WO2011080050A2 - Binding molecules - Google Patents

Binding molecules Download PDF

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Publication number
WO2011080050A2
WO2011080050A2 PCT/EP2010/069430 EP2010069430W WO2011080050A2 WO 2011080050 A2 WO2011080050 A2 WO 2011080050A2 EP 2010069430 W EP2010069430 W EP 2010069430W WO 2011080050 A2 WO2011080050 A2 WO 2011080050A2
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WIPO (PCT)
Prior art keywords
seq
antibody
hccl21
binding molecule
human
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PCT/EP2010/069430
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French (fr)
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WO2011080050A3 (en
Inventor
Michael Bardroff
Stefan Ewert
Ute Jaeger
Gabor Jarai
Christian Carsten Silvester Kunz
Katja Wiesehan
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Novartis Ag
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Priority to US28563209P priority Critical
Priority to US28563409P priority
Priority to US61/285,634 priority
Priority to US61/285,632 priority
Application filed by Novartis Ag filed Critical Novartis Ag
Publication of WO2011080050A2 publication Critical patent/WO2011080050A2/en
Publication of WO2011080050A3 publication Critical patent/WO2011080050A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55Fab or Fab'
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567Framework region [FR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present invention relates to binding molecules, in particular antibodies and immunoglobulins to human CCL21 (hCCL21) and their use in the treatment and/or prevention of human diseases and disorders, particularly those of a fibrotic nature. Pharmaceutical compositions, methods of manufacture and methods of treatment are also disclosed.

Description

Binding molecules

1. Field of the Invention

The present invention concerns binding molecules, in particular immunoglobulins and preferably antibodies, that bind with chemokine (c-c motif) ligand 21 (CCL21 ) and inhibit the interaction between CCL21 and its cognate receptor chemokine(c-c motif) receptor 7 (CCR7). The present invention also concerns processes for the manufacture of said binding molecules,

pharmaceutical compositions comprising said binding molecules and methods of treating and/or preventing disease using said binding molecules. Other aspects, objects and advantages of the present invention will be apparent from the description below.

2. Background of the Invention

CCL21 is a CC chemokine (synonyms: SLC, TCA4, 6Ckine exodus-2). Its main function in normal physiology has been proposed to be in lymphocyte homing. CCL21 attracts both T cells and dendritic cells to secondary lymphoid organs through its receptor CCR7. The effect of CCL21 appears to be essential for the priming of naive T cells in the initiation phase of the immune response.

In addition to their role in lymphoid tissues CCL21/CCR7 appears to play a role in non-lymphoid tissues. In fibrotic lung diseases and in particular in idiopathic pulmonary fibrosis (IPF) the CCL19/21 - CCR7 interaction is proposed to contribute to the pathogenesis in multiple ways. It may be a driving force for the migration of circulating CCR7+ fibrocytes, a potential precursor of lung fibroblasts/myofibroblasts to the lung. Furthermore it can also contribute to the activation and proliferation of fibroblasts/ myofibroblasts in the interstitium leading to extracellular matrix deposition and the development of fibrotic lesions.

CCL21 is highly expressed in high endothelial venules of lymph nodes and spleen. It is also found at lower levels in small intestine, thyroid gland, trachea, thymus, lung, bone marrow, liver and pancreas. CCL21 has been described as a constitutive chemokine but its expression is also known to be induced during different inflammatory conditions. mRNA of CCL21 and protein have been shown to be up-regulated in IPF lung tissue in fibrotic foci when compared to normal controls for CCL21 and CCL19 as well as for their cognate receptor CCR7. CCR7 mRNA levels have also been shown to be elevated in fibroblasts isolated from IPF versus control tissue. Functionally, CCL21 directs the movement of fibroblasts isolated from IPF lung tissue and promotes fibroblast proliferation. Fibroblasts secrete the precursors of the extracellular matrix. They provide a structural framework for any tissues, and play a critical role in wound healing.

Commercially available anti-CCL21 mAb (R&D systems) reduces fibrotic response in a humanized SCID mouse model of IPF. In this model human lung fibroblasts from IPF patients are transferred to SCID mice which develop a fibrotic response in the lung. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, Pierce et al. injected 1 .0 x 106 primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia(IPF/UIP), nonspecific interstitial pneumonia (NSIP), or histologically normal biopsies intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after IPF/UIP fibroblast injection, patchy interstitial fibrosis and increased

hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred NSIP fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization, of either human CCR7 or CCL21 with an anti-human CCL21 or an anti-CCR7 monoclonal isolated antibody (R&D systems), significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of UIP fibroblasts. Thus, this study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CCL21 and CCR7.

3. Summary of the InventionThe present invention therefore provides an isolated

immunoglobulin that binds (e.g. specifically binds) with CCL21 , particularly human CCL21

(hCCL21 ) and modulates (for example, inhibits) the interaction between CCL21 and its cognate receptor CCR7, and in particular human CCR7 (hCCR7).

The term "binding molecule" in the context of this invention generally means a biological molecule with a binding affinity for a target molecule. For the purposes of the present invention the target molecule is CCL21 , particularly human CCL21. Typically, the binding molecule will be an immunoglobulin, for example an antibody, fragment thereof, or derivative thereof. Alternative binding molecules are contemplated, including, but not limited to an Adnectin, Ankyrin, Avimer, Affibody, Anticalin, or Affilin.

The term "isolated" means throughout this specification, that the binding molecule

immunoglobulin, antibody or polynucleotide, as the case maybe, exists in a physical milieu distinct from that in which it may occur in nature.

In one embodiment of the invention there is provided a binding molecule that binds (e.g.

specifically binds) with CCL21 , particularly human CCL21 (hCCL21 ) and modulates (e.g.

inhibits) the interaction between hCCL21 and hCCR7.

Preferably, the binding molecule also binds (e.g. specifically binds) with cynomolgus CCL21 and does not bind (e.g. specifically bind) mouse CCL21 , or human CCL19. More preferably the binding molecule and neutralises the biological activity of human CCL21. Even more preferably the binding molecule inhibits the interaction between hCCL21 and hCCR7.

In preferred embodiments the binding molecule binds (e.g. specifically binds) with an epitope of human CCL21 (hCCL21 ), wherein the epitope: a) is comprised within the following amino acid residues from the sequence of CCL21 as defined with reference to SEQ ID No 53: i) aa33-aa49 and aa60-aa90, ii) aa33-aa49 and aa77-aa90, iii) aa35, aa38, aa39, aa48, aa62, aa64, aa65, aa67, aa74, aa77, aa82, aa86, aa87, aa90, iv) aa35, aa39, aa40, aa43-45, aa47, aa62, aa64, aa66, aa77, aa82-87, aa90, v) aa35, aa43, aa48, aa62, aa64, aa77, aa81 , aa82, aa84-88, aa90, vi) aa35, aa43, aa77, aa80-aa82, aa84-aa86, aa88-aa90, vii) aa43, aa81 , aa82, aa84, aa85, aa88, aa90, viii) aa43, aa81 , aa84, aa85, aa88, ix) aa38, aa64, aa74, aa82, aa86, aa90, x) aa35, aa43, aa64, aa81 , aa82, aa85, aa88, or xi) aa40, aa43, aa45, aa47, aa62, aa64, aa77, aa82, aa83, aa86, aa90; b) comprises at least one, two, three, four, five, or six of the amino acid residues as defined in any one of the groups (i) to (xi) listed above; or c) comprises the amino acid residues as defined in any one of the groups (i) to (xi) listed above.

The present invention also provides a binding molecule that binds (e.g. specifically binds) with CCL21 , particularly hCCL21 and inhibits (e.g. competitively) the binding to hCCL21 of the binding molecule as described above, preferably wherein the inhibition is detectable at comparable (e.g. equimolar) conditions.

In preferred embodiments the binding molecule is a) an immunoglobulin, or b) an Adnectin, Ankyrin, Avimer, Affibody, Anticalin, or Affilin.

In preferred embodiments the binding molecule is an immunoglobulin and the immunoglobulin is an antibody.

In preferred embodiments the binding molecule is isolated.

In preferred embodiments the binding molecule comprises CDRH3 of SEQ.I.D.NO:3 or 25 or 35 or 45. In more preferred embodiments the binding molecule comprises;

(a) CDRH1 of SEQ.I.D.NO:1 or 23 or 33 or 43; (b) CDRH2 of SEQ.I.D.NO:2 or 24 or 34 or 44;

(c) CDRH3 of SEQ.I.D.NO:3 or 25 or 35 or 45.

In even more preferred embodiments the binding molecule also comprises;

(d) CDRL1 of SEQ.I.D.NO:4 or 26 or 46

(e) CDRL2 of SEQ.I.D.NO:5 or 27 or 47 (f) CDRL3 of SEQ.I.D.NO:6 or 28 or 48.

In preferred embodiments the binding molecule is an antibody which comprises a heavy chain variable domain of SEQ.I.D.NO: 7 or 8 or 29 or 39 or 49. In more preferred embodiments the heavy chain variable region is combined with a light chain variable region to form an hCCL21 binding site.

In preferred embodiments the binding molecule comprises a light chain variable region of SEQ.I.D.NO: 9 or 10 or 30 or 40 or 50.

In preferred embodiments the binding molecule comprises a heavy chain of SEQ.I.D.NO:1 1 or 12 or 31 or 41 or 51. In preferred embodiments the binding molecule which comprises a light chain of SEQ.I.D.NO:13 or 14 or 32 or 42 or 52.

In preferred embodiments the binding molecule comprises

(a) a heavy chain of SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:13;

(b) a heavy chain of SEQ.I.D.NO:31 and a light chain of SEQ.I.D.NO:32. (c) a heavy chain of SEQ.I.D.NO:51 and a light chain of SEQ.I.D.NO:52. In more preferred embodiments the binding molecule comprises a heavy chain of SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:14.

In preferred embodiments the binding molecule comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:13. In preferred embodiments the binding molecule comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:14.

In particularly preferred embodiments the binding molecule is an intact antibody. More preferably, the antibody is a human, humanised or chimeric antibody (e.g. having a human IgG such as lgG1 or lgG4 constant region). Most preferably the antibody is a human antibody. In alternative preferred embodiments the binding molecule is an antibody fragment or a single variable domain. More preferably the antibody fragment is a ScFv, Fab, Fab , F(ab )2. Even more preferably the single variable domain is a dAb or VHH. In alternative preferred embodiments the binding molecule is a bispecific antibody comprising a first specificity to hCCL21 and a second specificity to e.g. hCCL19.

In preferred embodiments the binding molecule is an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. by inhibiting the interaction between hCCL21 and hCCR7) said antibody comprising at least a heavy chain variable region capable of being encoded by a polynucleotide having at least 90% (such as 95% or greater, e.g. 96%, 97%, 98% or 99%) identity to SEQ.I.D.NO:15 or 16.

In preferred embodiments the binding molecule is an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. by inhibiting the interaction between hCCL21 and hCCR7) said antibody comprising at least a heavy chain variable region capable of being encoded by a polynucleotide having at least 90% (such as 95% or greater, e.g. 96%, 97%, 98% or 99%) identity to SEQ.I.D.NO:15 or 16 and a light chain variable region capable of being encoded by a polynucleotide having at least 90% (such as 95% or greater, e.g. 96%, 97%, 98% or 99%) identity to SEQ.I.D.NO:17 or 18. Also provided is a polynucleotide encoding the heavy chain variable region of any one of the preceding claims.

Also provided is a polynucleotide encoding the light chain variable region of any one the preceding claims.

Also provided is a composition comprising both of the polynucleotides described above. Also provided is a polynucleotide wherein the heavy chain variable region polynucleotide is SEQ.I.D.NO:15 or 16.

Also provided is a polynucleotide wherein the light chain variable region polynucleotide is SEQ.I.D.NO:17 or 18.

Also provided is a polynucleotide encoding the heavy chain and/or the light chain of any one of the antibodies described above. Preferably the polynucleotide is SEQ.I.D.NO:19 or 20, or SEQ.I.D.NO:21 or 22.

In preferred embodiments the polynucleotide of the invention is at least 90% (such as 95% or greater, e.g. 96%, 97%, 97% or 99%) identical to SEQ.I.D.NO:15 or 16. In alternative preferred embodiments the polynucleotide of the invention is at least 90% (such as 95% or greater, e.g. 96%, 97%, 97% or 99%) identical to SEQ.I.D.NO:17 or 18. Also provided is a composition comprising both of these polynucleotides.

Also provided is a vector comprising the any one of the polynucleotides described above, or a stably transformed or transfected host cell comprising such a vector. Preferably the host cell of is a mammalian cell, more preferably the host cell is from a CHO, NS0, or Y2/0 cell line.

The invention also provides a method for the production of an antibody which method comprises culturing a host cell as described above under conditions permissive for the production said antibody. Preferably the method comprises culturing said host cell in serum-free media. More preferably the method further comprises recovering said antibody.

Also provided is a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) the binding molecule, immunoglobulin, antibody or antibody fragment as described above. The pharmaceutical composition may preferably provided in intravenously or sub- cutaneously administrable form. In preferred embodiments of the pharmaceutical composition the binding molecule, immunoglobulin, antibody or antibody fragment is in lyophilised form.

Also provided is the binding molecule, immunoglobulin, antibody or antibody fragment or a pharmaceutical composition as defined herein for use in treating and/or preventing (e.g.

inhibiting the clinical progression) of a human disease or disorder.

Preferably the binding molecule, immunoglobulin, antibody, antibody fragment or

pharmaceutical composition of the invention is for use in treating and/or preventing (e.g.

inhibiting the clinical progression) pulmonary fibrosis (e.g. IPF), the exacerbation of IPF, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post-ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof, in a mammalian patient (such as a human and/or a domesticated (e.g. companion) mammal.

Also provided is use of a binding molecule, immunoglobulin, antibody or antibody fragment, or a pharmaceutical composition according to the invention in the manufacture of a medicament for use in a method of treating and/or preventing (e.g. inhibiting the clinical progression) of a human disease or disorder.

Preferably such use of a binding molecule, immunoglobulin or antibody or pharmaceutical composition according is for the manufacture of a medicament for use when the method is a method of treating and/or preventing (e.g. inhibiting the clinical progression) pulmonary fibrosis (e.g. IPF), the exacerbation of IPF, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with

Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post-ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof, in a mammalian patient (such as a human and/or a domesticated (e.g. companion) mammal.

Also provided is a method of treating and/or preventing (e.g. inhibiting the clinical progression) in a human patient afflicted with pulmonary fibrosis (e.g. IPF), the exacerbation of IPF, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post-ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof which method comprises administering to said patient a therapeutically effective amount of the pharmaceutical composition of the invention.

Also provided is a complex comprising CCL21 , and a binding molecule, immunoglobulin, antibody or antibody fragment according to the invention. Preferably the CCL21 is human CCL21 or cynomolgus CCL21 . In preferred embodiments the binding molecule,

immunoglobulin, antibody or antibody fragment binds to CCL21 at an epitope as defined in above.

In one aspect of the invention, there is provided an isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7). In one embodiment of this aspect, the isolated antibody "specifically binds" with hCCL21 , that is, binds hCCL21 and other primate CCL21 (such as cynomolgus CCL21 (cCCL21 )) and inhibits the interaction between the CCL21 ligand and its cognate CCR7 receptor but does not cross-react to any appreciable extent with other human chemokine ligands.

The term "neutralises" and grammatical variations thereof means throughout this specification, that the biological activity of the target is reduced either totally or partially in the presence of the immunoglobulin or antibody, as the case maybe.

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH3 of

SEQ.I.D.NO:3.

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH3 of

SEQ.I.D.NO:25.

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH3 of

SEQ.I.D.NO:35. In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH3 of

SEQ.I.D.NO:45 In another aspect of the invention there is provided an isolated antibody that binds hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises three light chain CDRs and three heavy chain CDRs wherein;

CDRH 1 is set forth in SEQ.I.D.NO:1 CDRH2 is set forth in SEQ.I.D.NO:2

CDRH3 is set forth in SEQ.I.D.NO:3

CDRL1 is set forth in SEQ.I.D.NO:4

CDRL2 is set forth in SEQ.I.D.NO:5

CDRL3 is set forth in SEQ.I.D.NO:6 In another aspect of the invention there is provided an isolated antibody that binds hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises three light chain CDRs and three heavy chain CDRs wherein;

CDRH1 is set forth in SEQ.I.D.NO:23 CDRH2 is set forth in SEQ.I.D.NO:24

CDRH3 is set forth in SEQ.I.D.NO:25

CDRL1 is set forth in SEQ.I.D.NO:26

CDRL2 is set forth in SEQ.I.D.NO:27

CDRL3 is set forth in SEQ.I.D.NO:28 In another aspect of the invention there is provided an isolated antibody that binds hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises three light chain CDRs and three heavy chain CDRs wherein; CDRH1 is set forth in SEQ.I.D.NO:33

CDRH2 is set forth in SEQ.I.D.NO:34

CDRH3 is set forth in SEQ.I.D.NO:35

CDRL1 is set forth in SEQ.I.D.NO:36

CDRL2 is set forth in SEQ.I.D.NO:37 CDRL3 is set forth in SEQ.I.D.NO:38

In another aspect of the invention there is provided an isolated antibody that binds hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises three light chain CDRs and three heavy chain CDRs wherein; CDRH1 is set forth in SEQ.I.D.NO:43

CDRH2 is set forth in SEQ.I.D.NO:44

CDRH3 is set forth in SEQ.I.D.NO:45

CDRL1 is set forth in SEQ.I.D.NO:46

CDRL2 is set forth in SEQ.I.D.NO:47 CDRL3 is set forth in SEQ.I.D.NO:48

Throughout this specification, complementarity determining region ("CDR") is defined according to the Kabat definition with the exception of CDRH1 of SEQ.I.D.NO:1 which is the stretch of amino acids defined by a combination of both Kabat and Chothia definitions for this CDR.

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable domain of SEQ.I.D.NO:7 or 8 and a light chain variable region of SEQ.I.D.NO:9 or SEQ.I.D.NO: 10. In one embodiment of this aspect, the isolated antibody further comprises a heavy chain constant region (e.g. human heavy chain constant region) that maybe of an IgG isotype (such as lgG1 or lgG4).

