"ANTIBODIES AGAINST G-CSFR AND USES THEREOF"

RELATED APPLICATION

The present application claims priority from US Provisional Patent Application

No. 61/496,351 entitled "Antibodies against G-CSFR and uses thereof filed on 13 June 2011, the entire contents of which are hereby incorporated by referenced.

SEQUENCE LISTING

The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

The present disclosure relates to antibodies that bind to granulocyte-colony stimulating factor receptor (G-CSFR) receptor and uses thereof, e.g., in therapy.

BACKGROUND

Granulocyte colony- stimulating factor (G-CSF) is a major regulator of granulocyte production. G-CSF is produced by bone marrow stromal cells, endothelial cells, macrophages, and fibroblasts, and production is induced by inflammatory stimuli. G-CSF acts through the G-CSF receptor (G-CSFR), which is expressed on early myeloid progenitors, mature neutrophils, monocytes/macrophages, T and B lymphocytes and endothelial cells. Mice deficient in G-CSF or the G-CSFR exhibit marked neutropenia, demonstrating the importance of G-CSF in steady-state granulopoiesis. However, G-CSF appears to be dispensable for emergency granulopoiesis, e.g., in response to an infection. G-CSF increases the production and release of neutrophils, mobilizes hematopoietic stem and progenitor cell, and modulates the differentiation, lifespan, and effector functions of mature neutrophils. G-CSF may also exert effects on macrophages, including expansion of monocyte/macrophage numbers, enhancement of phagocytic function, and regulation of inflammatory cytokine and chemokine production. G-CSF has also been shown to mobilize endothelial progenitor cells and induce or promote angiogenesis.

While G-CSF is used therapeutically, e.g., to treat neutropenia and/or mobilize hematopoietic stem cells, it also has negative actions in some conditions, e.g., inflammatory conditions and/or cancer. For example, administration of G-CSF

exacerbates rheumatoid arthritis (RA), murine collagen-induced arthritis (CIA) and a passive transfer model of CIA in rats. G-CSF has been found in the serum and synovial fluid of RA patients. Furthermore, interleukin (IL)-1 and tumor necrosis factor a (TNFα), which are found at increased levels in patients suffering from RA, induce the production of G-CSF by human synovial fibroblasts and chondrocytes. Mice deficient in G-CSF are resistant to the induction of acute and chronic inflammatory arthritis.

G-CSF has also been shown to play a role in multiple sclerosis (MS). For example, G-CSF enhances adhesion of an auto-reactive T cell line model of MS to extracellular matrix as effectively as interferon γ and TNFα, which are known to exacerbate MS symptoms. Moreover, G-CSF deficient mice are resistant to development of experimental autoimmune encephalomyelitis (EAE).

G-CSF and G-CSFR have also been tied to cancer, with studies showing that this signaling pathway contributes to chemotherapy resistance, growth, survival, invasiveness and metastasis of various cancers. Moreover, G-CSF has been shown to induce to angiogenesis, a process important in the development of solid tumors.

It will be clear to the skilled person from the foregoing, that there is a need in the art for reagents that reduce the signaling of G-CSF through the G-CSFR. Exemplary agents will be suitable for use as therapeutics, e.g., to treat or prevent a G-CSF-mediated condition.

SUMMARY

The present inventors have produced a class of proteins comprising antibody binding sites (e.g., Fabs and antibodies) that bind to human G-CSFR (hG-CSFR) and potently neutralize G-CSF signaling, e.g., prevent formation of granulocytes from CD34+ bone marrow cells and/or prevent cell proliferation in response to G-CSF and/or reduce or prevent neutrophilia induced by administration of G-CSF. A class of proteins identified by the inventors also cross-react cynomolgus monkey G-CSFR (cynoG-CSFR), which facilitates pre-clinical studies with the proteins. A class of proteins identified by the inventors bind to hG-CSFR with high affinity. A class of proteins identified by the inventors are human antibodies, which are suitable for treatment of a variety of conditions.

