmaceutical compositions may also be administered orally, specifically those containing CPHPC.

Pharmaceutical compositions may comprise between lmg to lOg of the antibody, for example between 5 mg and 1 g of antibody. Alternatively, the composition may comprise between 5 mg and 500 mg, for example between 5 mg and 50 mg.

Methods for the preparation of such pharmaceutical compositions are well known to those skilled in the art. Pharmaceutical compositions may comprise between 1 mg to 10 g of antibody in unit dosage form, optionally together with instructions for use. Pharmaceutical compositions may be lyophilised (freeze dried) for reconstitution prior to administration according to methods well known or apparent to those skilled in the art. Where antibodies have an IgGl isotype, a chelator of copper, such as citrate (e.g. sodium citrate) or EDTA or histidine, may be added to the pharmaceutical composition to reduce the degree of copper-mediated degradation of antibodies of this isotype, see EP0612251. Pharmaceutical compositions may also comprise a solubiliser such as arginine base, a detergent/anti-aggregation agent such as polysorbate 80, and an inert gas such as nitrogen to replace vial headspace oxygen.

Effective doses and treatment regimes for administering the antibody are generally determined empirically and may be dependent on factors such as the age, weight and health status of the patient and disease or disorder to be treated. Such factors are within the purview of the attending physician. Guidance in selecting appropriate doses may be found in e.g. Smith et al (1977) Antibodies in human diagnosis and therapy, Raven Press, New York.

The dosage of antibody administered to a subject is generally between 1 μg/kg to 150 mg/kg, between 0.1 mg/kg and 100 mg/kg, between 0.5 mg/kg and 50 mg/kg, between 1 and 25 mg/kg or between 1 and 10 mg/kg of the subject's body weight. For example, the dose may be 10 mg/kg, 30 mg/kg, or 60 mg/kg. The antibody may be administered parenterally, for example subcutaneously, intravenously or intramuscularly.

If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

The antibody may be administered in a single large dose or in smaller repeated doses. The administration of a dose may be by slow continuous infusion over a period of from 2 to 24 hours, such as from 2 to 12 hours, or from 2 to 6 hours. This may result in reduced toxic side effects.

The administration of a dose may be repeated one or more times as necessary, for example, three times daily, once every day, once every 2 days, once a week, once a fortnight, once a month, once every 3 months, once every 6 months, or once every 12 months. The antibody may be administered by maintenance therapy, for example once a week for a period of 6 months or more. The antibody may be administered by intermittent therapy, for example for a period of 3 to 6 months and then no dose for 3 to 6 months, followed by administration of antibody again for 3 to 6 months, and so on in a cycle.

For example, the dose may be administered subcutaneously, once every 14 or 28 days in the form of multiple sub-doses on each day of administration.

Accordingly, the administration may use a pre-determined or routine schedule for administration, thereby resulting in a predetermined designated period of time between dose administrations. The schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. Any particular combination would be covered by the schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.

The pharmaceutical composition may comprise a kit of parts of the antibody together with other medicaments, optionally with instructions for use. For convenience, the kit may comprise the reagents in predetermined amounts with instructions for use.

Treatment can be therapeutic, prophylactic or preventative. The subject will be one who is in need thereof. Those in need of treatment may include individuals already suffering from a particular medical disease in addition to those who may develop the disease in the future.

The antibody described herein may also be used in methods of therapy. The term

"therapy" encompasses alleviation, reduction, or prevention of at least one aspect or symptom of a disease. For example, the antibody described herein may be used to ameliorate or reduce one or more aspects or symptoms of a disease described herein.

The antibody described herein is used in an effective amount for therapeutic, prophylactic or preventative treatment. A therapeutically effective amount of the antibody described herein is an amount effective to ameliorate or reduce one or more aspects or symptoms of the disease. The antibody described herein may also be used to treat, prevent, or cure the disease described herein.

The antibody described herein need not affect a complete cure, or eradicate every symptom or manifestation of the disease to constitute a viable therapeutic treatment. As is recognized in the pertinent field, drugs employed as therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a disease in order to constitute a viable prophylactic agent. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur (for example by delaying the onset of the disease) or worsen in a subject, is sufficient.

The antibodies described herein may be provided in a diagnostic kit comprising one or more antibodies, a detectable label, and instructions for use of the kit. For convenience, the kit may comprise the reagents in predetermined amounts with instructions for use.

In one embodiment the present invention relates to a process for producing an antibody in a single host cell, comprising the steps of:

(i) transforming said single host cell with a first DNA sequence encoding a heavy chain comprising polypeptide of SEQ ID NO: 11, 12, 13, 14 or 15; and a second DNA sequence encoding a light chain comprising a polypeptide of SEQ ID NO: 2; and

(ii) expressing said first DNA sequence and said second DNA sequence so that said antibody heavy and light chains are produced in said transformed single host cell.