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable domain of SEQ.I.D.NO:29 and a light chain variable region of SEQ.I.D.NO:30. In one embodiment of this aspect, the isolated antibody further comprises a heavy chain constant region (e.g. human heavy chain constant region) that maybe of an IgG isotype (such as lgG1 or lgG4).

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable domain of SEQ.I.D.NO:39 and a light chain variable region of SEQ.I.D.NO:40. In one embodiment of this aspect, the isolated antibody further comprises a heavy chain constant region (e.g. human heavy chain constant region) that maybe of an IgG isotype (such as lgG1 or lgG4). In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable domain of SEQ.I.D.NO:49 and a light chain variable region of SEQ.I.D.NO:50. In one embodiment of this aspect, the isolated antibody further comprises a heavy chain constant region (e.g. human heavy chain constant region) that maybe of an IgG isotype (such as lgG1 or lgG4).

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain having three heavy chain CDRs wherein;

CDRH 1 is set forth in SEQ.I.D.NO:1 CDRH2 is set forth in SEQ.I.D.NO:2 CDRH3 is set forth in SEQ.I.D.NO:3.

In another aspect of the invention there is provided an isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain having three heavy chain CDRs wherein;

CDRH 1 is set forth in SEQ.I.D.NO:23

CDRH2 is set forth in SEQ.I.D.NO:24

CDRH3 is set forth in SEQ.I.D.NO:25. In another aspect of the invention there is provided an isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain having three heavy chain CDRs wherein;

CDRH 1 is set forth in SEQ.I.D.NO:33 CDRH2 is set forth in SEQ.I.D.NO:34

CDRH3 is set forth in SEQ.I.D.NO:35.

In another aspect of the invention there is provided an isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain having three heavy chain CDRs wherein;

CDRH1 is set forth in SEQ.I.D.NO:43

CDRH2 is set forth in SEQ.I.D.NO:44

CDRH3 is set forth in SEQ.I.D.NO:45.

In some embodiments of these aspects of the invention, the heavy chain is combined with a light chain to form a hCCL21 binding site. In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a light chain having three light chain CDRs wherein; CDRL1 is set forth in SEQ.I.D.NO:4

CDRL2 is set forth in SEQ.I.D.NO:5

CDRL3 is set forth in SEQ.I.D.NO:6.

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a light chain having three light chain CDRs wherein;

CDRL1 is set forth in SEQ.I.D.NO:26

CDRL2 is set forth in SEQ.I.D.NO:27

CDRL3 is set forth in SEQ.I.D.NO:28 In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a light chain having three light chain CDRs wherein;

CDRL1 is set forth in SEQ.I.D.NO:36 CDRL2 is set forth in SEQ.I.D.NO:37

CDRL3 is set forth in SEQ.I.D.NO:38

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a light chain having three light chain CDRs wherein;

CDRL1 is set forth in SEQ.I.D.NO:46 CDRL2 is set forth in SEQ.I.D.NO:47

CDRL3 is set forth in SEQ.I.D.NO:48

In some embodiments of these aspects of the invention, the light chain is combined with a heavy chain to form a hCCL21 binding site. In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7),said isolated antibody comprises a heavy chain of SEQ.I.D.NO:1 1 or 12. In one embodiment of this aspect of the invention, the heavy chain is combined with a light chain to form an hCCL21 binding site. In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a light chain of SEQ.I.D.NO:13 or SEQ.I.D.NO:14 or 32 or 42 or 52. In some embodiments of this aspect of the invention, the light chain is combined with a heavy chain to form a hCCL21 binding site. In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:1 1 or 12 or 31 or 41 or 51 and a light chain of SEQ.I.D.NO:13 or SEQ.I.D.NO:14 or 32 or 42 or 52. In another aspect of the invention, there is provided a pharmaceutical composition comprising (for example as its sole therapeutically active ingredient) an isolated antibody or isolated immunoglobulin of the invention as set forth herein.

In another aspect of the invention, there is provided a pharmaceutical composition comprising (for example as its sole therapeutically active ingredient) an isolated antibody or isolated immunoglobulin of the invention as set forth herein for use in therapy, such as the prevention and/or treatment of human disease.

In another aspect of the invention, there is provided a pharmaceutical composition comprising (for example as its sole therapeutically active ingredient) an isolated antibody or isolated immunoglobulin of the invention as set forth herein for use in the treatment and/or prevention of a fibrotic disease or disorder in humans.

In another aspect of the invention, there is provided a pharmaceutical composition comprising (for example as its sole therapeutically active ingredient) an isolated antibody or isolated immunoglobulin of the invention as set forth herein for use in the treatment and/or prevention of a fibrotic disease or disorder such as liver, kidney or a pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF) in humans.

In another aspect of the invention, there is provided a method of treating and/or preventing (for example preventing or inhibiting clinical progression) a fibrotic disease or disorder (such as liver, kidney and/or a pulmonary fibrotic disease or disorder e.g. IPF) in a human subject which method comprises administering a therapeutically effective amount of an isolated antibody or immunoglobulin as set forth herein.

In another aspect of the invention there is provided an isolated polynucleotide encoding a heavy chain variable region having the heavy chain CDRs as set forth in SEQ.I.D.NO:1 , 2 and 3. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a heavy chain constant region thereby encoding a heavy chain comprising heavy chain CDRs as set forth in SEQ.I.D.NO:1 , 2 and 3.

In another aspect of the invention there is provided an isolated polynucleotide encoding a heavy chain variable region having the heavy chain CDRs as set forth in SEQ.I.D.NO:23, 24 and 25. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a heavy chain constant region thereby encoding a heavy chain comprising heavy chain CDRs as set forth in SEQ.I.D.NO:23, 24 and 25.

In another aspect of the invention there is provided an isolated polynucleotide encoding a heavy chain variable region having the heavy chain CDRs as set forth in SEQ.I.D.NO:33, 34 and 35. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a heavy chain constant region thereby encoding a heavy chain comprising heavy chain CDRs as set forth in SEQ.I.D.NO:33, 34 and 35.

In another aspect of the invention there is provided an isolated polynucleotide encoding a heavy chain variable region having the heavy chain CDRs as set forth in SEQ.I.D.NO:43, 44 and 45. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a heavy chain constant region thereby encoding a heavy chain comprising heavy chain CDRs as set forth in SEQ.I.D.NO:43, 44 and 45.

In another aspect of the invention, there is provided an isolated polynucleotide encoding a light chain variable region having the light chains set forth in SEQ.I.D.NO:4, 5 and 6. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a light chain constant region thereby encoding a light chain comprising light chain CDRs as set forth in SEQ.I.D.NO:4, 5 and 6.

In another aspect of the invention, there is provided an isolated polynucleotide encoding a light chain variable region having the light chains set forth in SEQ.I.D.NO:26, 27 and 28. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a light chain constant region thereby encoding a light chain comprising light chain CDRs as set forth in SEQ.I.D.NO:26, 27 and 28.

In another aspect of the invention, there is provided an isolated polynucleotide encoding a light chain variable region having the light chains set forth in SEQ.I.D.NO:36, 37 and 38. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a light chain constant region thereby encoding a light chain comprising light chain CDRs as set forth in SEQ.I.D.NO:36, 37 and 38.

In another aspect of the invention, there is provided an isolated polynucleotide encoding a light chain variable region having the light chains set forth in SEQ.I.D.NO:46, 47 and 48. In one embodiment of this aspect, the isolated polynucleotide is (operatively) linked with another isolated polynucleotide that encodes a light chain constant region thereby encoding a light chain comprising light chain CDRs as set forth in SEQ.I.D.NO:46, 47 and 48.

In another aspect of the invention there is provided an isolated polynucleotide (such as

SEQ. I. D.NO:15) encoding a heavy chain of SEQ. I. D. NO: 1 1 or SEQ.I.D.NO:12 or 31 or 41 or 51.

In another aspect of the invention there is provided an isolated polynucleotide (such as

SEQ.I.D.NO:16) encoding a light chain of SEQ.I.D.NO: 13 or SEQ.I.D.NO:14 or 32 or 42 or 52. In another aspect of the invention there is provided a composition comprising a first isolated polynucleotide (such as SEQ.I.D.NO:15 or 16) encoding a heavy chain of SEQ.I.D.NO: 1 1 or SEQ.I.D.NO:12 or 31 or 41 or 42 and a second isolated polynucleotide (such as

SEQ.I.D.NO:16) encoding a light chain of SEQ.I.D.NO: 13 or SEQ.I.D.NO:14 or 32 or 42 or 52. In another aspect of the invention there is provided an isolated polynucleotide encoding a heavy chain variable region of an isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 7 or 8 or 29 or 39 or 49. In a related aspect of the invention, there is also provided an isolated polynucleotide encoding a light chain variable region of an isolated antibody that binds (e.g. specifically binds) hCCL21 , said isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide capable of encoding SEQ.I.D.NO: 9 or 10 or 30 or 40 or 50. In a further related aspect of the invention there is provided a composition comprising an isolated polynucleotide encoding a heavy chain variable region of an isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 7 or 8 or 29 or 39 or 49 and an isolated polynucleotide encoding a light chain variable region of an isolated antibody that binds (e.g. specifically binds) hCCL21 , said isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to a isolated polynucleotide capable of encoding SEQ.I.D.NO: 9 or 10 or 30 or 40 or 50.

The term "identity" refers to the similarity between at least two different sequences. This identity can be expressed as a percent identity and determined by standard alignment algorithms, for example, the Basic Local Alignment Tool (BLAST) described by Altshul et al. ((1990) J. Mol. Biol., 215: 403-410); the algorithm of Needleman et al. ((1970) J. Mol. Biol., 48: 444-453); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4: 1 1 -17). A set of parameters may be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity is usually calculated by comparing sequences of similar length.

In another aspect of the invention there is provided a heavy chain variable region that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said heavy chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 7 or 8 or 29 or 39 or 49. The heavy chain variable region of this aspect maybe combined with a light chain variable region such as SEQ.I.D.NO:13 or SEQ.I.D.NO:14 or 30 or 40 or 50 to form an antigen binding site that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7).

In another aspect of the invention there is provided an isolated antibody heavy chain that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said heavy chain capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 1 1 or 12 or 31 or 41 or 51 . The heavy chain of this aspect maybe combined with a light chain such as SEQ.I.D.NO:13 or SEQ.I.D.NO:14 or 32 or 42 or 52 to form an antigen binding site that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7).

In another aspect of the invention there is provided an isolated antibody that binds (e.g.

specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprising;

(a) a heavy chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 7 or 8 or 29 or 39 or 49 and

(b) a light chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 9 or 10 or 30 or 40 or 50. In one embodiment of this aspect of the invention the isolated antibody further comprises a heavy chain constant region (particularly a human heavy chain constant region of an IgG isotype such as lgG1 or lgG4) and/or a light chain constant region (particularly a human lambda or kappa light chain constant region). In another aspect of the invention, there is provided a stably transformed or transfected host cell comprising an isolated polynucleotide of invention as described herein.

In another aspect of the invention there is provided a process for the production of an isolated antibody of the invention, said process comprising culturing a host cell as described herein under conditions permissive for the production of an isolated antibody of the invention. 4. Brief Description of the Drawings

Figure 1 : Inhibition of recombinant hCCL21 -mediated migration of H9 cells (non germlined and control IgGs).

Figure 2: Inhibition of recombinant hCCL21 -mediated migration of H9 cells (germlined IgGs).

Figure 3: Inhibition of recombinant cCCL21 -mediated migration of H9 cells (non-germlined and control IgGs)

Figure 4: Inhibition of recombinant cCCL21 -mediated migration of H9 cells (germlined IgGs).

Figure 5: Inhibition of natural hCCL21 -mediated migration of H9 cells (germlined and non- germlined IgGs).

Figure 6: Inhibition of recombinant hCCL21 -mediated migration of primary human T cells (non- germlined and control IgGs)

Figure 7: Inhibition of migration of recombinant hCCL21 -mediated primary human T cells (germlined IgGs)

Figure 8: Inhibition of CCL21 mediated chemotaxis by MOR06935_g3-23_g3j

Figure 9: Inhibition of CCL21 mediated chemotaxis by MOR08378_g5-51_g2a2 Figure 10: Inhibition of CCL21 mediated chemotaxis by MOR08382_g3-23_gL6

Figure 1 1 : Inhibition of CCL21 mediated chemotaxis by MOR08389_g3-23_g3j Figure 12: Inhibition of CCL21 induced calcium mobilization by MOR06935_g3-23_g3j in stably transfected CHO-K1 cells

Figure 13: The sequence and predicted secondary structure of huCCL21 , helices and beta sheets marked (part A) and the 3D model of huCCL21 , disulfide bridge between Cys31 -Cys57, and Cys32-Cys75 marked (part B)

Figure 14: "Dot blot" showing strong and consistent binding of huCCL21 by MOR06935_g3- 23_g3j under native conditions, or following treatment of huCCL21 by heat, heat and reducing conditions, or heat, reducing conditions and SDS detergent.

Figure 15: Antigen mutation screening, library design (part A) and colony filter screening results (part B and C). In part B, circles indicate no binding between Fab antl-CCL21 and the relevant CCL21 variant, square boxes indicate weak binding.

Figure 16: H/DxMS and AMS results for MOR06935_g3-23_g3j (also known as QBP359) summarized and mapped onto the CCL21 structural model. Part A shows regions identified by H/DxMS are indicated in red and yellow. Part B shows residues identified by AMS are in red and yellow, red indicates strong indication of involvement in binding, yellow probable indication of involvement in binding (all these residues belong to the regions previously identified by H/DxMS). Part C shows the data as for part B, but with colouring removed for residues that are part of the hydrophobic core and are not exposed on the surface of the molecule. Part D shows the residues identified by AMS which are not part of the hydrophobic core are compared to the mouse sequence. Residues in cyan color differ from human to mouse (K43S, D77N, L81 G, Q85N, Q88R, H89R), the same positions are identical between human and cynomolgus sequences.

Figure 17: AMS results summarized for all four tested antibodies and mapped onto the CCL21 structural model. Residues identified by AMS that affect binding are colored in red and yellow as shown in slide 4. Residues which are part of the hydrophobic core are in grey. Part A shows results for MOR08382_g3-23_gl_6. Part B shows results for MOR08389_g3-23_g3j. Part C shows results for MOR06935_g3-23_g3j (also known as QBP359). Part D shows results for MOR08378_g5-51 g2a2

5. Detailed Description of the Invention. The present invention provides a binding molecule which is preferably an immunoglobulin, and more preferably an antibody or antibody fragment. The binding molecule may also be a non- antibody molecule, such as an Adnectin, Ankyrin, Avimer, Affibody, Anticalin, or Affilin. The binding molecule, immunoglobulin, antibody, antibody fragment etc. of the present invention may be in any of a number of formats well known to the skilled person. These formats include intact antibodies, various isolated antibody fragments such as domain antibodies and other engineered formats as described below. In preferred forms of all embodiments of the invention described herein, antibodies of the present invention are provided as a monoclonal

population.5.1.1.1 - Intact antibodies Intact antibodies include heteromultimeric glycoproteins comprising at least two heavy and two light chains. Aside from IgM, intact antibodies are usually heterotetrameric glycoproteins of approximately 150 to 200 KDa, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond while the number of disulfide linkages between the heavy chains of different

immunoglobulin isotypes varies. Each heavy and light chain also has intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant regions. Each light chain has a variable domain (VL) and a constant region at its other end; the constant region of the light chain is aligned with the first constant region of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. The light chains of antibodies from most vertebrate species can be assigned to one of two types called Kappa or Lambda based on the amino acid sequence of the constant region. Depending on the amino acid sequence of the constant region of their heavy chains, human antibodies can be assigned to five different classes, IgA, IgD, IgE, IgG and IgM. IgG and IgA can be further subdivided into subclasses, lgG1 , lgG2, lgG3 and lgG4; and lgA1 and lgA2. Species variants exist with mouse and rat having at least lgG2a, lgG2b. The variable domain of the isolated antibody confers binding specificity upon the isolated antibody with certain regions displaying particular variability called complementarity determining regions (CDRs). The more conserved portions of the variable region are called Framework regions (FR). The variable domains of intact heavy and light chains each comprise four FR connected by three CDRs. The CDRs in each chain are held together in close proximity by the FR regions and with the CDRs from other chain contribute to the formation of the antigen binding site of antibodies. The constant regions are not directly involved in the binding of the isolated antibody to the antigen but exhibit various effector functions such as participation in isolated antibody dependent cell-mediated cytoxicity (ADCC), phagocytosis via binding to Fcyr receptor, half-life/clearance rate via neonatal Fc receptor (FcRn) and complement dependent cytoxicity via the C1 q component of the complement cascade.

In one embodiment of the invention there is provided an isolated intact antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprising a CDRH3 of SEQ.I.D.NO:3.

In another embodiment of the invention there is provided an isolated intact antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7) which isolated antibody comprises a heavy chain variable region comprising CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, and CDRH3 of SEQ.I.D.NO:3 together with a light chain that combine to form a CCL21 binding site.

In another embodiment of the invention there is provided an isolated intact antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7) which isolated antibody comprises a heavy chain variable region comprising CDRH1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25 or CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35 or CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45 together with a light chain that combine to form a CCL21 binding site. In another embodiment there is provided an isolated intact antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7) which isolated antibody comprises: a heavy chain variable region comprising CDRH1 of SEQ.I.D.NO:1 , CDRH2 of

SEQ.I.D.NO:2, and CDRH3 of SEQ.I.D.NO:3 together with a light chain variable region comprising CDRL1 of SEQ.I.D.NO:4, CDRL2 of SEQ.I.D.NO:5 and CDRL3 of

SEQ.I.D.NO:6 that combine to form a CCL21 binding site or a heavy chain variable region comprising CDRH1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25 together with a light chain variable region comprising CDRL1 of SEQ.I.D.NO:26, CDRL2 of SEQ.I.D.NO:27 and CDRL3 of SEQ.I.D.NO:28 that combine to form a CCL21 binding site or a heavy chain variable region comprising CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35 together with a light chain variable region comprising CDRL1 of SEQ.I.D.NO:36, CDRL2 of SEQ.I.D.NO:37 and CDRL3 of SEQ.I.D.NO:38 that combine to form a CCL21 binding site or a heavy chain variable region comprising CDRH1 of SEQ.I.D.NO:43, CDRH2 of

SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45 together with a light chain variable region comprising CDRL1 of SEQ.I.D.NO:46, CDRL2 of SEQ.I.D.NO:47 and CDRL3 of SEQ.I.D.NO:48 that combine to form a CCL21 binding site.