The present disclosure provides a protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds to hG-CSFR and neutralizes G-CSF signaling, and wherein the protein inhibits growth of colony forming units -granulocytes (CFU-G) from CD34+ bone marrow cells grown in the presence of G-CSF with an IC50 of at least about 0.2nM. For example, the IC50 is 0.1nM or less, for

example. 0.09nM or less, or 0.08nM or less, or 0.07nM or less, or 0.06nM or less or0.05nM or less. In one example, the IC50 is 0.04nM or less. In another example, the IC50 is 0.02nM or less. Methods for assessing IC50 of a protein in such an assay are described herein. For example, the IC50 is determined in the presence of 10ng/ml of hG-CSF.

In one example, the IC50 is determined by culturing CD34+ bone marrow cells in the presence of 10ng/ml stem cell factor and 10ng/ml hG-CSF. For example, the cells are grown in semi-solid cell culture medium. In one example, the CFU-G are enumerated after 14 days of culture.

The present disclosure additionally or alternatively provides a protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds to both human and cynomolgus monkey G-CSFR with a similar affinity and neutralizes G-CSF signaling. Such proteins are advantageous since they facilitate pre-clinical studies in non-human mammals.

In one example, the affinity of the protein is determined using a biosensor, e.g., by surface plasmon resonance. For example, the ligand binding region or soluble hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-Fc is immobilized and the affinity of the protein of the disclosure is determined.

The present disclosure additionally provides a provides a protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds specifically to the same epitope in hG-CSFR as that bound by C1.2 (comprising a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region ( VL) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5).

The present disclosure additionally or alternatively provides a protein comprising an antigen binding site of an antibody, wherein (i) the protein competitively inhibits binding of C1.2 (comprising a VH comprising a sequence set forth in SEQ ID NO: 2 and a VL comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5) to hG-CSFR; (ii) the protein neutralizes G-CSF signaling; and (iii) the level of binding of the protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1 ;

(b)the histidine at position 237 of SEQ ID NO: 1 ;

(c) the methionine at position 198 of SEQ ID NO: 1 ;

(d) the tyrosine at position 172 of SEQ ID NO: 1 ;

(e) the leucine at position 171 of SEQ ID NO:1 ; or

(f) the leucine at position 111 of SEQ ID NO:1

is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.

The present disclosure additionally or alternatively provides a protein comprising an antigen binding site of an antibody, wherein (i) the protein competitively inhibits binding of C1.2 (comprising a VH comprising a sequence set forth in SEQ ID NO: 2 and a VL comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5) to hG-CSFR; (ii) the protein neutralizes G-CSF signaling; and (iii) preferentially binds to a polypeptide of SEQ ID NO: 1 relative to its ability to bind to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1 ;

(b)the histidine at position 237 of SEQ ID NO: 1 ;

(c) the methionine at position 198 of SEQ ID NO: 1 ;

(d) the tyrosine at position 172 of SEQ ID NO: 1 ;

(e) the leucine at position 171 of SEQ ID NO: 1 ; or

(f) the leucine at position 111 of SEQ ID NO: 1.

The present disclosure additionally or alternatively provides a protein comprising an antigen binding site of an antibody, wherein (i) the protein binds to hG-CSFR; (ii) the protein neutralizes G-CSF signaling; and (iii) the level of binding of the protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1

(b) the histidine at position 237 of SEQ ID NO: 1 ;

(c) the methionine at position 198 of SEQ ID NO: 1 ;

(d) the tyrosine at position 172 of SEQ ID NO: 1 ;

(e) the leucine at position 171 of SEQ ID NO:1 ; or

(f) the leucine at position 111 of SEQ ID NO:1

is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.

The present disclosure additionally or alternatively provides a protein comprising an antigen binding site of an antibody, wherein (i) the protein binds to hG-CSFR; (ii) the protein neutralizes G-CSF signaling; and (iii) preferentially binds to a polypeptide of SEQ ID NO: 1 relative to its ability to bind to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1

(b) the histidine at position 237 of SEQ ID NO: 1 ;

(c) the methionine at position 198 of SEQ ID NO: 1 ;

(d) the tyrosine at position 172 of SEQ ID NO: 1 ;

(e) the leucine at position 171 of SEQ ID NO:1 ; or

(f) the leucine at position 1 1 1 of SEQ ID NO: 1.