Furthermore, this process can be carried out such that said first and second DNA sequences are present in different vectors or said first and second DNA sequences are present in a single vector. Fc gamma receptor proteins containing an avi tag with biotinylation and streptavidin-Horse Radish peroxidase (SA-HRP) were purchased from Sino Biologicals and Invitrogen, respectively (FcyRI-avi-biotin, MW=34.4 kDa, Sino Biological, Cat. No. 10256-H27H-B, Lot: LC09AP2305; FcyRlla(H131)-avi-biotin, MW=33 kDa ,Sino Biological, Cat. No. 10374-H27H1-B, Lot: LC09MC0602; FcyRllla (V158)-avi- biotin, MW=48 kDa , Sino Biological, Cat. No. 10389-H27H1-B, Lot: LC09MC1103; SA-HRP, MW=100 kDa, Invitrogen, Cat. No. S911, Lot: 1711869). To prepare the Fc gamma receptor tetramers, Fc gamma receptor was mixed with SA-HRP at 5 to 1 molar ratio and incubated at room temperature for 30 minutes. 100 ul of 100 ug/ml pan-ELR antibodies were added to ELISA plates (from Vendor) and incubate at 37°C for 1 h followed by PBS wash for 4 times. To block the ELISA plates, 200 μΙ of 1%BSA in PBST was added and incubated at 37°C for 1.5 h followed by PBS wash for 4 times. 100 ul of 3-fold serially diluted FcyR-avi-biotin/ SA-HRP complex (starting concentration is 20 μg/mL) was added to and incubate at 37°C for 1 h followed by washing the plate with PBST for 4 times. The binding was detected by incubating with 100 μΙ/well TMB for 10 minutes, followed by quench with 50 μΙ/well IN HCI, and record of the optical absorption at 450nm (OD450) on a Molecular device spectra M5E plate reader. Binding data were plotted and analyzed using Graphpad (PRISM) program. TABLE 1 : list of pan-ELR antibodies and the corresponding SEQ ID NOs

AMINO ACID SEQUENCES

pan-ELR-1-1 heavy chain (SEQ ID NO:l)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGY TFYSENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHE ALHNHYTQKSLSLSPGK

pan-ELR light chain ( SEQ ID NO: 2)

DIQMTQSPSSLSASVGDRVTITCQASQDIESYLSWYQQKPGKAPKLLIYYATRLADG VPSRFSGSGSGQDYT LTISSLQPEDFATYYCLQHGESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW CLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

pan-ELR Fc disabled heavy chain (SEQ ID NO: 3)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGY TFYSENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKK VEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHE ALHNHYTQKSLSLSPGK . pan-ELR IgG4 (PE) heavy chain, pan-ELR-2-1 (SEQ ID NO: 4)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFY SENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKR VESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALH NHYTQKSLSLSLGK

pan-ELR IgG4 (PE) N297A heavy chain (SEQ ID NO: 5)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGY TFYSENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKR VESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFASTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALH NHYTQKSLSLSLGK

pan-ELR IgG4 (PE) N297S heavy chain (SEQ ID NO: 6)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFY SENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKR VESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFSSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALH NHYTQKSLSLSLGK

Fc variant 3-la (SEQ ID NO: 7)

CPPCPAPPAAASSVFLFPPKPKDTLMI SRTPEVTCVWDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TFRVVSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLS LSPGK

Fc variant 3-lb (SEQ ID NO: 8)

CPPCPAPPAAAPSVFLFPPKPKDTLMI SRTPEVTCVWDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLS LSPGK

Fc variant 3-lc (SEQ ID NO: 9)

CPPCPAPPAAASSVFLFPPKPKDTLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLS LSPGK Fc variant 3-2 (SEQ ID NO: 10)

CPPCPAPPAAAPSVFLFPPKPKDTLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLS LSLGK pan-ELR-3-la heavy chain (SEQ ID NO: 11)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFY SENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVD HKPSNTKVDKT VERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVWDVSAEDPEVQFNWYV DGVEVHNAKTK PREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHN HYTQKSLSLSPGK pan-ELR-3-lb heavy chain (SEQ ID NO: 12)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFY SENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVD HKPSNTKVDKT VERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVWDVSAEDPEVQFNWYV DGVEVHNAKTK PREEQFNSTFRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHN HYTQKSLSLSPGK pan-ELR-3-lc heavy chain (SEQ ID NO: 13)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFY SENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVD HKPSNTKVDKT VERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYV DGVEVHNAKTK PREEQFNSTFRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHN HYTQKSLSLSPGK pan-ELR-3-2 heavy chain (SEQ ID NO: 14)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFY SENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKR VESKYGPPCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYV DGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHN HYTQKSLSLSLGK

pan-ELR-3-1 heavy chain variant (SEQ ID NO 15)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGY TFYSENFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVD HKPSNTKVDKT VERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVWDVSAEDPEVQFNWYV DGVEVHNAKTK PREEQFNSTFRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHN HYTQKSLSLSPGK