Typically, the intact isolated antibody comprises a primate, and in particular a human constant region of an IgG isotype, e.g. lgG1 or lgG4 and is an isolated human, humanised or chimeric antibody as described below. The isolated intact antibody may display lytic or non-lytic actvity (i.e. may have CDC and/or ADCC activity).

5.1.1.1.1 Human antibodies Human antibodies may be produced by a number of methods known to those of skill in the art. Human antibodies can be made by the hybridoma method using human myeloma or mouse- human heteromyeloma cells lines see Kozbor J. Immunol 133, 3001 , (1984) and Brodeur, Monoclonal Isolated antibody Production Techniques and Applications, pp51 -63 (Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice both of which utilize human V region repertories (see Winter G, (1994), Annu. Rev. Immunol. 12, 433- 455, Green LL (1999), J. Immunol. Methods 231 , 1 1 -23).

Several strains of transgenic mice are now available wherein their mouse immunoglobulin loci has been replaced with human immunoglobulin gene segments (see Tomizuka K, (2000) PNAS 97, 722-727; Fishwild DM (1996) Nature Biotechnol. 14, 845-851. Mendez MJ, 1997, Nature Genetics, 15, 146-156). Upon antigen challenge such mice are capable of producing a repertoire of human antibodies from which antibodies of interest can be selected. Of particular note is the Trimera™ system (see Eren R et al, (1988) Immunology 93:154-161 ) where human lymphocytes are transplanted into irradiated mice, the Selected Lymphocyte Isolated antibody System (SLAM, see Babcook et al, PNAS (1996) 93: 7843-7848) where human (or other species) lymphocytes are effectively put through a massive pooled in vitro isolated antibody generation procedure followed by deconvulated, limiting dilution and selection procedure and the Xenomouse™ (Abgenix Inc). An alternative approach is available from Morphotek Inc using the Morphodoma™ technology.

Phage display technology can be used to produce human antibodies (and fragments thereof), see McCafferty; Nature, 348, 552-553 (1990) and Griffiths AD ei a/ (1994) EMBO 13: 3245- 3260. According to this technique isolated antibody V domain genes are cloned in frame into either a major or minor coat of protein gene of a filamentous bacteriophage such as M13 or fd and displayed (usually with the aid of a helper phage) as function isolated antibody fragments on the surface of the phage particle. Selections based on the function properties of the isolated antibody result in selection of the gene encoding the isolated antibody exhibiting these properties. The phage display technique can be used to select antigen specific antibodies from libraries made from human B cells taken from individuals afflicted with a disease or disorder or alternatively from unimmunized human donors (see Marks; J Mol Bio 222, 581 -591 , 1991 ). Where an intact human isolated antibody is desired comprising an Fc domain it is necessary redone the phage displayed derived fragment into a mammalian expression vectors comprising the desired constant regions and establishing stable expressing cell lines.

The technique of affinity maturation (Marks; Bio/technol 10, 779-783 (1992)) may be used to provide binding affinity wherein the affinity of the primary human isolated antibody is improved by sequentially replacing the H and L chain V regions with naturally occurring variants and selecting on the basis of improved binding affinities. Variants of this technique such as 'epitope imprinting' are now also available, see WO 93/06213. See also Waterhouse; Nucl Acids Res 21 , 2265-2266 (1993).

In one embodiment of the invention there is provided an isolated intact human antibody that binds (e.g. specifically binds) with hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a CDRH3 of SEQ.I.D.NO:3 or SEQ.I.D.NO:25 or SEQ.I.D.NO:35 or SEQ.I.D.NO:45. The isolated antibody may comprise a human constant region, e.g. an IgG constant region (such as an lgG1 or lgG4). In another embodiment of the invention there is provided an isolated intact human antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRH3 of SEQ.I.D.NO:3 or CDRH1 of

SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25 or CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35 or CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45. The isolated antibody may comprise a IgG constant region (e.g. of an lgG1 or lgG4 isotype)

In another embodiment of the invention there is provided an isolated intact human antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises; (a) CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRH3 of SEQ.I.D.NO:3;

(b) CDRL1 of SEQ.I.D.NO:4, CDRL2 of SEQ.I.D.NO:5, CDRL3 of SEQ.I.D.NO:6 or

(c) CDRH 1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25;

(d) CDRL1 of SEQ.I.D.NO:26, CDRL2 of SEQ.I.D.NO:27 and CDRL3 of SEQ.I.D.NO:28 or

(e) CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35; (f) CDRL1 of SEQ.I.D.NO:36, CDRL2 of SEQ.I.D.NO:37 and CDRL3 of SEQ.I.D.NO:38 or

(g) CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45;

(h) CDRL1 of SEQ.I.D.NO:46, CDRL2 of SEQ.I.D.NO:47 and CDRL3 of SEQ.I.D.NO:48.

The isolated antibody may comprise a human IgG constant region (e.g. of an lgG1 or lgG4 isotype).

In another embodiment of the invention there is provided an isolated intact human antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 or SEQ.I.D.NO:29 or SEQ.I.D.NO:39 SEQ.I.D.NO:49. The isolated antibody may comprise a IgG constant region (e.g. of an lgG1 or lgG4 isotype). In another embodiment of the invention there is provided an isolated intact human antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable region of SEQ.I.D.NO:8. The isolated antibody may comprise a IgG constant region (e.g. of an lgG1 or lgG4 isotype).

In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 and a light chain variable region of SEQ.I.D.NO:9 or a heavy chain variable region of SEQ.I.D.NO:29 and a light chain variable region of SEQ.I.D.NO:30 or a heavy chain variable region of SEQ.I.D.NO:39 and a light chain variable region of SEQ.I.D.NO:40 or a heavy chain variable region of SEQ.I.D.NO:49 and a light chain variable region of SEQ.I.D.NO:50. The isolated antibody may comprise a human IgG constant region (e.g. of an lgG1 or lgG4 isotype). In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 and a light chain variable region of SEQ.I.D.NO:10. The isolated antibody may comprise a IgG constant region (e.g. of an lgG1 or lgG4 isotype). In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable region of SEQ.I.D.NO:8 and a light chain variable region of SEQ.I.D.NO:9. The isolated antibody may comprise a IgG constant region (e.g. of an lgG1 or lgG4 isotype). In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain variable region of SEQ.I.D.NO:8 and a light chain variable region of SEQ.I.D.NO:10. The isolated antibody may comprise a IgG constant region (e.g. of an lgG1 or lgG4 isotype). In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:1 1 or of SEQ.I.D.NO:31 or of SEQ.I.D.NO:41 or of SEQ.I.D.NO:51.

In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:12.

In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:13 or a heavy chain of SEQ.I.D.NO:31 and a light chain of

SEQ.I.D.NO:32 or a heavy chain of SEQ.I.D.NO:41 and a light chain of SEQ.I.D.NO:42 or a heavy chain of SEQ.I.D.NO:51 and a light chain of SEQ.I.D.NO:52.

In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:14.

In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:13. In another embodiment of the invention there is provided an isolated intact human antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:14.

5.1.1.1.2 Chimaeric and Humanised Antibodies

The use of intact non-human antibodies in the treatment of human diseases or disorders carries with it the potential for the now well established problems of immunogenicity, that is the immune system of the patient may recognises the non-human intact isolated antibody as non-self and mount a neutralising response. This is particularly evident upon multiple administration of the non-human isolated antibody to a human patient. Various techniques have been developed over the years to overcome these problems and generally involve reducing the non-human immunogenicity signature in the intact isolated antibody whilst retaining the relative ease in obtaining non-human antibodies from an immunised animal, e.g. mouse, rat or rabbit. Broadly two approaches have been used to achieve this. The first are chimeric (sometimes "chimaeric") antibodies, which generally comprise a non-human (e.g. rodent such as mouse) variable domain fused to a human constant region. Because the antigen-binding site of an isolated antibody is localised within the variable regions the chimeric isolated antibody retains its binding affinity for the antigen but acquires the effector functions of the human constant region and are therefore able to perform effector functions such as described supra. Chimeric antibodies are typically produced using recombinant DNA methods. DNA encoding the antibodies (e.g. cDNA) is isolated and sequenced using conventional procedures (e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the H and L chains of the isolated antibody of the invention, e.g. DNA encoding SEQ ID NO 1 , 2, 3, 4, 5 and 6 described supra). Hybridoma cells serve as a typical source of such DNA. Once isolated, the DNA is placed into expression vectors which are then transfected into host cells such as E.Coli, COS cells, CHO cells or myeloma cells that do not otherwise produce immunoglobulin protein to obtain synthesis of the isolated antibody. The DNA may be modified by substituting the coding sequence for human L and H chains for the corresponding non-human (e.g. murine) H and L constant regions see e.g. Morrison; PNAS 81 , 6851 (1984).

The second approach involves the generation of humanised antibodies wherein the non-human content of the isolated antibody is reduced by humanizing the variable regions. Two techniques for humanisation have gained popularity. The first is humanisation by CDR grafting. CDRs build loops close to the isolated antibody's N-terminus where they form a surface mounted in a scaffold provided by the framework region. Antigen-binding specificity of the isolated antibody is mainly defined by the topography and by the chemical characteristics of its CDR surface. These features are in turn determined by the conformation of the individual CDRs, by the relative disposition of the CDRs, and by the nature and disposition of the side chains of the residues comprising the CDRs. A large decrease in immunogenicity can be achieved by grafting only the CDRs of a non-human (e.g. murine) antibodies ('donor' antibodies) onto human framework ('acceptor framework') and constant regions (see Jones et al (1986) Nature 321 , 522-525 and Verhoeyen M et al (1988) Science 239, 1534-1536). However, CDR grafting per se may not result in the complete retention of antigen-binding properties and it is frequency found that some framework residues (sometimes referred to as 'backmutations') of the donor isolated antibody need to be preserved in the humanised compound if significant antigen-binding affinity is to be recovered (see Queen C et al (1989) PNAS 86, 10, 029-10,033, Co, M ei al (1991 ) Nature 351 , 501 -502). In this case, human V regions showing the greatest sequence homology to the non- human donor isolated antibody are chosen from a database in order to provide the human framework (FR). The selection of human FRs can be made either from human consensus or individual human antibodies. Where necessary key residues from the donor isolated antibody are substituted into the human acceptor framework to preserve CDR conformations. Computer modelling of the isolated antibody may be used to help identify such structurally important residues, see W099/48523.

Alternatively, humanisation may be achieved by a process of 'veneering'. A statistical analysis of unique human and murine immunoglobulin heavy and light chain variable regions revealed that the precise patterns of exposed residues are different in human and murine antibodies, and most individual surface positions have a strong preference for a small number of different residues (see Padlan EA, et al; (1991 ) Mol Immunol 28, 489-498 and Pedersen JT et al (1994) J Mol Biol 235; 959-973). Therefore it is possible to reduce the immunogenicity of a non-human Fv by replacing exposed residues in its framework regions that differ from those usually found in human antibodies. Because protein antigenicity may be correlated with surface accessibility, replacement of the surface residues may be sufficient to render the mouse variable region 'invisible' to the human immune system (see also Mark GE et al (1994) in Handbook of

Experimental Pharmacology vol 1 13: The pharmacology of monoclonal Antibodies, Springer- Verlag, pp 105-134). This procedure of humanisation is referred to as 'veneering' because only the surface of the isolated antibody is altered, the supporting residues remain undisturbed.

In one embodiment of the invention there is provided an isolated chimeric antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7). In one embodiment of the invention there is provided an isolated chimeric antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said chimeric antibody comprises a CDRH3 of SEQ.I.D.NO:3 or

SEQ.I.D.NO:25 or SEQ.I.D.NO:35 or SEQ.I.D.NO:45.

In another embodiment of the invention there is provided an isolated chimeric antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRH3 of SEQ.I.D.NO:3 or CDRH1 of

SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25 or CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35 or CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45. The isolated antibody may comprise a human IgG constant region (e.g. of an lgG1 or lgG4 isotype)

In another embodiment of the invention there is provided an isolated chimeric antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises;

(a) CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRH3 of SEQ.I.D.NO:3; (b) CDRL1 of SEQ.I.D.NO:4, CDRL2 of SEQ.I.D.NO:5, CDRL3 of SEQ.I.D.NO:6 or

(c) CDRH1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25;

(d) CDRL1 of SEQ.I.D.NO:26, CDRL2 of SEQ.I.D.NO:27 and CDRL3 of SEQ.I.D.NO:28 or

(e) CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35;

(f) CDRL1 of SEQ.I.D.NO:36, CDRL2 of SEQ.I.D.NO:37 and CDRL3 of SEQ.I.D.NO:38 or (g) CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45;

(h) CDRL1 of SEQ.I.D.NO:46, CDRL2 of SEQ.I.D.NO:47 and CDRL3 of SEQ.I.D.NO:48.

The isolated antibody may comprise a human IgG constant region (e.g. of an lgG1 or lgG4 isotype). In one embodiment of the invention there is provided an isolated intact humanised antibody that binds (e.g. specifically binds) with hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises a CDRH3 of SEQ.I.D.NO:3 or SEQ.I.D.NO:25 or SEQ.I.D.NO:35 or SEQ.I.D.NO:45. The isolated antibody may comprise a human constant region of an IgG isotype (such as an lgG1 or lgG4).

In another embodiment of the invention there is provided an isolated intact humanised antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRH3 of SEQ.I.D.NO:3 or CDRH1 of

SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25 or CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35 or CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45. The isolated antibody may comprise a human IgG constant region (e.g. of an lgG1 or lgG4 isotype)

In another embodiment of the invention there is provided an isolated intact humanised antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said isolated antibody comprises; (a) CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRH3 of SEQ.I.D.NO:3;

(b) CDRL1 of SEQ.I.D.NO:4, CDRL2 of SEQ.I.D.NO:5, CDRL3 of SEQ.I.D.NO:6 or

(c) CDRH 1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25;

(d) CDRL1 of SEQ.I.D.NO:26, CDRL2 of SEQ.I.D.NO:27 and CDRL3 of SEQ.I.D.NO:28 or

(e) CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35; (f) CDRL1 of SEQ.I.D.NO:36, CDRL2 of SEQ.I.D.NO:37 and CDRL3 of SEQ.I.D.NO:38 or

(g) CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45;

(h) CDRL1 of SEQ.I.D.NO:46, CDRL2 of SEQ.I.D.NO:47 and CDRL3 of SEQ.I.D.NO:48.

The isolated antibody may comprise a human IgG constant region (e.g. of an lgG1 or lgG4 isotype).

5.1.1.1.3 Bispecific antibodies A bispecific isolated antibody is an isolated antibody having binding specificities for at least two different epitopes. Methods of making such antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two

immunoglobulin H chain-L chain pairs, where the two H chains have different binding

specificities, (see Millstein et al, Nature 305, 537-539 (1983), WO93/08829 and Traunecker et al, EMBO, 10, 1991 , 3655-3659). Because of the random assortment of H and L chains, a potential mixture of ten different isolated antibody structures are produced of which only one has the desired binding specificity. An alternative approach involves fusing the variable domains with the desired binding specificities to heavy chain constant region comprising at least part of the hinge region, CH2 and CH3 regions. It is preferred to have the CH1 region containing the site necessary for light chain binding present in at least one of the fusions. DNA encoding these fusions and, if desired, the L chain are inserted into separate expression vectors and are the contransfected into a suitable host organism. It is possible though to insert the coding sequences for two or all three chains into one expression vector. In one preferred approach, the bispecific isolated antibody is composed of an H chain with a first binding specificity in one arm and an H-L chain pair, providing a second binding specificity in the other arm, see WO94/04690. Also see Suresh et al, Methods in Enzymology 121 , 210, 1986.

Thus in one aspect of the invention there is provided an isolated bispecific antibody that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7) and a second different antigen (such as CCL19). In one embodiment, the isolated bispecific antibody comprises a CDRH3 of SEQ.I.D.NO:3 or SEQ.I.D.NO:25 or

SEQ.I.D.NO:35 or SEQ.I.D.NO:45. In another embodiment, the bispecific antibody comprises a CDRH 1 of SEQ.I.D.NO:1 , a CDRH2 of SEQ.I.D.NO:2 and a CDRH3 of SEQ.I.D.NO:3 or CDRH1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25 or CDRH 1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35 or CDRH 1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45. In a further embodiment of this aspect, the isolated antibody comprises a CDRH1 of SEQ.I.D.NO:1 , a CDRH2 of SEQ.I.D.NO:2 and a CDRH3 of SEQ.I.D.NO:3, CDRL1 of SEQ.I.D.NO:4, CDRL2 of SEQ.I.D.NO:5 and CDRL3 of SEQ.I.D.NO:6 or CDRH1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, and CDRH3 of SEQ.I.D.NO:25, CDRL1 of SEQ.I.D.NO:26, CDRL2 of

SEQ.I.D.NO:27 and CDRL3 of SEQ.I.D.NO:28 or CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34, and CDRH3 of SEQ.I.D.NO:35, CDRL1 of SEQ.I.D.NO:36, CDRL2 of

SEQ.I.D.NO:37 and CDRL3 of SEQ.I.D.NO:38 or CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, and CDRH3 of SEQ.I.D.NO:45, CDRL1 of SEQ.I.D.NO:46, CDRL2 of

SEQ.I.D.NO:47 and CDRL3 of SEQ.I.D.NO:48. In a further embodiment, the isolated bispecific antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 or 8 and a light chain variable region of SEQ.I.D.NO:9 or 10. In another further embodiment, the isolated bispecific antibody comprises a heavy chain variable region of SEQ.I.D.NO:29 and a light chain variable region of SEQ.I.D.NO:30, or the isolated bispecific antibody comprises a heavy chain variable regione of SEQ.I.D.NO:39 and a light chain variable region of SEQ.I.D.NO:40, or the isolated bispecific antibody comprises a heavy chain variable regione of SEQ.I.D.NO:49 and a light chain variable region of SEQ.I.D.NO:50.