In one example, the level of binding of the protein to the polypeptide comprising the alanine substitution is reduced by at least about 10 fold or 20 fold or 50 fold or 100 fold or 150 fold or 200 fold compared to the binding of the protein to the polypeptide of SEQ ID NO: 1. Preferably, the level of binding of the protein to the polypeptide comprising the alanine substitution is reduced by at least about 50 fold. Preferably, the level of binding of the protein to the polypeptide comprising the alanine substitution is reduced by at least about 60 fold.

In one example, the antigen binding site of the protein does not detectably bind to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the arginine at position 287 of SEQ ID NO: 1 .

In one example, the level of binding is assessed using a biosensor, e.g., by surface plasmon resonance. For example, the protein is immobilized and the level of binding to a polypeptide of SEQ ID NO: 1 or to a form of the polypeptide comprising an alanine substitution is determined.

Additional forms of a polypeptide comprising the amino acids of SEQ ID NO: 1 with or without other substitutions bound or not significantly bound or not detectably bound by a protein of the present disclosure are described herein and are to be taken to apply mutatis mutandis to the present examples of the disclosure.

In one example, the antigen binding site cross-reacts with:

(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ ID NO: 1 ; and/or

(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of SEQ ID NO: 1 .

In one example, the antigen binding site additionally cross-reacts with a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the leucine at position 169 of SEQ ID NO: 1

In one example, the protein competitively inhibits the binding of C1.2 (comprising a VH comprising a sequence set forth in SEQ ID NO: 2 and a VL comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH

comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5) to one or more of:

(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine at a position 167 of SEQ ID NO: 1 ; and/or

(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of SEQ ID NO: 1.

In one example, a protein described herein according to any example binds to an epitope comprising residues within one or two or three or four regions selected from

111-115, 170-176, 218-234 and/or 286-300 of SEQ ID NO: 1.

In one example, upon binding of a protein described herein according to any example to a polypeptide of SEQ ID NO: 1 and cleavage using proteolytic enzymes remains bound to one or two or three or four peptides comprising or consisting of amino acids 111-115 of SEQ ID NO: 1 or amino acids 170-176 of SEQ ID NO: 1 or amino acids 218-234 of SEQ ID NO: 1 or amino acids 286-300 of SEQ ID NO: 1.

In one example, the protein binds to a conformational epitope.

The present disclosure additionally or alternatively provides a protein that binds to hG-CSFR and neutralizes G-CSF signaling, the protein comprising at least one of:

(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 6, a CDR2 comprising a sequence set forth in SEQ ID NO: 7 and a CDR3 comprising a sequence at least about 55% identity to the sequence set forth in SEQ ID NO: 8;

(ii) a VH comprising a sequence at least about 80%, such as 85% or 90% or 91 % or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 2 and/or 4;

(iii) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 9, a CDR2 comprising a sequence set forth in SEQ ID NO: 10 and a CDR3 comprising a sequence at least about 33% identity to the sequence set forth in SEQ ID NO: 11 ; and (iv) a VL comprising a sequence at least about 80%, such as 85% or 90% or 91 % or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%identical to a sequence set forth in SEQ ID NO: 3 and/or 5.

Such a protein can comprise any one or more of the functional activities described herein, e.g., preferential binding to a polypeptide of SEQ ID NO: 1 relative to the level of binding of a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1

(b)the histidine at position 237 of SEQ ID NO: 1 ;

(c) the methionine at position 198 of SEQ ID NO: 1 ;

(d) the tyrosine at position 172 of SEQ ID NO: 1 ;

(e) the leucine at position 171 of SEQ ID NO:1 ; or

(f) the leucine at position 111 of SEQ ID NO: 1.

In one example, the percentage identity at (ii) is at least about 95%.

In one example, the percentage identity at (iv) is at least about 94%.

In one example, differences between the recited sequence and the protein are substitutions.

The skilled artisan will be capable of determining sites for mutations to a protein of the disclosure, e.g., within a framework region of a variable region containing protein. Moreover, the inventors have identified numerous sites in a VH CDR3 and a VL CDR3 that can be mutated as well as numerous mutations that maintain activity of a protein of the disclosure. For example a mutation, e.g., a substitution is within one or more (e.g., 2 or 3 or 4) of the four C-terminal residues of HCDR3 and/or one or more (e.g., 2 or 3 or 4 or 5 or 6) of the N-terminal or C-terminal residues of LCDR3. In one example, the N-terminal five amino acids of VH CDR3 are LGELG. In one example, the three N-terminal amino acids of VL CDR3 are QQS and/or the three C-terminal amino acids of VL CDR3 are PLT.

In one example, the VH comprises a CDR3 comprising a sequence LGELG X1X2X3X4, wherein:

X1 is selected from the group consisting of tryptophan, glutamine, methionine, serine, phenylalanine, glutamic acid and histidine and/or is a neutral amino acid, such as tryptophan, glutamine or methionine, for example, the amino acid is tryptophan;

X2 is an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, serine, glycine and isoleucine, for example is phenylalanine, tyrosine, methionine or serine, for example, the amino acid is phenylalanine;

X3 is an amino acid selected from the group consisting of aspartic acid, methionine, glutamine, serine, leucine, valine, arginine and histidine, for example, is proline, glutamic acid, alanine, leucine, phenylalanine or tyrosine, for example, the amino acid is aspartic acid; and

X4 is any amino acid or an amino acid selected from the group consisting of proline, glutamic acid, alanine, leucine, phenylalanine, tyrosine, threonine, asparagine, aspartic acid, serine, glycine, arginine, and lysine, for example, the amino acid is proline.

In one example, the VL comprises a CDR3 comprising a sequence X1X2X3X4X5X6X7X8X9, wherein:

X1 is an amino acid selected from the group consisting of glutamine, glutamic acid, histidine, alanine and serine and/or is a hydrophilic amino acid, such as glutamine or glutamic acid, for example, the amino acid is glutamine;

X2 is an amino acid selected from the group consisting of glutamine, valine, phenylalanine, asparagine and glutamic acid, for example, the amino acid is glutamine; X3 is an amino acid selected from the group consisting of serine and glycine, for example, the amino acid is serine;

X4 is an amino acid selected from the group consisting of tryptophan, methionine, phenylalanine, tyrosine, isoleucine and leucine, for example, the amino acid is tryptophan or tyrosine;

X5 is any amino acid or an amino acid selected from the group consisting of glutamic acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine, alanine, arganine, histidine, tyrosine, lysine and threonine, for example, the amino acid is serine;

Χ6 is an amino acid selected from the group consisting of tyrosine, methionine, isoleucine and threonine, for example, the amino acid is methionine, tyrosine or threonine;

X7 is an amino acid selected from the group consisting of proline, alanine, histidine, glycine and lysine, for example the amino acid is proline;

X8 is an amino acid selected from the group consisting of leucine, glutamine, methionine, alanine, phenylalanine, isoleucine, lysine, histidine and glycine, for example, the amino acid is leucine;

X9 is any amino acid or an amino acid selected from the group consisting of threonine, phenylalanine, tyrosine, methionine, lysine, serine, histidine, proline, tryptophan, isoleucine, glutamine, glycine and valine, for example, the amino acid is threonine.

The present disclosure additionally or alternatively provides a protein (e.g., an antibody) that binds to hG-CSFR and neutralizes G-CSF signaling, the protein comprising at least one variable region of an antibody selected from the group consisting of:

(i) a VH comprising an amino acid sequence set forth in SEQ ID NO: 2;

(ii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 3;

(iii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4;

(iv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 5;

(v) a VL comprising an amino acid sequence set forth in SEQ ID NO: 14;

(vi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 15;

(vii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 16;

(viii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 17;

(ix) a VL comprising an amino acid sequence set forth in SEQ ID NO: 18;

(x) a VL comprising an amino acid sequence set forth in SEQ ID NO: 19;

(xi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 20;

(xii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 21 ;

(xiii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 22;

(xiv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 23;

(xv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 24;

(xvi) a VL comprising an amino acid sequence set forth in SEQ ID NO: 25;

(xvii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 26;

(xviii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 27;

(xix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 28;

(xx) a VL comprising an amino acid sequence set forth in SEQ ID NO: 29;

(xxi) a VL comprising an amino acid sequence set forth in SEQ ID NO: 30;

(xxii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 31 ;

(xxiii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 32;

(xxiv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 33;