5.1.1.1.4 Isolated antibody Fragments and Domain antibodies.

In certain embodiments of the invention there is provided isolated antibody fragment which binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7). Such fragments may be functional antigen binding fragments of intact and/or human, humanised, chimaeric antibodies such as Fab, Fab', F(abi)2, Fv, ScFv fragments of the antibodies described herein.

Traditionally, such fragments are produced by the proteolytic digestion of intact antibodies by e.g. papain digestion (see for example WO 94/29348) but may be produced directly from recombinantly transformed host cells. For the production of ScFv, see Bird et al; (1988) Science, 242, 423-426. In addition, isolated antibody fragments may be produced using a variety of engineering techniques as described below.

FV fragments appear to have lower interaction energy of their two chains than Fab fragments. To stabilise the association of the VH and VL domains, they have been linked with peptides (Bird et al, (1988) Science, 242, 423-426, Huston et al, PNAS, 85, 5879-5883), disulphide bridges (Glockshuber et al, (1990) Biochemistry, 29, 1362-1367) and 'knob in hole' mutations (Zhu et al (1997), Protein Sci., 6, 781 -788). ScFv fragments can be produced by methods well known to those skilled in the art (see Whitlow ei a/ (1991 ), Methods companion Methods Enzymol, 2, 97-105 and Huston ei a/ (1993) Int Rev Immunol 10, 195-217. ScFv may be produced in bacterial cells such as E.Coli but are more preferably produced in eukaryotic cells. One disadvantage of ScFv is the monovalency of the product, which precludes an increased avidity due to polyvalent binding, and their short half-life. Attempts to overcome these problems include bivalent (ScFv')2 produced from ScFv containing an additional C terminal cysteine by chemical coupling (Adams ei a/ (1993) Can Res 53, 4026-4034 and McCartney ei a/ (1995) Protein Eng, 8, 301 -314) or by spontaneous site-specific dimerization of ScFv containing an unpaired C terminal cysteine residue (see Kipriyanov et al (1995) Cell. Biophys 26, 187-204). Alternatively, ScFv can be forced to form multimers by shortening the peptide linker to 3 and 12 residues to form 'diabodies' (see Holliger ei a/ PNAS (1993), 90, 6444-6448). Reducing the linker still further can result in ScFV trimers ('triabodies', see Kortt ei a/ (1997) Protein Eng, 10, 423-433) and tetramers ('tetrabodies', see Le Gall et al (1999) FEBS Lett, 453, 164-168).

Construction of bialent ScFV compounds can also be achieved by genetic fusion with protein dimerzing motifs to form 'miniantibodies' (see Pack ei a/ (1992) Biochemistry 31 , 1579-1584) and 'minibodies' (see Hu ei a/ (1996), Cancer Res. 56, 3055-3061 ). ScFv-Sc-Fv tandems ((ScFV)2) may also be produced by linking two ScFV units by a third peptide linger, (see Kurucz et al (1995) J Immunol, 154, 4576-4582). Bispecific diabodies can be produced through the noncovalent association of two single chain fusion products consisting of VH domain from one isolated antibody connected by a short linker to the VL domain of another isolated antibody, (see Kipriyanov et al (1998), Int J Can 77, 763-772). The stability of such bispecific diabodies can be enhanced by the introduction of disulphide bridges or 'knob in hole' mutations as described supra or by the formation of single chain diabodies (ScDb) wherein two hydrid ScFv fragments are connected through a peptide linker (see Kontermann et al (1999) J Immunol Methods 226, 179-188). Tetravalent bispecific compounds are available by e.g fusing a ScFv fragment to the CH3 domain of an IgG compound or to a Fab fragment through the hinge region (see Coloma ei a/ (1997) Nature Biotechnol, 15, 159-163). Alternatively, tetravalent bispecific compounds have been created by the fusion of bispecific single chain diabodies (see Alt et al (1999) FEBS Lett 454, 90-94). Smaller tetravalent bispecific compounds can also be formed by the dimerization of either ScFv-ScFv tandems with a linker containing a helix-loop-helix motif (DiBi miniantibodies, see Muller et al (1998) FEBS Lett 432, 45-49) or a single chain compound comprising four isolated antibody variable domains (VH and VL) in an orientation preventing intramolecular pairing (tandem diabody, see Kipriyanov et al, (1999) J Mol Biol 293, 41 -56). Bispecific Fab')2 fragments can be created by chemical coupling of Fab' fragments or by heterodimerization through leucine zippers (see Shalaby et al (1992) J Exp Med 175, 217-225 and Kostelny et al (1992), J Immunol 148 1547-1553). In one embodiment there is provided an isolated antibody fragment (e.g. ScFv, Fab, Fab', F(ab')2) which binds with hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7). Depending on the format, the isolated fragment comprises CDRH3 of SEQ.I.D.NO:3 and one or more of CDRH1 of SEQ.I.D.NO:1 , CDRH2 of SEQ.I.D.NO:2, CDRL1 of SEQ.I.D.NO:4, CDRL2 of SEQ.I.D.NO:5, CDRL3 of SEQ.I.D.NO:6 or the isolated fragment comprises CDRH3 of SEQ.I.D.NO:25 and one or more of CDRH1 of SEQ.I.D.NO:23, CDRH2 of SEQ.I.D.NO:24, CDRL1 of SEQ.I.D.NO:26, CDRL2 of

SEQ.I.D.NO:27, CDRL3 of SEQ.I.D.NO:28 or the isolated fragment comprises CDRH3 of SEQ.I.D.NO:35 and one or more of CDRH1 of SEQ.I.D.NO:33, CDRH2 of SEQ.I.D.NO:34,

CDRL1 of SEQ.I.D.NO:36, CDRL2 of SEQ.I.D.NO:37, CDRL3 of SEQ.I.D.NO:38 or the isolated fragment comprises CDRH3 of SEQ.I.D.NO:45 and one or more of CDRH1 of SEQ.I.D.NO:43, CDRH2 of SEQ.I.D.NO:44, CDRL1 of SEQ.I.D.NO:46, CDRL2 of SEQ.I.D.NO:47, CDRL3 of SEQ.I.D.NO:48 or. Depending on the format, the isolated antibody fragment may comprise either a variable heavy chain region of SEQ.I.D.NO:7 or 8 together with a variable light chain region of either SEQ.I.D.NO:9 or 10. In another further embodiment, the isolated antibody fragment may comprise a heavy chain variable region of SEQ.I.D.NO:29 and a light chain variable region of SEQ.I.D.NO:30, or the isolated antibody fragment comprises a heavy chain variable regione of SEQ.I.D.NO:39 and a light chain variable region of SEQ.I.D.NO:40, or the isolated antibody fragment comprises a heavy chain variable regione of SEQ.I.D.NO:49 and a light chain variable region of SEQ.I.D.NO:50.

In another aspect of the invention there is provided an isolated antibody which is a single variable domain (e.g. a dAb or VHH) that binds hCCL21 and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7). In some embodiments of the invention the single variable domain comprises a CDRH3 of SEQ.I.D.NO:3 or

SEQ.I.D.NO:25 or SEQ.I.D.NO:35 or SEQ.I.D.NO:45. In other embodiments, the single variable domain comprises a CDRH1 of SEQ.I.D.NO:1 , a CDRH2 of SEQ.I.D.NO:2.and a CDRH3 of SEQ.I.D.NO:3 or CDRH1 of SEQ.I.D.NO:23, a CDRH2 of SEQ.I.D.NO:24.and a CDRH3 of SEQ.I.D.NO:25 or CDRH1 of SEQ.I.D.NO:33, a CDRH2 of SEQ.I.D.NO:34.and a CDRH3 of SEQ.I.D.NO:35 or CDRH1 of SEQ.I.D.NO:43, a CDRH2 of SEQ.I.D.NO:44.and a CDRH3 of SEQ.I.D.NO:45. The term "single variable domain" refers to an antibody variable domain (VH, VHH, VL) that specifically binds an antigen or epitope independently of a different V region or domain. An single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other, different variable regions or variable domains where the other regions or domains are not required for antigen binding by the single variable domain (i.e., where the single variable domain binds antigen independently of the additional variable domains). A "domain antibody" or "dAb" is the same as a "single variable domain" which is capable of binding to an antigen as the term is used herein. A single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid VHH dAbs. Camelid VHH are single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanised according to standard techniques available in the art, and such domains are still considered to be "domain antibodies" according to the invention. As used herein "VH" includes camelid VHH domains.

5.1.1.1.5 Heteroconjugate antibodies

Heteroconjugate antibodies also form an embodiment of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies formed using any convenient cross-linking methods. See, for example, US 4,676,980.

5.1.1.1.6 Other modifications

The interaction between the Fc region of an isolated antibody and various Fc receptors (FcyR) is believed to mediate the effector functions of the isolated antibody which include isolated antibody-dependent cellular cytotoxicity (ADCC), fixation of complement, phagocytosis, and half-life/clearance of the isolated antibody. Various modifications to the Fc region of antibodies of the invention may be carried out depending on the desired property. For example, specific mutations in the Fc region to render an otherwise lytic isolated antibody, non-lytic is detailed in EP 0629 240B 1 and EP0307434B2 or one may incorporate a salvage receptor binding epitope into the isolated antibody to increase serum half-life, see US 5. 739, 277. There are five currently recognised human Fey, FcyR (I), FCyRllb, FcyRllla and neonatal FcRn. Shields et al, (2001 ) J Biol Chem 276, 6591 -6604 demonstrated that a common set of lgG1 residues is involved in binding all FcyRs, while FCyRII and FcyRIII utilize distinct sites outside of this common set. One group of lgG1 residues reduced binding to all FcyRs when altered to alanine: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain and clustered near the hinge joining CH1 and CH2. While FcyRI utilizes only the common set of lgG1 residues for binding, FcyRII and FcyRIII (e.g. Glu-293). Some variants showed improved binding to FcyRII or FcyRIII but did not affect binding to the other receptor (e.g. Ser-267Ala improved binding to FcyRII but binding to FcyRIII was unaffected). Other variants exhibited improved binding to DcyRII or FcyRIII with reduction in binding to the other receptor (e.g. Ser298Ala improved binding to FcyRIII and reduced binding to FcyRII). For FcyRllla, the best binding lgG1 variants had combined alanine substitutions at Ser-298, Glu-333 and Ls-334. The neonatal FcRn receptor is believed to be involved in both isolated antibody clearance and the transcytosis across tissues (see Junghans RP (1997) Immunol Res 16, 2957 and Ghetie et al (2000) Annu Rev Immunol 18, 739-766). Human lgG1 residues determined to interact directly with human

FcRn included Ile253, Ser254, Lys288, Thr307, Gln31 1 , Asn434 and His435. Switches at any of these positions described in this section may enable increased serum half-life and/or altered effector properties of antibodies of the invention and therefore forms an embodiment of the invention.

Other modifications include glycosylation variants of the antibodies of the invention.

Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on isolated antibody function, particularly effector functioning such as those described above, see for example, Boyd ei a/ (1996), Mol Immunol 32, 131 1 -1318.

Glycosylation variants of the therapeutic antibodies or antigen binding fragments thereof of the present invention 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 side for enzymatic attachment of carbohydrate moieties and may therefore be used to manipulate the glycosylation of an isolated 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- galactrosyltransferace 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, Science (2004), 303, 371 ; Sears et al, Science (2001 ), 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. Thus the invention contemplates a plurality of isolated (monoclonal) antibodies (which may be of the IgG isotype, e,g. lgG1 ) as herein described comprising a defined number (e.g. 7 or less, for example 5 or less such as two or a single) glycoform(s) or said antibodies or antibody fragment. Further embodiments of the invention include isolated antibodies or antibody fragments as described herein 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 IL et al (2000) Int J Pharmaceut 198; 83-95). The reader may assume that all antibody and antibody fragments (including domain antibodies) aspects and embodiments described herein are specifically and individually contemplated to be coupled to a non- proteinaeous polymer such as PEG, and each forms an embodiment of the invention.

5.2 Alternatives to antibodies

Whilst the present invention is primarily directed to embodiments relating to antibodies that are specific for huCCL21 it will be apparent to the skilled reader that alternative binding molecules which possess equivalent binding specificity and inhibitory activity may also be provided on the basis of the teaching of the teaching herein.

It is to be understood that where the term "antibody" is used in a generic sense in the context of the present invention (for example to refer to a specific binding partner for huCCL21 that possesses the appropriate binding specificity and inhibitory activity) the following antibody alternatives are also contemplated as being suitable for the purposes of the present invention, mutatis mutandis. 5.2.1 Adnectins - Compound Therapeutics

The adnectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands. (US 6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional isolated antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG.

Because of this structure, the non-immunoglobulin isolated antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based compounds can be used as scaffolds where the loop regions of the compound can be replaced with CDRs of the invention using standard cloning techniques. Accordingly, in some embodiments there is provided an adnectin compound that binds (e.g. specifically binds) with and neutralises the biological activity of hCCL21 (e.g. inhibits the interaction between hCCL21 and hCCR7). . 5.2.2 Ankyrin - Molecular Partners

This technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel ohelices and a β-turn. Binding of the variable regions is mostly optimized by using ribosome display. Accordingly, in some embodiments there is provided an ankyrin compound that binds (e.g. specifically binds) with and neutralises the biological activity of hCCL21 (e.g. inhibits the interaction between hCCL21 and hCCR7)

5.2.3 Maxybodies/Avimers - Avidia Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, US20040175756;

US20050053973; US20050048512; and US20060008844. Accordingly, in some embodiments there is provided an avidia compound that binds (e.g. specifically binds) with and neutralises the biological activity of hCCL21 (e.g. inhibits the interaction between hCCL21 and hCCR7)

5.2.4 Protein A - Affibody

Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants (See e.g., US 5,831 ,012). Affibody® compounds mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of Affibody® compounds is similar to that of an isolated antibody.

Accordingly, in some embodiments there is provided an affibody compound that binds (e.g. specifically binds) with and neutralises the biological activity of hCCL21 (e.g. inhibits the interaction between hCCL21 and hCCR7)

5.2.5 Anticalins - Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target compounds of different shape with high affinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing "anticalins" is PCT WO 199916873. Accordingly, in some embodiments there is provided an anticalin compound that binds (e.g. specifically binds) with and neutralises the biological activity of hCCL21 (e.g. inhibits the interaction between hCCL21 and hCCR7)

5.2.6 Affilin - Scil Proteins Affilin™ compounds are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small compounds. New Affilin™ compounds can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin™ compounds do not show any structural homology to immunoglobulin proteins. Scil Proteins employs two Affilin™ scaffolds, one of which is gamma crystalline, a human structural eye lens protein and the other is "ubiquitin" superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in WO200104144 and examples of "ubiquitin-like" proteins are described in WO2004106368. Accordingly, in some embodiments there is provided an affilin compound that binds (e.g.

specifically binds) with and neutralises the biological activity of hCCL21 (e.g. inhibits the interaction between hCCL21 and hCCR7) 6. Production Methods

Therapeutic immunoglobulins and antibodies of the invention maybe produced as a polyclonal population but are more preferably produced as a monoclonal population (that is as a substantially homogenous population of identical antibodies directed against a specific antigenic binding site). It will of course be apparent to those skilled in the art that a population implies more than one isolated antibody entity. 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 KJJ (2000) Genet. Eng. News 20:1 - 55, mice (see Pollock et al) or plants (see Doran PM, (2000) Curr.Opinion Biotechnol. 1 1 , 199-204, Ma JK-C (1998), Nat.Med. 4; 601 -606, Baez J e. al, BioPharm (2000) 13: 50-54, Stoger E et al; (2000) Plant Mol.Biol. 42:583-590). Antibodies may also be produced by chemical synthesis. However, antibodies of the invention are typically produced using recombinant cell culturing technology as well known to those skilled in the art. An isolated polynucleotide encoding the isolated antibody is typically inserted into a replicable vector such as a plasmid for further cloning (amplification) or expression. One useful expression system is a glutamate synthetase system (such as sold by Lonza Biologies), particularly where the host cell is CHO or NSO (see below). Isolated polynucleotide encoding the isolated antibody maybe readily isolated and sequenced using conventional procedures (e.g. oligonucleotide probes). Vectors that may be used include plasmid, virus, phage, transposons, minichromsomes of which plasmids are a typical embodiment. 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 light and/or heavy chain isolated polynucleotide so as to facilitate expression. Isolated polynucleotide encoding the light and heavy chains may be inserted into separate vectors and transfected into the same host cell or, if desired both the heavy chain and light chain can be inserted into the same vector for transfection into the host cell. Thus according to one aspect of the present invention there is provided a process of constructing a vector encoding the light and/or heavy chains of an isolated antibody or antigen binding fragment thereof of the invention, which method comprises inserting into a vector, a isolated polynucleotide encoding either a light chain and/or heavy chain of an isolated antibody of the invention.

Vectors of the invention (such as plasmids) may further comprise a number of sequences necessary or desirable for facilitating expression of an antibody of the invention and these are described further below.

6.1 Signal sequences

Antibodies of the present invention maybe 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 recognised and processed by the host cell. For prokaryotic host cells, the signal sequence may be an alkaline phosphatase, penicillinase, or heat stable enterotoxin II leaders. For yeast secretion the signal sequences may be a yeast invertase leader, [alpha] 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 are available. Typically the signal sequence is ligated in reading frame to DNA encoding the isolated antibody of the invention.

6.2 Origin of replication

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.