(xxv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 34;

(xxvi) a VL comprising an amino acid sequence set forth in SEQ ID NO: 35 ;

(xxvii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 36;

(xxviii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 37;

(xxix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 38;

(xxx) a VL comprising an amino acid sequence set forth in SEQ ID NO: 39;

(xxxi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 40;

(xxxii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 41 ;

(xxxiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 42;

(xxxiv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 43;

(xxxv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 44;

(xxxvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 45 ;

(xxxvii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 46;

(xxxviii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 47 (xxix) a VL comprising an amino acid sequence set forth in SEQ ID NO: 48; (xl) a VH comprising an amino acid sequence set forth in SEQ ID NO: 49; (xli) a VL comprising an amino acid sequence set forth in SEQ ID NO: 50; (xlii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 51 ; (xliii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 52; (xliv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 53;

(xlv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 54;

(xlvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 55;

(xlvii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 56;

(xlviii)a VH comprising an amino acid sequence set forth in SEQ ID NO: 57;

(xlix) a VL comprising an amino acid sequence set forth in SEQ ID NO: 58;

(l) a VH comprising an amino acid sequence set forth in SEQ ID NO: 59;

(li) a VL comprising an amino acid sequence set forth in SEQ ID NO: 60;

(lii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 61 ;

(liii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 62; and

(liv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 63.

In one example, a protein described herein comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain. The skilled artisan will understand that the antigen binding domain comprises the binding site of the antibody.

In one example, the VH and the VL are in a single polypeptide chain. For example, the protein is:

(i) a single chain Fv fragment (scFv);

(ii) a dimeric scFv (di-scFv); or

(iii) at least one of (i) and/or (ii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3.

In one example, the VL and VH are in separate polypeptide chains.

For example, the protein is:

(i) a diabody;

(ii) a triabody;

(iii) a tetrabody;

(iv) a Fab;

(v) a F(ab')2;

(vi) a Fv; or

(vii) one of (i) to (vi) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3.

The foregoing proteins (described in the previous two lists) can also be referred to as antigen binding domains of antibodies.

In one example, the protein is an antibody. In one example, the antibody is a naked antibody.

In one example, a protein is chimeric, de-immunized, humanized, human or primatized.

In one example, the protein or antibody is human.

The present disclosure additionally or alternatively provides an antibody that binds to hG-CSFR and neutralizes G-CSF signaling, the antibody comprising:

(i) a VH comprising an amino acid sequence set forth in SEQ ID NO: 2 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 3;

(ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 5;

(iii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 14;

(iv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 16;

(v) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 17;

(vi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 18;

(vii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 20 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 19;

(viii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 21 ;

(ix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 22;

(x) a VH comprising an amino acid sequence set forth in SEQ ID NO: 24 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 23;

(ix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 25;

(x) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 26;

(xi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 28 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 27;

(xii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 29;

(xiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 31 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 30;

(xiv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 33 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 32;

(xv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 34;

(xvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 36 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 35;

(xvii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 37;

(xviii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 40 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 39;

(xix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 42 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 41 ;

(xx) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 43;

(xxi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 45 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 44;

(xxii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 46;

(xxiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 47;

(xxiv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 49 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 48;

(xxv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 51 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 50;

(xxvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 53 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 52;

(xxvii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 54;

(xxviii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 55 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 5;

(xxix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 57 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 56;

(xxx) a VH comprising an amino acid sequence set forth in SEQ ID NO: 59 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 58;

(xxxi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 61 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 60;

(xxxii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 62;

(xxxiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 63; and

(xxxix) a VH comprising three CDRs of a VH set forth in any one or more of (i) to (xxxiii) and a VL comprising three CDRs of a VL set forth in any one or more of (i) to (xxxiii).

Sequences of exemplary VH and VL are described in Table 3, wherein the recited VH or VL CDR3 sequence is substituted for the corresponding sequence in the VH or VL of C1.2 or C1.2G as described herein.

In one example, the present disclosure provides an antibody that binds to hG-CSFR and neutralizes G-CSF signaling, the antibody comprising:

(i) a VH comprising an amino acid sequence set forth in SEQ ID NO: 2 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 3; or

(ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 5.