6.3 Selection marker

Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins e.g. ampicillin, neomycin, methotrexate or tetracycline or (b) complement auxotrophic deficiencies or supply nutrients not available in the complex media. The selection scheme may involve arresting growth of the host cell. Cells, which have been successfully transformed with the genes encoding the therapeutic isolated antibody of the present invention, survive due to e.g. drug resistance conferred by the selection marker. Another example is the so-called DHFR selection marker wherein transformants are cultured in the presence of methotrexate. In typical embodiments, cells are 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). A suitable selection gene for use in yeast is the trpl gene, see Stinchcomb et al Nature 282, 38, 1979.

6.4 Promoters

Suitable promoters for expressing antibodies of the invention are operably linked to

DNA isolated polynucleotide encoding the isolated antibody of the invention. 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.

6.5 Enhancer element

Where appropriate, e.g. for expression in higher eukaroytics, an enhancer element operably linked to the promoter element in a vector may be used. Suitable 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 bp100-270), cytomegalovirus early promoter enhancer, polyma enhancer, baculoviral enhancer or murine lgG2a locus (see WO04/009823). The enhancer is preferably located on the vector at a site upstream to the promoter. 6.6 Host cells

Suitable host cells for cloning or expressing vectors encoding antibodies of the invention 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 (EP183, 070, see also Peng et al J.Biotechnol. 108 (2004) 185-192), Candida, Thchoderma reesia

(EP244, 234J, Penicillin, Tolypocladium and Aspergillus hosts such as A.nidulans and A.niger are also contemplated.

Although Prokaryotic and yeast host cells are specifically contemplated by the invention, preferably however, host cells of the present invention are higher eukaryotic cells. Suitable 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 (BHK) (ATCC CRL.1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO. CRL 1573), Chinese hamster ovary cells CHO (e.g. CHO-K1 , ATCC NO: CCL 61 , DHFR-CHO cell line such as DG44 (see Urlaub et a/, (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), HELA cells, canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), Hep G2 and myeloma or lymphoma cells e.g. NSO (see US 5,807,715), Sp2/0, YO. Thus in one embodiment of the invention there is provided a stably transformed host cell comprising a vector encoding a heavy chain and/or light chain of the isolated antibody or antigen binding fragment thereof as herein described. Preferably such host cells comprise a first vector encoding the light chain and a second vector encoding said heavy chain. 6.7 Bacterial fermentation

Bacterial systems may be used for the expression of non-immunoglobulin therapeutic proteins described above. Bacterial systems are also particularly suited for the expression of isolated antibody fragments. Such fragments are localised intracellular or within the periplasma. Insoluble periplasmic 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 Cu pit PM ei a/ (1999) Lett Appl Microbiol, 29, 273-277. 6.8 Cell Culturinq Methods.

Host cells transformed with vectors encoding the isolated antibodies of the invention or antigen binding fragments or domain antibodies of the invention 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 it is preferred for large scale production that stirred tank reactors are used particularly for suspension cultures. Preferably the stirred tankers are 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 it is preferred that 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 maybe used as growth substrates for anchorage dependent cell lines or the cells maybe 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), it is preferred that such host cells are cultured in synthetic serum -free media such as disclosed in Keen ei a/ (1995) Cytotechnology 17:153-163, or commercially available media such as ProCHO-CDM or UltraCHO(TM) (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 K et al (1995) in Animal Cell technology: Developments towards the 21 st century (Beuvery E.G. ei a/ eds), pp619-623, Kluwer Academic publishers).

Antibodies of the invention 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 isolated antibodies of the invention 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). In the first instance, 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. In one embodiment, the antibodies of the invention, following various clarification steps, are captured using Protein A or G affinity chromatography followed by further chromatography steps such as ion exchange and/or HA chromatography, anion or cation exchange, size exclusion

chromatography and ammonium sulphate precipitation. Typically, various virus removal steps are also employed (e.g. nanofiltration using e.g. a DV-20 filter). Following these various steps, a purified (preferably monoclonal) preparation comprising at least 75mg/ml or greater e.g.

10Omg/ml or greater of the isolated antibody of the invention or antigen binding fragment thereof is provided and therefore forms an embodiment of the invention. Suitably such preparations are substantially free of aggregated forms of antibodies of the invention.

7. Epitope mapping In order to develop a clearer picture of the binding relationship between the binding molecules, in particular antibodies, of the invention, epitope mapping studies were carried out. The object was to determine the site of binding of the candidate monoclonal antibodies on the target huCCL21 molecule. In particular, the objective was to identify the specific amino acid residues of huCCL21 that are involved in the binding interaction between the antibodies and target. Epitope mapping was generally carried out using state of the art techniques available to the skilled person. In particular, bioinformatic techniques were used to predict the secondary structure of huCCL21 . In addition, NMR and Xray structures for related molecules were used to construct a three dimensional model for huCCL21. Western blotting analysis was used to investigate the linearity of the epitope. Hydrogen/Deuterium exchange mass spectrometry and antigen mutation screening were used to probe the structure of CCL21 for amino acid residues which influence antibody binding, i.e. are implicated in the relevant epitopes. Human, mouse and cynomolgus CCL21 sequences and affinity measurements were compared. Further details are provided in the Examples.

8. Pharmaceutical compositions

The invention provides pharmaceutical compositions comprising the binding molecule, immunoglobulin, antibody, antibody fragment of the invention formulated together with a pharmaceutically acceptable carrier. The compositions can additionally contain other therapeutic agents that are suitable for treating or preventing a human disease or disorder noted below. Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (particularly low molecular weight chemical entities) may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The composition should be sterile and fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug

Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of an antibody of the invention described herein is employed in the pharmaceutical compositions of the invention. They are typically formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

A physician can start doses of the antibodies of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the present invention, for the treatment of a fibrotic disease or disorder described herein vary depending upon many different factors, including means of

administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. For administration with an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1 -10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months.

Antibodies of the invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of therapeutic protein in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibodies of the invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show longer half life than that of chimeric antibodies and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic (preventative) applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

In accordance therefore with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a CDRH3 of

SEQ.I.D.NO:3 or SEQ.I.D.NO:25 or SEQ.I.D.NO:35 or SEQ.I.D.NO:45. The composition may further comprise a pharmaceutically acceptable carrier. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein. In accordance therefore with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises; (a) CDRH1 of SEQ.I.D.NO:1

(b) CDRH2 of SEQ.I.D.NO:2

(c) CDRH3 of SEQ.I.D.NO:3

(d) CDRL1 of SEQ.I.D.NO:4

(e) CDRL2 of SEQ.I.D.NO:5 (f) CDRL3 of SEQ.I.D.NO:6.

In another embodiment of the invention the pharmaceutical composition comprises (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises; CDRH1 of SEQ.I.D.NO:23, CDRH2 of

SEQ.I.D.NO:24, CDRH3 of SEQ.I.D.NO:25, CDRL1 of SEQ.I.D.NO:26, CDRL2 of

SEQ.I.D.NO:27, CDRL3 of SEQ.I.D.NO:28 or CDRH1 of SEQ.I.D.NO:33, CDRH2 of

SEQ.I.D.NO:34, CDRH3 of SEQ.I.D.NO:35, CDRL1 of SEQ.I.D.NO:36, CDRL2 of

SEQ.I.D.NO:37, CDRL3 of SEQ.I.D.NO:38 or CDRH1 of SEQ.I.D.NO:43, CDRH2 of

SEQ.I.D.NO:44, CDRH3 of SEQ.I.D.NO:45, CDRL1 of SEQ.I.D.NO:46, CDRL2 of

SEQ.I.D.NO:47, CDRL3 of SEQ.I.D.NO:48.

Typically the composition will be in a form suitable for intravenous or subcutaneous

administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g.

human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 or of SEQ.I.D.NO:29 or of SEQ.I.D.NO:39 or of SEQ.I.D.NO:49.

Typically the composition will be in a form suitable for intravenous or subcutaneous

administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain variable region of SEQ.I.D.NO:8. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 and a light chain variable region of SEQ.I.D.NO:9 or a heavy chain variable region of SEQ.I.D.NO:29 and a light chain variable region of SEQ.I.D.NO:30 or a heavy chain variable region of SEQ.I.D.NO:39 and a light chain variable region of SEQ.I.D.NO:40 or a heavy chain variable region of SEQ.I.D.NO:49 and a light chain variable region of

SEQ.I.D.NO:50. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain variable region of SEQ.I.D.NO:7 and a light chain variable region of SEQ.I.D.NO:10. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain variable region of SEQ.I.D.NO:8 and a light chain variable region of SEQ.I.D.NO:9. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain variable region of SEQ.I.D.NO:8 and a light chain variable region of SEQ.I.D.NO:10. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain of SEQ.I.D.NO:1 1 or of SEQ.I.D.NO:31 or of SEQ.I.D.NO:41 or of SEQ.I.D.NO:51. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain of SEQ.I.D.NO:12. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain of

SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:13 or a heavy chain of SEQ.I.D.NO:31 and a light chain of SEQ.I.D.NO:32 or a heavy chain of SEQ.I.D.NO:41 and a light chain of

SEQ.I.D.NO:42 or a heavy chain of SEQ.I.D.NO:51 and a light chain of SEQ.I.D.NO:52.

Typically the composition will be in a form suitable for intravenous or subcutaneous

administration. In other embodiments, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein.

In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain of

SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:14. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the

composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein. In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:13. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the

composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody as described herein. In accordance with an embodiment of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:14. Typically the composition will be in a form suitable for intravenous or subcutaneous administration. In other embodiments, the

composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody (e.g. of the heavy chain variable region) as described herein. In accordance with an aspect of the invention there is provided a pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprising;

(a) a heavy chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 7 or 8 and

(b) a light chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 9 or 10. In one embodiment of this aspect, the pharmaceutical composition comprises (e.g. as its sole therapeutically active ingredient) an antibody that binds with hCCL21 (e.g. specifically binds) and neutralises the biological activity thereof (e.g. inhibits the interaction between hCCL21 and hCCR7), said antibody comprising:

(a) a heavy chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 29 and

(b) a light chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 30 or

(c) a heavy chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 39 and

(d) a light chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 40 or (e) a heavy chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 49 and

(f) a light chain variable region capable of being encoded by an isolated polynucleotide having at least 90% identity (e.g. 95% or greater such as 96%, 97%, 98% or 99%) to an isolated polynucleotide encoding SEQ.I.D.NO: 59.

Typically the composition will be in a form suitable for intravenous or subcutaneous

administration. In some embodiments of this aspect of the invention, the composition maybe in lyophilized form. The antibody of this embodiment of the invention maybe monoclonal and maybe an intact antibody (e.g. human, humanised or chimeric) or an antibody fragment or single variable domain antibody (of the heavy chain variable region as described for this embodiment of the invention).

9. Clinical uses. Immunoglobulins and antibodies of the invention may be used in therapeutic methods for the treatment of various human diseases and/or disorders. Of particular interest are fibrotic diseases, particularly chronic fibrotic disease e.g. particularly hepatic fibrosis (such as liver cirrhosis), kidney (such as diabetic kidney disease), lung (such as IPF) and other tissue fibrosis. Immunoglobulins and antibodies of the invention maybe used to treat a clinically established disease or disorder and/or to prevent or inhibit the progression of the disease/disorder in a human patient.

In accordance with one aspect of the invention there is provided a method of treating and/or preventing pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity

pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with

Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post-ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof which method comprises administrating a therapeutically effective amount of an immunoglobulin or antibody as described herein. Particular embodiments of this aspect include methods of treating and/or preventing pulmonary fibrosis, COPD, chronic renal disease.

Pharmaceutical compositions as described in section 7 of this specification for use in therapy and in particular for use in the treatment and/or prevention of pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post-ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof form another aspect of the invention. Particular embodiments of this aspect include pharmaceutical compositions for use in treating and/or preventing pulmonary fibrosis (such as IPF), the exacerbation of IPF, COPD, chronic renal disease, chronic hepatic fibrosis. The reader of this specification may assume that all pharmaceutical compositions embodiments described in section 7 of this specification are individually and specifically contemplated to be used in any of the diseases/disorders described herein and each forms a separate embodiment of the invention.

10. Exemplification

Sequences of antibodies of the invention, together with a sequence correlation table are described towards the end of this specification.

10.1 Functional characterization of antibodies of the invention.

10.1.1 Materials Recombinant human CCL21 (Invitrogen, Cat. No. PHC1473) H9 cells (ECACC, Cat. No. 85050301 )

24-well chemotaxis plates, δμηη pore size (BD Biosciences, Cat. No. 351 185)

24-well chemotaxis plates, 3μηι pore size (BD Biosciences, Cat. No. 351 183)

Human CCL21 DuoSet ELISA (R&D Systems, Cat. No. DY366) Ex-cell Hybri-Max medium (SAFC Biosciences, Cat. No. s2897)

Amicon Ultra-15 centrifugal filter units (Millipore, Cat. No. UFC901024)

Human CCL21 Ultimate ORF Entry clone (Invitrogen, Cat. No. IOH1 1392)

Lipofectamine 2000 (Invitrogen, Cat. No. 1 1668-027)

Human CD3+ T cell Enrichment Column (R&D Systems, Cat. No. HTC-500) Anti-CCL21 antibody (AbCam, Cat. No. ab10350) 10.1.2 Cell culture

H9 cells were maintained in RPMI 1640 supplemented with 10% FBS, 2mM L-Glutamine and 100U/mL/100 g/ml_ Pen/Strep.

HEK-293 cells were maintained in MEM supplemented with 10% FBS, 2mM L-Glutamine and 100U/ml_/100 g/mL Pen/Strep.

CH0-K1 cells were maintained in DMEM-F12 with 10% FBS, 2mM L-Glutamine and

100U/mL/100μg/mL Pen/Strep. Stable transfectants were cultured in the presence of 400μg mL zeocin (human CCR7) or 850 μg mL geneticin (cynomolgus monkey CCR7).

10.1.3 Generation and quantification of CCL21 produced in human cells Human CCL21 in the pENT221 vector was purchased from Invitrogen and recombined with pcDNA-Dest40 via Gateway® cloning technology (Invitrogen) to generate a mammalian expression vector (NPL010662). HEK-293T cells were transfected using Lipofectamine 2000 (Invitrogen) and transfected cells selected with 250μg mL geneticin. Single cell sorting by Mo- Flo was used to generate clones which were assayed for CCL21 secretion by ELISA. The clone with highest secretion was grown to confluence in poly-D-lysine coated flasks and growth media replaced with Hybri-max Excel media. Flasks were incubated for 72h and supernatants concentrated by Ambicon 10KDa molecular weight cut off filters. The CCL21 content of the resultant solution was determined by ELISA.

10.1.4 Human T cell chemotaxis assays This assay was used to measure the neutralizing potency of anti-CCL21 IgGs with either 100nM recombinant human CCL21 , recombinant cynomolgus monkey CCL21 (synthesized inhouse) or human CCL21 secreted and isolated from human cells (= "natural" ligand). Natural ligand was found to result in a background level of migration mediated by factors present in the tissue culture medium used to culture the transfected cells from which the protein was harvested. This baseline was determined and subtracted from CCL21 -mediated migration by the use of a commercially available neutralizing anti-CCL21 antibody control (AbCam) used at

20μg mL which is sufficient to completely inhibit migration induced by recombinant CCL21 . The assay was also adapted to analyze migration of primary T cells. PBMCs were isolated from whole blood by ficoll gradient and T cells purified using R&D Systems T Cell Enrichment Column according to the manufacturer's instructions. 5x105 primary human T cells/well were then used in chemotaxis assays as described above using assay plates with a 3μηι pore size and 250nM recombinant human CCL21 .

10.1.5 Calcium mobilization assays The development and validation of a CCL21 mediated FLIPR assay using CHO-K1 cells was developed. Stably transfected clones #22 (human CCR7) and #2 (cynomolgus monkey CCR7) were selected and cells primed with 100nM ATP prior to stimulation with 1 μΜ CCL21.

10.1.6 Results

IgGs selected for functional analysis Eight antibodies from 4 parental frameworks (FW) were selected for IgG conversation and functional characterization following screening of multiple Fabs in two rounds of affinity maturation. Of these molecules, IgGs with the VA3A H3 framework (MOR06935, MOR08383 and MOR08389) were found to contain a VL mixed framework mutation and were consequently repaired. An additional 10 IgGs were generated by the germlining of MOR08376, MOR08377, MOR08378, MOR08382, MOR08389 and the framework repaired MOR06935. These antibodies were also sequence optimized using the GeneArt software. All IgGs analyzed are described in Table 1. MOR06935 is a Fab with a VH of SEQ.I.D.NO:8 and a light chain of SEQ.I.D.NO:10. The germlined version of MOR06935 is a Fab with a VH of SEQ.I.D.NO:7 and a VL of

SEQ.I.D.NO:9. Table 1 : IgGs Analyzed for anti-CCL21 activity in chemotaxis assays

Identification Number Framework Comment

MOR08382 VK3A H3

MOR07337 VK4A H5

MOR08376 VA2/VH5

MOR08377 VA2/VH5 Identification Number Framework Comment

MOR08378 VA2/VH5

MOR08951 VA3/VH3 FW repaired MOR08383

MOR08952 VA3/VH3 FW repaired MOR06935

MOR08953 VA3/VH3 FW repaired MOR08389

FW repaired germlined

MOR06935_g3-23_g3j VA3A H3

MOR06935

FW repaired germlined

MOR06935_g3-23_g3l VA3A H3

MOR06935

FW repaired germlined

MOR06935_g3-23_g3r VA3A H3

MOR06935

MOR08378 g5-51_g2a2_opt VA2A H5 germlined MOR08378

MOR08376_g5-51_g2a2_opt VA2A H5 germlined MOR08376

MOR8382_g3-23_gL6_opt VK3A H3 germlined MOR08382

MOR8389_g3-23_g3j_opt VA3A H3 germlined MOR08389

MOR8377_g5-51_g2a2_opt VA2A H5 germlined MOR08377

Anti-lysozyme control IgGi

MOR03207

antibody

Inhibition of recombinant hCCL21 -mediated migration of H9 cells

Anti-CCL21 IgGs were analyzed for their ability to inhibit migration of H9 cells in response to recombinant human CCL21 in chemotaxis assays (Figures 1 & 2). All IgGs fully inhibited migration when used at 2μΜ (the highest concentration tested corresponding to a molar ratio of 20:1 antibody:ligand) with IC50 values in the range of 13-101 nM (Table 2). The exception to this was MOR07337 which only showed 78% inhibition at 2μΜ and a higher IC50 value of 140nM. Inhibition at a 1 :1 molar ratio of lgG:CCL21 ranged from 61 -100% (Table 3) again with the exception of MOR07337 which only resulted in a 19% inhibition of migration. The control anti- lysozyme IgG MOR03207 did not have a significant inhibitory activity even at 2μΜ.

10.1.7 Inhibition of recombinant cCCL21 -mediated migration of H9 cells

Anti-CCL21 IgGs were analyzed for their ability to inhibit migration of H9 cells in response to recombinant cynomolgus monkey CCL21 in chemotaxis assays (Figures 3 and 4). All IgGs completely inhibited migration when used at 2μΜ with the exception of MOR07337 which caused a significantly weaker reduction of just 35% at this concentration. IC50 values were in the range of 5-93nM (Table 2) and inhibition at a 1 :1 molar ratio ranged from 61 -100% (Table 3) for all IgGs tested with the exception of MOR07337 which inhibited migration by only 21 % at this ratio. The unexpected inhibition by the control anti-lysozyme IgG MOR03207 observed at 2μΜ appears to be non-specific as no inhibition was observed at any other concentration.

10.1.8 Inhibition of natural hCCL21 -mediated migration of H9 cells

Anti-CCL21 IgGs were analyzed for their ability to inhibit migration of H9 cells in response to natural human CCL21 in chemotaxis assays (Figure 5). All IgGs completely inhibited migration when tested at a concentration of 2μΜ, had IC50 values in the range of 34-130nM (Table 2) and inhibited migration at a 1 :1 molar ratio from 49-93% (Table 3). This differs from assays using recombinant CCL21 where MOR07337 proved less potent than other selected IgGs. The activity of IgGs MOR08367 and 08377 and their derivatives were not tested against natural CCL21. The less favorable DAS characteristics of these IgGs and the desire to conserve parental diversity led to other antibodies being prioritized for testing with the natural protein which was of limited availability. This issue also necessitated the testing of germlined derivatives of MOR08389, 08378 and 08382 in dose response curves limited to four points. The control IgG MOR03207 showed a higher level of non-specific inhibition when used at 200nM or 2μΜ against natural CCL21 than that seen against recombinant human or cynomolgus monkey protein. However, at a 1 :1 molar ratio the observed inhibition was non-significant.

10.1.9 Inhibition of recombinant hCCL21 -mediated migration of primary human T cells

Anti-CCL21 IgGs were analyzed for their ability to inhibit migration of primary human T cells in response to recombinant human CCL21 in chemotaxis assays (Figures 6 and 7). All IgGs completely inhibited migration at a concentration of 2μΜ with the exception of MOR07337 which was slightly less potent resulting in only 91 % inhibition. IC50 values for all IgGs other than MOR07337 were in the range of 15-160nM (Table 2) and inhibition at a 1 :1 molar ratio ranged from 78-100% (Table 3). MOR07337 proved less strongly neutralizing with an IC50 value of 287nM and just 35% inhibition of migration at a 1 :1 molar ratio. The control anti-lysozyme IgG MOR03207 showed weak inhibition of hCCL21 mediated chemotaxis at 2μΜ but not at any other concentration.

10.1.10 Results summary tables

The IC50 values, percentage inhibition at a concentration of 2μΜ and percentage inhibition at a 1 :1 molar ratio for all IgGs in each of the chemotaxis assays are summarized in Tables 2 and 3. All IgGs behaved very similarly with the exception of MOR07337 which was consistently less potent than the other candidates. Germlining of IgGs caused no detectable alteration to their neutralizing capacity as compared to parental antibodies.

Table 2: ICsn values (nM) for inhibition of CCL21 -mediated chemotaxis by IgGs

Figure imgf000066_0001
MOR06935_g3-23_g3l 42 47 48 81

MOR06935_g3-23_g3r 39 80 22 49

MOR08378 g5-51_g2a2_opt 49.7 6.6 20.1 69

MOR08376_g5-51_g2a2_opt 69.7 6.5 24.6 n.d.

MOR08382_g3-23_gL6_opt 23.7 6.8 7.1 66

MOR08389_g3-23_g3j_opt 54.1 9.7 8.4 34

MOR08377_g5-51_g2a2_opt 12.7 7.6 21.3 n.d.

No No Weak

MOR03207 inhibition No inhibition inhibition inhibition

Table 3: Percentage inhibition of migration of CCL21 -mediated chemotaxis with IgGs at 2μΜ or l OOnM (1 :1 molar ratio)

Figure imgf000067_0001
MOR8951 100/61 100/84 100/61 100/58

MOR8952 100/72 100/78 100/63 100/49

MOR8953 100/70 100/90 100/62 100/57

MOR06935_g3-23_g3j 100/61 100/86 100/96 100/77

MOR06935_g3-23_g3l 100/78 100/87 100/74 100/68

MOR06935_g3-23_g3r 100/79 100/77 100/100 100/93

MOR08378 g5-51_g2a2_opt 100/87 100/100 100/100 100/64

MOR08376_g5-51_g2a2_opt 97/93 100/100 100/96 n.d.

MOR08382_g3-23_gL6_opt 97/97 100/100 100/100 100/70

MOR8389_g3-23_g3j_opt 94/94 100/100 100/100 100/82

MOR8377_g5-51_g2a2_opt 100/100 100/100 100/100 n.d.

MOR03207 24/2 37/0 81/0 54/4

10.1.11 Functional analysis of selected IgG

The functional data presented here and our goal to preserve structural diversity among the IgG candidates was used to select various IgGs. These are MOR06935_g3-23_g3j, MOR08378_g5- 51_g2a2, MOR08389_g3-23_g3j and MOR08382_g3-23_gl_6 representing 3 different frameworks. The synthesis of these IgGs was performed in large scale and tested in chemotaxis assays (Figures 8 to 12). IC50 values for MOR06935 ranged from 1 1 -45 nM in the four assays and inhibition at a 1 :1 molar ratio ranged from 81 -100% (Table 4). These values are comparable with data generated previously with MOR06935_g3-23-g3j. Table 4 : Inhibition of CCL21 mediated chemotaxis by lead and back-up candidates (ICsn and % inhibition at 1 :1 molar ratio)

Figure imgf000069_0001

Inhibition of CCL21 -mediated calcium mobilization in CHO-K1 cells transfected with human or cynomolgus monkey CCR7

Chemotaxis assays described herein utilize cells expressing the human CCR7 receptor. In order to demonstrate that the interaction between the cynomolgus monkey ligand and receptor and that of the human ligand and receptor is equivalent, a comparison was made of the ability of MOR06935_g3-23_g3j to inhibit calcium mobilization at the cynomolgus monkey (c) or human (h) CCR7 receptor. Transfected CHO-K1 cells were stimulated either with recombinant human or cynomolgus monkey CCL21 and calcium release measured by FLIPR (Figure 12). IC50 values were similar in all assays with the exception of inhibition of the cCCL21 at the receptor from the same species, where MOR06935_g3-23_g3j appears in the region of five-fold more potent (Table 5). This result can be accounted for by differences in receptor expression levels between different clones and batch variation in biological activity of recombinant cCCL21 , both of which may result in reduced responsiveness.

Table 5 IC50 values (nM) for inhibition of CCL21 -mediated calcium mobilization by

MOR06935_g3-23_g3j CCL21 CCR7 IC5o values (nM)

Human Human 341

Human Cynomolgus monkey 252

Cynomolgus monkey Human 21 1

Cynomolgus monkey Cynomolgus monkey 62

10.2.1 Epitope Mapping

Epitope mapping studies were carried out for each of the four main candidate antibodies MOR06935_g3-23_g3j, MOR08378_g5-51_g2a, MOR08382_g3_23_gL6 and MOR08389_g3- 23-g3j.

10.2.1.1 Structural model of huCCL21

State of the art bioinformatics methods permit the prediction of the secondary structure of huCCL21 on the basis of its known amino acid sequence

(MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKWRSYRKQEPSLGCSIPAILFL PRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTE RSQTPKGP). Figure 13A provides the sequence and predicted secondary structure for huCCL21.

NMR and Xray structures from chemokines related to CCL21 (CCL1 , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL1 1 , CCL13, CCL14, CCL15, CCL17, CCL20) were compared. The fold of the protein core for all these chemokines is very similar: the N-terminal region preceding the very conserved CC motif is disordered, it follows a loop which terminate in a 3io helix and then the helical turn is followed by 3 stranded β-sheet. The loop connecting β1 and β2 strands is not always defined as the rest of the fold. The β3 strand is then connected to a regular amphipathic a-helix. The helix is packed against the β-sheet and a number of hydrophobic interactions are formed between the β-sheet and helix. The C-terminal region follows, in CCL21 this region is very long compared to other CC chemokines. In particular, the Xray structure of CCL20 was used as a template for a 3 dimensional structural model. Not the entire CCL21 sequence could be modeled. The model consists of the amino acid residues staring with D30 and finishing with K92 (i.e. the sequence

DCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDK). Thus, the long C-terminal region (aa 93-134) containing the two additional cysteine residues is missing from the structural model.

The structural model is shown in Figure 13B. The N terminal residue is in the model is represented by D30. The C terminal residue in the model is represented by K92. The disulfide bonds between C31 -C57 and C32-C75 are also indicated as dashed lines. 10.2.1.2 Western blotting data

A dot blot was carried out using standard methodology. Reactivity of MOR06935_g3-23_g3j with huCCL21 was assessed. huCCL21 was bound to the membrane in (i) native form, (ii) following heat treatment to 95°C, (iii) heat treatment to 95°C in combination with reducing conditions (0.1 M TCEP), or (iv) heat treatment to 95°C, reducing conditions (0.1 M TCEP) and SDS detergent treatment to provide a comparison between antibody sensitivity for a native target and for a progressively more denatured target. Bound antibody was detected using a labeled secondary antibody (anti-human IgG).

Binding between MOR06935_g3-23_g3j and huCCL21 was seen under all conditions tested. This indicates a strong linear component to the epitope bound since the interaction between the antibody and antigen are not greatly influenced by treatments to disrupt the structure of the antigen (huCCL21 ).

10.2.1.3 H/DxMS mapping

Hydrogen/Deuterium exchange Mass Spectrometry was used to probe huCCL21 for information regarding the epitope for MOR06935_g3-23_g3j. H/DxMS mapping relies upon the mass difference between 'normal' hydrogen atoms and the 'heavy' isotope 'deuterium' which also comprises a neutron in addition to the single proton present in the normal hydrogen nucleus. Upon transfer from water to a deuterium based solvent system (heavy water), a protein will experience an increase in mass as the protein's hydrogen atoms become gradually replaced with deuterons (i.e. the heavier isotope of hydrogen). The likelihood of a hydrogen/deuterium exchange event is largely determined by protein structure and solvent accessibility. The Hydrogen/Deuterium Exchange Mass Spectrometry (H/DxMS) technology is used to measure exchange and as a consequence protein structure and solvent accessibility.

When a binding partner binds to an antibody target (e.g. antigen/antibody interaction), experimentally observable changes in hydrogen/deuterium exchange rate may be observed. Surface regions that exclude solvent upon complex formation exchange much more slowly. Solvent excluded regions are useful for deducing the location of a binding site. For instance in the case of an antigen-antibody interaction, these changes might highlight the location of the epitope. An additional effect to take into account is other perturbations that might result from the binding of the antibody to the antigen. Increased deuteron incorporation after antibody binding might represent either increased protection due to direct binding of the antibody to this region or indirect perturbation of the structure (allosteric changes) because of antibody binding. These two effects cannot be distinguished very easily, though the strongest effect observed is often attributed to direct protection from the antibody.

The location of increased deuteron incorporation after antibody binding may be deduced by digestion of the target protein following hydrogen/deuterium exchange (e.g. with a suitable enzyme such as pepsin) and then mass spectrometry to determine the mass of the relevant fragments.

Experimental set-up: In the labeling experiment, the antigen (huCCL21 ) was on-exchanged in solution for a predetermined time and then off-exchanged in the antibody column for the same duration. The epitope becomes deuterated during the on-exchange as it is exposed to the solvent and may retain deuterium during the off-exchange as it may be protected by the antibody (MOR06935_g3-23_g3j). On the other hand, regions which are not affected by antibody binding may be deuterated during the on-exchange but will lose deuterium during the off-exchange. In the labeling experiment, antigen is fully deuterated in solution and then affinity captured by column conjugated antibody. Then an H20 based solvent is passed through the column and non-protected deuterons are washed away.

In the control experiment, column captured material (i.e. the complex between huCCL21 and antibody) is deuterated and then washed with an H20 based solvent. Very little deuterium should remain in the control sample since the presence of the bound antibody should prevent deuteration of the epitope site. The only sites which will become deuterated are those which will be accessible to the H20 based solvent. Accordingly, there is no protection for bound deterons.

The eluted antigen (huCCL21 ) may then be digested into fragments (for example using a suitable protease) and subject to HPLC and mass spectrometry. By comparing the H/D-Ex patterns of the labeling and control experiments, the epitope is revealed as that area of the antigen retaining deuterium.

The results of this analysis, when conducted with MOR06935_g3-23_g3j, are provided in table 7 below.

In addition, the control data (obtained in the absence of antibody) was used to provide support for the structural model. Patterns of deuteration under these conditions (on exchange experiments without antibody at 23C and pH 7.0, five time points 30, 100, 300, 1 ,000 and 3,000 seconds) agreed with the structural model derived from sequence comparison and modeling.

10.2.1.4 Antigen Mutation Screening (AMS)

General: The idea of AMS is to identify residues involved in binding by generating variants of the target proteins and testing the binding of the antibody to the variant. Factors to take into account include the possibility that the mutation indirectly affects binding by changing the structure of the target, or that the substitution of a single amino acid residue might be difficult to detect if the overall contribution to binding at that location is small.

Two assay formats were tested: - ELISA with E.coli lysate.

Colony filter screening (CFS).

Library design: All positions in huCCL21 were substituted with alanine except prolines, glycines, cysteines and alanines. The resultant 77 huCCL21 variants were expressed with a DsbA signal sequence and a C-terminal flag-his tag (Plasmid RC86/1 ). Substitution of K43 or Q88 for alanine resulted in significantly reduced expression of huCCL21. CCL21 was expressed in soluble form in E.coli and experiments were carried out with the unpurified soluble proteins in order to avoid any potential difficulties in expression, purification and subsequent refolding of the target CCL21 variant.

ELISA: Plate coated with Fabs ^g/ml). Binding with CCL21 -FLAG wt (serial dilution of cell lysate). Detection with AP-conjugated anti-FLAG. The signal was strongest for MOR06935_g3- 23_g3j. Plate coated with cell lysate. Detection with AP-conjugated anti-FLAG. Expression level was variable.

Colony Filter Screening (CFS): Bacterial colonies expressing FLAG tagged huCCL21 variants from the library generated above were inoculated on plates on LB agar with glucose.

Expression of huCCL21 was under the control of an IPTG sensitive promoter. Therefore expression of huCCL21 is repressed on the first plate. Colonies were arranged in a regular grid pattern for ease of analysis. Thus the 'address' of each variant in the grid was known. Positive control colonies expressing wild type CCL21 were also included in the grid.

Following a suitable period of time to permit establishment and growth of the individual colonies, a pair of uncoated membranes were used to lift colonies representative bacteria off from the original agar plate (any suitable membrane may be used, for example PVDF). Briefly, the membrane was laid over the colonies for a sufficient length of time for some of the individual bacteria in the colony to adhere to the membrane (i.e. preserving the same pattern as the original colony inoculation onto the first plate). Each membrane was then laid onto a fresh agar dish containing LB and IPTG in order to induce expression of huCCL2 in the transferred bacteria.

Following a sufficient length of time to permit expression of CCL21 the membranes were then prepared for western blot analysis. The first membrane was probed using a Fab anti CCL21 derived from each of the four monoclonal antibodies defined above. The second membrane was probed using an anti FLAG antibody. The first membrane represents a probe for the specificity of the antibody in question for each of the mutant variants of huCCL21 in the library. The second membrane represents a positive control for expression of the huCCL21 variant associated with the colony in question.

Thus, comparison of the presence of huCCL21 (as indicated by a positive anti FLAG signal on the second membrane, with the strength of the signal observed for anti-CCL21 binding for each mutant provides an indication of the influence each amino acid residue in huCCL21 has in respect of the binding of each of the four antibodies. That is, the stronger the reduction in the signal seen on the first membrane for a given colony, the more significant the contribution of the relevant amino acid residue to the epitope that is bound by that antibody. Where the strength of signal observed for anti-FLAG and anti-CCL21 probes is comparable, the amino acid substitution represented in that member of the library is considered not to affect binding of the anti-CCL21 antibody to CCL21 .

Representative results are depicted in Figure 15B and 15C. Figure 15B depicts the first membrane showing anti-CCL21 binding strength. Figure 15C depicts anti-FLAG binding. The leftmost column for each membrane (enclosed in a long rectangular box) is made up from colonies expressing wild type CCL21 as a positive control. In Figure 15B, 'colonies' enclosed in circles show an absence of anti-CCL21 binding. 'Colonies' enclosed in square boxes indicated reduced binding.

A full summary of the AMS results obtained by CFS is provided in table 8 below.

10.2.1.5 Interpretation of the structural model in the light of mapping data

Another factor that was considered when mapping the epitopes for the four antibodies tested was the similarity/differences between human, cynomolgus monkey and mouse CCL21 sequences. For example, it was known that the antibodies in question bind to human and cynomolgus sequences, but not to mouse. Accordingly, amino acid residues that are conserved in human and cynomolgus CCL21 , but not conserved between human and mouse are more likely to be involved in the epitope. A sequence comparison for the key portions of human, cynomolgus and mouse CCL21 is provided in table 6 below.

Table 6 Comparison of human, cynomolgus and mouse CCL21 sequence

Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001

Figure imgf000078_0001

Antibodies absent in the species variant are indicated using "-"

On the basis of the primary sequence, predicted secondary structure and the 3D model, and in combination with the further epitope mapping data provided by H/DxMS and antigen mutation screening (AMS) and also comparison between the binding results obtained using CCL21 from different species, it was possible assess the accessibility of each amino acid residue to the external environment/possible binding partners. It was also possible to judge the likelihood that each amino acid residue forms part of the hydrophobic core of the molecule. This is indicated in table 7 below.

Table 7 Overview of structural model of huCCL21

Figure imgf000078_0002
Figure imgf000079_0001
Figure imgf000080_0001
134 P Not in model

The data provided by H/DxMS and AMS is summarised in table 8 below. H/DxMS data was obtained for MOR06935_g3-23_g3j in two separate experiments and is in close agreement. AMS data is shown for each of the four antibodies of interest: mAb 1 = MOR08378_g5-51 g2a2, mAb 2 = MOR06935_g3-23_g3j, mAb 3 = MOR08389_g3-23_g3j and mAb 4 = MOR08382_g3- 23_gl_6. Where H/DxMS or AMS data suggests a strong likelihood of an involvement for the residue in question, the term "Affects" is used below. Where an effect is observed, but less strongly, the term "Probable" is used. "N/A" indicates that the method used cannot address this amino acid residue. "?" indicates that data are not available.

Table 8 Summary of H/DxMS and AMS data

H/DxMS AMS

Position Amino Experiment Experiment mAb 1 mAb 2 mAb 3 mAb 4 no. acid 1 2

24 S N/A N/A N/A N/A

25 D

26 G N/A N/A N/A N/A

27 G N/A N/A N/A N/A

28 A N/A N/A N/A N/A

29 Q N/A N/A N/A N/A

30 D

31 C N/A N/A N/A N/A

32 C N/A N/A N/A N/A

33 L Affects

34 K Affects

35 Y Affects Probable Probable Affects Probable

36 S Affects Affects

37 Q Affects Affects

38 R Affects Affects Affects

39 K Affects Affects Probable Probable

40 I Affects Affects Affects

41 P Affects Affects N/A N/A N/A N/A

42 A Affects Affects N/A N/A N/A N/A

43 K Affects Affects Affects Affects Affects

44 V Affects Affects Probable

45 V Affects Affects Affects

46 R Affects Affects S Affects Affects

Y Affects Probable Probable Probable Affects

R Affects

K

Q

E

P N/A N/A N/A N/A

S

L

G N/A N/A N/A N/A

C N/A N/A N/A N/A

S

I

P Probable N/A N/A N/A N/A

A Probable N/A N/A N/A N/A

I Probable Probable Probable Probable Affects

L Probable ? ? ? ?

K Probable Affects Affects Affects Affects

L Probable Probable

P Probable Probable N/A N/A N/A N/A

R Probable Probable Probable Probable

K Probable Probable

R Probable Probable

S Probable Probable

Q Probable Probable

A Probable Probable N/A N/A N/A N/A

E Probable Probable

L Probable Probable Affects

C N/A N/A N/A N/A

A N/A N/A N/A N/A

D Affects Probable Probable Probable Affects

P Affects Affects N/A N/A N/A N/A

K Affects Affects

E Affects Affects Probable

L Affects Affects Affects Affects

W Affects Affects Affects Affects Affects Affects

V Affects Affects

Q Affects Affects Probable Probable

Q Affects Affects Affects Probable

L Affects Affects Probable Probable Affects

M Affects Probable Probable Probable

Q Affects Affects Affects Probable

H Affects Affects Probable Probable

L Affects Affects Affects Affects Probable Affects

D

K

T

P N/A N/A N/A N/A 95 S

96 P N/A N/A N/A N/A

97 Q

98 K

99 P N/A N/A N/A N/A

100 A N/A N/A N/A N/A

101 Q

102 G

103 C N/A N/A N/A N/A

104 R

105 K

106 D Probable

107 R

108 G N/A N/A N/A N/A

109 A N/A N/A N/A N/A

1 10 S

1 1 1 K

1 12 T

1 13 G N/A N/A N/A N/A

1 14 K

1 15 K

1 16 G N/A N/A N/A N/A

1 17 K

1 18 G N/A N/A N/A N/A

1 19 S

120 K

121 G N/A N/A N/A N/A

122 C N/A N/A N/A N/A

123 K

124 R

125 T

126 E

127 R

128 S

129 Q

130 T

131 P N/A N/A N/A N/A

132 K

133 G

134 P N/A N/A N/A N/A

Thus AMS data helps to provide a detailed view of the specific amino acid residues that are of particularly great significance in determining antigen/antibody interactions, within the wider context of the more general epitope identification provided by H/DxMS. By considering these data in combination with structural prediction, species (hu/cyno/mouse CCL21 ) specificity and sequence differences, and modeling data it is possible to identify amino acid residues that are likely to be part of the relevant epitope with an increasingly great degree of certainty. Therefore, the advantages of the individual mapping techniques used are able to complement one another, whilst any potential drawbacks for a single mapping technique are mitigated against. Comparison and combination of the data from all techniques permits a sophisticated and refined view of the epitope to be obtained and allows a high degree of confidence in predicting the contribution of any amino acid residue in huCCL21 to the epitope. Accordingly, it is possible to identify those amino acid residues that strongly contribute to the epitope, those that might have a more minor contribution and those that appear to have no contribution.

A progressive process for developing an increasingly detailed model of the epitope for mAb MOR06935_g3-23_g3j (aka QBP359) is illustrated in Figure 16. H/DxMS data, AMS data and a consideration of the structural model of CCL21 (and species variation) are considered in turn (and illustrated in Figure 16A-D). The individual amino acid residues of particular interest are therefore indentified with increasing specificity.

The conclusions of an equivalent process for each of the antibodies tested are illustrated in Figure 17. MOR06935_g3-23_g3j and MOR08389_g3-23_g3j have a very similar pattern in AMS. There is also a good overlap between the regions recognized by MOR06935_g3-23_g3j and those recognized by MOR08382_g3-23_gl_6. MOR08378_g5-51 g2a2 seems to be most different to MOR06935_g3-23_g3j in the pattern of the residues affecting binding observed by AMS. However, CCL21 is a small protein so we would not exclude a partial overlap of the epitope recognized by MOR08378_g5-51 g2a2 and MOR06935_g3-23_g3j.

10.2.2 Crossreactivity

The binding affinities as well as the kinetic rate constants of the anti-CCL21 human lgG1 antibody QBP359 to its human and cynomolgus target were determined using surface plasmon resonance (Biacore). The binding of CCL21 to QBP359 was recorded in a capture assay. The antibodies were captured on a protein A surface and CCL21 served as the analyte in solution. The resulting sensorgrams were fitted to a 1 :1 interaction model. A dissociation equilibrium constant KD of 30±4.5pM for human CCL21 and 26±9pM for cynomolgus CCL21 was determined. Very fast association rate constants of almost 107M-1 s-1 were observed for both antigens: 6.8±0.6 x 107M-1 s-1 for human CCL21 and 9.1 ±0.6 x 107M-1 s-1 for cynomolgus CCL21 .

Binding affinities of huCCL21 and cynoCCL21 to QBP359 were measured using a capture assay on a Biacore T100 instrument. In brief, 1 :2 dilution series of the analytes (huCCL21 and cynoCCL21 , respectively) were prepared and injected on the reference flow cell (Fc1 ) and on the measuring flow cells (Fc2) in parallel. The chip surface was regenerated before each analyte injection. For huCCL21 three independent experiments were performed, and two for cynoCCL21 .

Initial experiments were used to optimise the assay conditions for the antigen. In particular the buffer (HBS-N) conditions were optimised by adding 0.025%(w/v) BSA and 30mM citrate to PBS.

The binding affinities at equilibrium of huCCL21 and cynoCCL21 to anti-QBP359 were determined by surface plasmon resonance using the Biacore technology. For the binding of huCCL21 and cynoCCL21 to QBP359 a 1 :1 binding was observed. An identical affinity of approximately 30pM was determined for both huCCL21 (30±4.5pM) and cynoCCL21 (26±9pM) in three and two independent experiments, respectively. Very fast association rate constants of almost 107M-1 S-1 were observed for both antigens: 6.8±0.6 x 107M-1 s-1 for huCCL21 and 9.1 ±0.6 x 107M-1 s-1 for cynoCCL21. The binding affinities and the kinetic rate constants are summarized in Table 9 below. Corresponding standard deviations (which were determined in three independent experiments for huCCL21 and two for cynoCCL21 ) demonstrate that the rate constants could be determined reproducibly

Therefore, comparison shows close agreement between human and cynomolgus binding affinity. Separate experiments determined that mouse CCL21 does not bind to the antibody. Similarly, human CCL19 has been shown not to bind to any of the antibodies of the present invention. Accordingly, these antibodies are specific for an epitope(s) present in the human/cynomolgus version of CCL21 , but not the mouse form of the protein or in human CCL19.

Similarity between epitope mapping data for other antibodies discussed above would suggest similar specificity should be observed for each of the antibodies tested herein. Table 9 Binding affinities and kinetic rate constants of huCCL21 and cyno CCL21 to QBP359 measured by Biacore

Figure imgf000086_0001

10.3 Discussion

We have developed fully human anti-CCL21 antibodies to neutralize CCR7-mediated responses. This may lead to reduced fibroblast activation and fibrocyte recruitment in patients with IPF and therefore may prove effective in reducing the severity of fibrosis. Functional analysis of 8 antibodies and their germlined derivatives from 4 different parental frameworks revealed that 7 of these candidates were fully neutralizing as demonstrated by their ability to cause complete or near complete inhibition of CCL21 at equimolar concentrations in chemotaxis assays. Their inhibition of CCL21 is specific as demonstrated by the lack of inhibition by the anti-lysozyme control antibody MOR03207. One of the candidates, MOR07337, was

consistently weaker in all assays with the exception of the inhibition of natural CCL21 . This was also the only candidate with the VK4A H5 framework but the existing diversity of IgGs which neutralize in all 4 assays lead us to prioritize them ahead of MOR07337 given the inferior behavior of this antibody in 3 out of 4 assays. Germlined IgGs of the MOR08376, MOR08377, MOR08378, MOR08382 and MOR08389 families were generated in sufficient quantities to permit functional testing in calcium mobilization assays in addition to chemotaxis analysis (Table 5). In agreement with chemotaxis data all IgGs fully inhibited both cynomolgus and human CCL21 -mediated calcium release with similar potency. In summary, MOR06935_g3-23_g3j, MOR08378_g5-51_g2a2, MOR08389_g3-23_g3j and MOR08382_g3-23_gL6 represent 3 different frameworks and are fully neutralizing in CCL21 mediated functional assays in both human and cyno systems with comparable potency. The epitopes for each antibody have been mapped by a range of techniques including in silico modeling and structural predictions, linear epitope blotting, H/DxMS, AMS, and affinity measurements. H/DxMS data indicates the general regions of interest in the epitope mapping exercise, AMS data helps to identify particular amino acid residues that have an influence in antibody binding (directly or indirectly). The structural model for the target molecule allows for an assessment of the amino acid residues that are likely to be exposed on the surface of the molecule and thus, likely to be accessible to binding molecules, such as antibodies.

Thus, in combination, all of these epitope mapping data also permit the identification of those amino acid residues that are most likely to be involved in the interaction between the target (i.e. CCL21 ) and the binding molecule (e.g. antibody).

Sequence Correlation Table

Figure imgf000088_0001
23 CDRH1, MOR08378

24 CDRH2, MOR08378

25 CDRH3, MOR08378

26 CDRL1, MOR08378

27 CDRL2, MOR08378

28 CDRL3, MOR08378

29 VH, MOR08378

30 VL, MOR08378

31 Heavy chain, MOR08378

32 Light chain, MOR08378

33 CDRH1, MOR08382

34 CDRH2, MOR08382

35 CDRH3, MOR08382

36 CDRL1, MOR08382

37 CDRL2, MOR08382

38 CDRL3, MOR08382

39 VH, MOR08382

40 VL, MOR08382

41 Heavy chain, MOR08382

42 Light chain, MOR08382

43 CDRH1, MOR08389

44 CDRH2, MOR08389

45 CDRH3, MOR08389

46 CDRL1, MOR08389 47 CD L2, MOR08389

48 CDRL3, MOR08389

49 VH, MOR08389

50 VL, MOR08389

51 Heavy chain, MOR08389

52 Light chain, MOR08389

53 Human CCL21

54 Portion of CCL21 covered by three dimensional model

55 Portion of Cynomolgus CCL21 for comparison with human molecule (starting at amino acid 24)

56 Portion of Mouse CCL21A for comparison with human molecule (starting at amino acid 24)

57 Portion of Mouse CCL21B for comparison with human molecule (starting at amino acid 24)

SEQ . I . D . NO : 1

GFTFNSYSMS

SEQ . I . D . NO : 2

AISMEGYSKSYADSVKG

SEQ . I . D . NO : 3

GYGYYFDV

SEQ . I . D . NO : 4

GGDSIGSQYAS

SEQ . I . D . NO : 5

DDNERPS

SEQ . I . D . NO : 6

GSWDSNPTHV

SEQ . I . D . NO : 7

EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYSMSWVRQAPGKGLEWVSAISMEGYSKSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYYFDVWGQGTLVTVSS

SEQ . I . D . NO : 8

QVQLVESGGGLVQPGGSLRLSCAASGFTFNSYSMSWVRQAPGKGLEWVSAISMEGYSKSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYYFDVWGQGTLVTVSS

SEQ . I . D . NO : 9

SYELTQPLSVSVALGQTARITCGGDSIGSQYASWYQQKPGQAPVLVIYDDNERPSGIPERFSGSNSGNTA TLTISRAQAGDEADYYCGSWDSNPTHVFGGGTKLTVLG

SEQ. I .D. NO: 10

DIELTQPPSVSGSPGQSITISCSGDSIGSQYASWYQQKPGQAPWVIYDDNERPSGIPERFSGSNSGNTA TLTISGTQAEDEADYYCGSWDSNPTHVFGGGTKLTVLG

SEQ. I .D. NO: 11

EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYSMSWVRQAPGKGLEWVSAISMEGYSKSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ. I .D.NO: 12

QVQLVESGGGLVQPGGSLRLSCAASGFTFNSYSMSWVRQAPGKGLEWVSAISMEGYSKSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ. I .D.NO: 13

SYELTQPLSVSVALGQTARITCGGDSIGSQYASWYQQKPGQAPVLVIYDDNERPSGIPERFSGSNSGNTA TLTISRAQAGDEADYYCGSWDSNPTHVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLI SDF YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS

SEQ. I .D.NO: 14

DIELTQPPSVSGSPGQSITISCSGDSIGSQYASWYQQKPGQAPWVIYDDNERPSGIPERFSGSNSGNTA TLTISGTQAEDEADYYCGSWDSNPTHVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLI SDF YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS

SEQ. I .D.NO: 15

GAGGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCCTGCGCCG CCTCCGGCTTCACCTTCAACTCCTACTCCATGTCCTGGGTGCGCCAGGCTCCTGGCAAAGGACTGGAGTG GGTGTCCGCCATCTCCATGGAGGGCTACTCCAAGTCCTACGCCGACTCCGTGAAGGGCCGGTTCACCATC TCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAATAGTCTGAGAGCCGAGGACACCGCCGTGT ACTACTGCGCCAGGGGCTACGGCTACTACTTCGACGTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTC C

SEQ. I .D.NO: 16

CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGG CCTCCGGATTTACCTTTAATTCTTATTCTATGTCTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTG GGTGAGCGCTATTTCTATGGAGGGTTATTCTAAGTCTTATGCTGATTCTGTTAAGGGTCGTTTTACCATT TCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGT ATTATTGCGCGCGTGGTTATGGTTATTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTC A

SEQ. I .D.NO: 17

TCCTACGAGCTGACCCAGCCTCTGTCTGTCTCTGTCGCTCTGGGCCAGACCGCCCGGATCACCTGTGGCG GCGACTCCATCGGCTCCCAGTACGCCTCCTGGTATCAGCAGAAACCTGGACAGGCCCCTGTGCTGGTGAT CTACGACGACAACGAACGCCCATCTGGAATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCC ACCCTGACCATCTCCAGAGCCCAGGCCGGCGACGAGGCCGACTACTACTGCGGCTCCTGGGACTCCAACC CTACCCACGTGTTTGGCGGCGGAACAAAGCTGACCGTCCTGGGA

SEQ . I . D . NO : 18

GATATCGAACTGACCCAGCCGCCTTCAGTGAGCGGCTCACCAGGTCAGAGCATTACCATCTCGTGTAGCG GCGATTCTATTGGTTCTCAGTATGCTTCTTGGTACCAGCAGAAACCCGGGCAGGCGCCAGTTGTTGTGAT TTATGATGATAATGAGCGTCCCTCAGGCATCCCGGAACGCTTTAGCGGATCCAACAGCGGCAACACCGCG ACCCTGACCATTAGCGGCACTCAGGCGGAAGACGAAGCGGATTATTATTGCGGTTCTTGGGATTCTAATC CTACTCATGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGT

SEQ . I . D . NO : 19

GAGGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCCTGCGCCG CCTCCGGCTTCACCTTCAACTCCTACTCCATGTCCTGGGTGCGCCAGGCTCCTGGCAAAGGACTGGAGTG GGTGTCCGCCATCTCCATGGAGGGCTACTCCAAGTCCTACGCCGACTCCGTGAAGGGCCGGTTCACCATC TCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAATAGTCTGAGAGCCGAGGACACCGCCGTGT ACTACTGCGCCAGGGGCTACGGCTACTACTTCGACGTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTC CGCCTCCACCAAGGGCCCAAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGA CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGAC CGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAG GTGGACAAGAGAGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCCGAGC TGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCC CGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGT CCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC CCTGCCAGCCCCAATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACC CTGCCACCCAGCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACC CAAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGT GCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGC AACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGT CCCCAGGCAAGTGATGA

SEQ. I .D. NO: 20

CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGG CCTCCGGATTTACCTTTAATTCTTATTCTATGTCTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTG GGTGAGCGCTATTTCTATGGAGGGTTATTCTAAGTCTTATGCTGATTCTGTTAAGGGTCGTTTTACCATT TCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGT ATTATTGCGCGCGTGGTTATGGTTATTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTC AGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAG GTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAC TCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC TGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAA

SEQ . I . D . NO : 21

TCCTACGAGCTGACCCAGCCTCTGTCTGTCTCTGTCGCTCTGGGCCAGACCGCCCGGATCACCTGTGGCG GCGACTCCATCGGCTCCCAGTACGCCTCCTGGTATCAGCAGAAACCTGGACAGGCCCCTGTGCTGGTGAT CTACGACGACAACGAACGCCCATCTGGAATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCC ACCCTGACCATCTCCAGAGCCCAGGCCGGCGACGAGGCCGACTACTACTGCGGCTCCTGGGACTCCAACC CTACCCACGTGTTTGGCGGCGGAACAAAGCTGACCGTCCTGGGACAGCCTAAGGCCGCTCCTTCCGTGAC CCTGTTCCCTCCTTCCTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCTCCGACTTC TACCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGACTCCTCCCCTGTGAAGGCCGGCGTGGAGACAACCA CCCCTTCCAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCTACCTGTCCCTGACCCCTGAGCAGTGGAA GTCCCACCGGTCCTACTCTTGCCAGGTGACCCACGAGGGCTCCACCGTGGAGAAAACCGTGGCCCCTACC GAGTGCTCCTAGTGA

SEQ . I . D . NO : 22

GATATCGAACTGACCCAGCCGCCTTCAGTGAGCGGCTCACCAGGTCAGAGCATTACCATCTCGTGTAGCG GCGATTCTATTGGTTCTCAGTATGCTTCTTGGTACCAGCAGAAACCCGGGCAGGCGCCAGTTGTTGTGAT TTATGATGATAATGAGCGTCCCTCAGGCATCCCGGAACGCTTTAGCGGATCCAACAGCGGCAACACCGCG ACCCTGACCATTAGCGGCACTCAGGCGGAAGACGAAGCGGATTATTATTGCGGTTCTTGGGATTCTAATC CTACTCATGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCAC TCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTC TACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCA CACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAA GTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACA GAATGTTCA

SEQ. I .D. NO: 23

GYSFNNYWIS

SEQ . I . D . NO : 24

I IDPTNSQTTYSPSFQG

SEQ. I .D. NO: 25

DGYPIFDY

SEQ . I . D . NO : 26

TGTSSDLGGYNYVS

SEQ. I .D. NO: 27 DVNYRPS

SEQ . I . D . NO : 28

SSYTYYSNSGV

SEQ . I . D . NO : 29

EVQLVQSGAEVKKPGESLKISCKGSGYSFNNYWISWVRQMPGKGLEWMGI IDPTNSQTTYSPSFQGQVTI SADKSISTAYLQWSSLKASDTAMYYCARDGYPIFDYWGQGTLVTVSS

SEQ. I. D. NO: 30

QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNYRPSGVSNRFSGSKSG NTASLTI SGLQAEDEADYYCSSYTYYSNSGVFGGGTKLTVLG

SEQ. I. D. NO: 31

EVQLVQSGAEVKKPGESLKISCKGSGYSFNNYWISWVRQMPGKGLEWMGI IDPTNSQTTYSPSFQGQVTI SADKSISTAYLQWSSLKASDTAMYYCARDGYPIFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ . I . D . NO : 32

QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNYRPSGVSNRFSGSKSG NTASLTI SGLQAEDEADYYCSSYTYYSNSGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS

SEQ. I. D. NO: 33

GFTFSSYWLN SEQ. I. D. NO: 34

YISNQGDDTSYADSVKG SEQ. I. D. NO: 35

DYQYIGDSFDI SEQ. I. D. NO: 36

RASQYVSYVSLA SEQ. I. D. NO: 37

DASNRAT SEQ.I.D.NO:38

QQYDDLSYT SEQ.I.D.NO:39

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWLNWVRQAPGKGLEWVSYISNQGDDTSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARDYQYIGDSFDIWGQGTLVTVSS

SEQ.I.D.NO:40

EIVLTQSPATLSLSPGERATLSCRASQYVSYVSLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQYDDLSYTFGQGTKVEIKR

SEQ.I.D.NO:41

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWLNWVRQAPGKGLEWVSYISNQGDDTSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARDYQYIGDSFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ.I.D.NO:42

EIVLTQSPATLSLSPGERATLSCRASQYVSYVSLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQYDDLSYTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

SEQ.I.D.NO:43

GFTFNSYSMS SEQ.I.D.NO:44

AISMEGYSKSYADSVKG

SEQ.I.D.NO:45

GYGYYFDV

SEQ . I . D . NO : 46

GGDSIGSQYAS

SEQ. I. D. NO: 47 DDNERPS

SEQ.I.D.NO:48

SSWDMDAVNV SEQ . I . D . NO : 49

EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYSMSWVRQAPGKGLEWVSAISMEGYSKSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYYFDVWGQGTLVTVSS

SEQ. I. D. NO: 50

SYELTQPLSVSVALGQTARITCGGDSIGSQYASWYQQKPGQAPVLVIYDDNERPSGIPERFSGSNSGNTA TLTISRAQAGDEADYYCSSWDMDAVNVFGGGTKLTVLG

SEQ. I. D. NO: 51

EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYSMSWVRQAPGKGLEWVSAI SMEGYSKSYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ . I . D . NO : 52

SYELTQPLSVSVALGQTARITCGGDSIGSQYASWYQQKPGQAPVLVIYDDNERPSGIPERFSGSNSGNTA TLTISRAQAGDEADYYCSSWDMDAVNVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDF YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS

SEQ. I. D. NO: 53

MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFL

PRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTE

RSQTPKGP

SEQ. I. D. NO: 54

DCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDK SEQ. I. D. NO: 55 SDGGAQDCCLKYSQRKIPAKWRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQL MQHLDKTPTPRKPVQGCRKDRGVPKNGKKGKGCKRTEQSQTPKGP

SEQ.I.D.NO:56

SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNL MRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG

SEQ.I.D.NO:57

SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFBPRKHSKPELCANPEEGWVQNL MRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG

Claims

Claims
1 . A binding molecule that binds (e.g. specifically binds) with CCL21 , particularly human CCL21 (hCCL21 ) and modulates (e.g. inhibits) the interaction between hCCL21 and hCCR7.
2. The binding molecule of claim 1 , wherein said binding molecule also binds (e.g. specifically binds) with cynomolgus CCL21 and does not bind (e.g. specifically bind) mouse CCL21 , or human CCL19.
3. The binding molecule of claim 1 or 2 that binds (e.g. specifically binds) with CCL21 , particularly hCCL21 (hCCL21 ) and neutralises the biological activity thereof.
4. The binding molecule of claim 3 that binds (e.g. specifically binds) with CCL21 , particularly hCCL21 (hCCL21 ) and inhibits the interaction between hCCL21 and hCCR7.
5. The binding molecule of any one of the preceding claims that binds (e.g. specifically binds) with an epitope of human CCL21 (hCCL21 ), wherein the epitope: a) is comprised within the following amino acid residues from the sequence of CCL21 as defined with reference to SEQ ID No 53: i) aa33-aa49 and aa60-aa90, ii) aa33-aa49 and aa77-aa90, iii) aa35, aa38, aa39, aa48, aa62, aa64, aa65, aa67, aa74, aa77, aa82, aa86, aa87, aa90, iv) aa35, aa39, aa40, aa43-45, aa47, aa62, aa64, aa66, aa77, aa82-87, aa90, v) aa35, aa43, aa48, aa62, aa64, aa77, aa81 , aa82, aa84-88, aa90, vi) aa35, aa43, aa77, aa80-aa82, aa84-aa86, aa88-aa90, vii) aa43, aa81 , aa82, aa84, aa85, aa88, aa90, viii) aa43, aa81 , aa84, aa85, aa88, ix) aa38, aa64, aa74, aa82, aa86, aa90, x) aa35, aa43, aa64, aa81 , aa82, aa85, aa88, or xi) aa40, aa43, aa45, aa47, aa62, aa64, aa77, aa82, aa83, aa86, aa90; b) comprises at least one, two, three, four, five, or six of the amino acid residues as defined in any one of the groups (i) to (xi) listed above; or c) comprises the amino acid residues as defined in any one of the groups (i) to (xi) listed above.
6. The binding molecule of any one of the preceding claims wherein the binding molecule is a) an immunoglobulin, or b) an Adnectin, Ankyrin, Avimer, Affibody, Anticalin, or Affilin.
7. The binding molecule of claim 6 or 7 wherein the binding molecule is an immunoglobulin and the immunoglobulin is an antibody.
8. The binding molecule of any one of the preceding claims wherein the binding molecule is isolated.
9. The binding molecule of any one of the preceding claims wherein the binding molecule comprises CDRH3 of SEQ.I.D.NO:3 or 25 or 35 or 45.
10. The binding molecule of any one of claims 1 to 8 wherein the binding molecule comprises;
(a) CDRH1 of SEQ.I.D.NO:1 or 23 or 33 or 43;
(b) CDRH2 of SEQ.I.D.NO:2 or 24 or 34 or 44;
(c) CDRH3 of SEQ.I.D.NO:3 or 25 or 35 or 45.
1 1. The binding molecule of claim 10 further comprising;
(d) CDRL1 of SEQ.I.D.NO:4 or 26 or 46
(e) CDRL2 of SEQ.I.D.NO:5 or 27 or 47
(f) CDRL3 of SEQ.I.D.NO:6 or 28 or 48.
12. The binding molecule of any one of claims 1 to 8 wherein the antibody comprises a heavy chain variable domain of SEQ.I.D.NO: 7 or 8 or 29 or 39 or 49.
13. The binding molecule of claim 12 wherein the heavy chain variable region is combined with a light chain variable region to form an hCCL21 binding site.
14. The binding molecule of any one of claims 1 to 8 comprising a light chain variable region of SEQ.I.D.NO: 9 or 10 or 30 or 40 or 50.
15. The binding molecule of any one of claims 1 to 8 which comprises a heavy chain of SEQ.I.D.NO:1 1 or 12 or 31 or 41 or 51.
16. The binding molecule of any one of claims 1 to 8 which comprises a light chain of
SEQ.I.D.NO:13 or 14 or 32 or 42 or 52.
17. The binding molecule of claim 16 which comprises
(a) a heavy chain of SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:13;
(b) a heavy chain of SEQ.I.D.NO:31 and a light chain of SEQ.I.D.NO:32.
(c) a heavy chain of SEQ.I.D.NO:51 and a light chain of SEQ.I.D.NO:52
18. The binding molecule of claim 16 which comprises a heavy chain of SEQ.I.D.NO:1 1 and a light chain of SEQ.I.D.NO:14.
19. The binding molecule of claim 16 which comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:13.
20. The binding molecule of claim 16 which comprises a heavy chain of SEQ.I.D.NO:12 and a light chain of SEQ.I.D.NO:14.
21. The binding molecule of any one of claims 1 to 20 wherein the binding molecule is an intact antibody.
22. The antibody of claim 21 which is a human, humanised or chimeric antibody (e.g. having a human IgG such as lgG1 or lgG4 constant region).
23. The antibody of claim 22 which is a human antibody.
24. The binding molecule of any one of claims 1 to 20 which is an antibody fragment or a single variable domain.
25. The antibody of claim 24 wherein the antibody fragment is a ScFv, Fab, Fab , F(ab )2.
26. The antibody of claim 24 wherein the single variable domain is a dAb or VHH.
27. The binding molecule of any one of the preceding claims wherein the binding molecule is a bispecific antibody comprising a first specificity to hCCL21 and a second specificity to e.g. hCCL19.
28. An isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. by inhibiting the interaction between hCCL21 and hCCR7) said antibody comprising at least a heavy chain variable region capable of being encoded by a polynucleotide having at least 90% (such as 95% or greater, e.g. 96%, 97%, 98% or 99%) identity to SEQ.I.D.NO:15 or 16.
29. An isolated antibody that binds (e.g. specifically binds) hCCL21 and neutralises the biological activity thereof (e.g. by inhibiting the interaction between hCCL21 and hCCR7) said antibody comprising at least a heavy chain variable region capable of being encoded by a polynucleotide having at least 90% (such as 95% or greater, e.g. 96%, 97%, 98% or 99%) identity to SEQ.I.D.NO:15 or 16 and a light chain variable region capable of being encoded by a polynucleotide having at least 90% (such as 95% or greater, e.g. 96%, 97%, 98% or 99%) identity to SEQ.I.D.NO:17 or 18.
30. A binding molecule that binds (e.g. specifically binds) with CCL21 , particularly hCCL21 and inhibits (e.g. competitively) the binding to hCCL21 of the binding molecule of any one of the preceding claims, preferably wherein the inhibition is detectable at comparable (e.g. equimolar) conditions.
31. A polynucleotide encoding the heavy chain variable region of any one of the preceding claims.
32. A polynucleotide encoding the light chain variable region of any one the preceding claims.
33. A composition comprising the polynucleotides of claims 31 and 32.
34. A polynucleotide of any one of claims 31 or 33 wherein the heavy chain variable region polynucleotide is SEQ.I.D.NO:15 or 16.
35. A polynucleotide of either claim 32 or 33 wherein the light chain variable region
polynucleotide is SEQ.I.D.NO:17 or 18.
36. A polynucleotide encoding the heavy chain of any one of claims 1 to 30.
37. A polynucleotide encoding the light chain of any one of claims 1 to 30.
38. A composition comprising the polynucleotides of claim 36 and 37.
39. A polynucleotide of either claim 36 or 38 which is SEQ.I.D.NO:19 or 20.
40. A polynucleotide of either claim 37 or 38 which is SEQ.I.D.NO:21 or 22.
41. A polynucleotide encoding a heavy chain variable region which binds hCCL21 , said polynucleotide being at least 90% (such as 95% or greater, e.g. 96%, 97%, 97% or 99%) identical to SEQ.I.D.NO:15 or 16.
42. A polynucleotide encoding a light chain variable region (e.g. which may bind hCCL21 ), said polynucleotide being at least 90% (such as 95% or greater, e.g. 96%, 97%, 97% or 99%) identical to SEQ.I.D.NO:17 or 18.
43. A composition comprising the polynucleotides of claims 41 and 42.
44. A vector comprising the polynucleotide of any one of claims 31 to 43.
45. A stably transformed or transfected host cell comprising the polynucleotide of claim 44.
46. The host cell of claim 45 which is a mammalian cell.
47. The host cell of claim 46 which is from a CHO, NS0, or Y2/0 cell line.
48. A method for the production of an antibody which method comprises culturing a host cell according to any one of claims 45 to 47 under conditions permissive for the production said antibody.
49. The method of claim 48 which comprises culturing said host cell in serum-free media.
50. The method of claim 48 or 49 further comprising recovering said antibody.
51. A pharmaceutical composition comprising (e.g. as its sole therapeutically active ingredient) the binding molecule, immunoglobulin, antibody or antibody fragment according to any one of claims 1 to 30.
52. A pharmaceutical composition of claim 51 in intravenously or sub-cutaneously
administrable form.
53. A pharmaceutical composition of claim 51 or 52 wherein the binding molecule,
immunoglobulin, antibody or antibody fragment is in lyophilised form.
54. A the binding molecule, immunoglobulin, antibody or antibody fragment or a pharmaceutical composition of any one of claims 51 to 53 for use in treating and/or preventing (e.g. inhibiting the clinical progression) of a human disease or disorder.
55. The binding molecule, immunoglobulin, antibody, antibody fragment or pharmaceutical composition of claim 54 for use in treating and/or preventing (e.g. inhibiting the clinical progression) pulmonary fibrosis (e.g. IPF), the exacerbation of IPF, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post- ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof, in a mammalian patient (such as a human and/or a domesticated (e.g. companion) mammal.
56. Use of a binding molecule, immunoglobulin, antibody or antibody fragment according to any one of claims 1 to 30, or a pharmaceutical composition of any one of claims 51 to 53 in the manufacture of a medicament for use in a method of treating and/or preventing (e.g. inhibiting the clinical progression) of a human disease or disorder.
57. Use of a binding molecule, immunoglobulin or antibody or pharmaceutical composition according to claim 56 wherein the method is a method of treating and/or preventing (e.g. inhibiting the clinical progression) pulmonary fibrosis (e.g. IPF), the exacerbation of IPF, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post-ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof, in a mammalian patient (such as a human and/or a domesticated (e.g. companion) mammal.
58. A method of treating and/or preventing (e.g. inhibiting the clinical progression) in a human patient afflicted with pulmonary fibrosis (e.g. IPF), the exacerbation of IPF, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal disease, hypersensitivity pneumonitis, respiratory bronchiolitis interstitial lung disease, schistosoma mansoni infection, primary pulmonary hypertension caused by plexiform lesions, lung manifestation of herpes virus associated -diseases, dermatological manifestations of herpes virus associated diseases; keloid scarring, lupus, nephrogenic fibrosing dermopathy, fibrosing lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis congestive heart failure and other post- ischemic conditions, post-surgical scarring of the abdomen, post-surgical scarring of wide angle glaucoma trabeculotomy, and any combinations thereof which method comprises administering to said patient a therapeutically effective amount of the pharmaceutical composition of any one of claims 49 to 51 .
59. A complex comprising CCL21 , and a binding molecule, immunoglobulin, antibody or antibody fragment according to any one of claims 1 to 30,
60. A complex according to claim 59 wherein the CCL21 is human CCL21 or cynomolgus CCL21.
61. A complex according to claim 59 or 60 wherein the binding molecule, immunoglobulin, antibody or antibody fragment binds to CCL21 at an epitope as defined in claim 5.
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