The present disclosure additionally or alternatively provides an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65. In one example, the antibody binds to hG-CSFR and neutralizes G-CSF signaling.

The present disclosure additionally or alternatively provides an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65. In one example, the antibody binds to hG-CSFR and neutralizes G-CSF signaling.

The present disclosure additionally or alternatively provides an antibody comprising one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID NO: 65. In one example, the antibody binds to hG-CSFR and neutralizes G-CSF signaling.

Reference herein to a protein or antibody that "binds to" hG-CSFR provides literal support for a protein or antibody that "binds specifically to" hG-CSFR.

In one example, a protein or antibody described herein does not significantly bind to mouse G-CSFR and/or does not detectably bind to mouse G-CSFR.

In one example, a protein or antibody described herein according to any example competitively inhibits binding of C1.2 and/or C1.2G to hG-CSFR or a cell expressing same or SEQ ID NO: 1 or a soluble hG-CSFR (e.g., comprising amino acids 1-311 of SEQ ID NO: 1 fused to a Fc region of an antibody).

In one example, a protein or antibody described herein binds to a ligand binding region of hG-CSFR and a ligand binding region of cynoG-CSFR with similar affinity. In one example, the protein binds to soluble hG-CSFR and soluble cynoG-CSFR with similar affinity. In one example, the protein binds to a polypeptide comprising SEQ ID NO: 1 and to a polypeptide comprising SEQ ID NO: 67 with similar affinity. In one example, the protein binds to hG-CSFR-Fc and cynoG-CSFR-Fc as described herein with similar affinity. In one example, the affinity is at least about 2nM, for example, at least about 1.5nM, such as at least about 1.2nM, 1.1nM or 1nM. In one example, the 0.5nM, such as, at least about 0.46nM or 0.45nM or 0.40nM or 0.39nM. In another example, the affinity is at least about 0.1nM, such as at least about 0.09nM, for example, at least about 0.08nM. In one example, the level of binding is assessed using a biosensor, e.g., by surface plasmon resonance. For example, the ligand binding region or soluble hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-Fc is immobilized and the level of binding to a protein of the disclosure is determined.

In another example, the protein of the disclosure is immobilized on, for example, a biosensor and the level of binding of the ligand binding region or soluble hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-Fc is determined. For example, the level of binding to the extracellular domain of hG-CSFR or cynoG-CSFR is determined. . In accordance with this example, the affinity of the protein for the extracellular domain of cynoG-CSFR is at least about InM, such as at least about 0.9nM, for example, at least about 0.75nM. For example, the affinity is at least about 0.7nM, such as at least about 0.6nM, for example, at least about 0.5nM. In one example, the affinity is about 0.5nM. Alternatively, or additionally, the affinity of the protein for the extracellular domain of hG-CSFR is at least about 7nM or 6nM or 5nM, such as at least about 4nM, for example, at least about 3nM, e.g., at least about 2.5nM. For example, the affinity is at least about 2.4 or 2.5nM.

The present disclosure also provides antigen binding domains or antigen binding fragments of the foregoing antibodies.

In one example, a protein or antibody as described herein comprises a constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In one example, the protein or antibody comprises an IgG4 constant region with a proline at position 241 (according to the numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and/or 1991)).

The C-terminal lysine of the heavy chain constant region of a whole antibody of the disclosure may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, whole antibodies may comprise antibody populations with all C-terminal lysine residues removed, antibody populations with no C-terminal lysine residues removed, and antibody populations having a mixture of antibodies with and without the C-terminal lysine residue. In some examples, the antibody populations may additionally comprise antibodies in which the C-terminal lysine residue is removed in one of the heavy chain constant regions. Similarly, a composition of whole antibodies may comprise the same or a similar mix of antibody populations with or without the C-terminal lysine residue.

In one example, the stabilized constant region comprises a sequence from position 1 19 to position 445 of SEQ ID NO: 64. In one example, the stabilized constant region comprises a sequence from position 119 to position 444 of SEQ ID NO: 68. In one example a protein or antibody as described herein or a composition of a protein or antibody as described herein, comprises a heavy chain constant region, including a stabilized heavy chain constant region, comprising a mixture of sequences fully or partially with or without the C-terminal lysine residue.

In one example, an antibody of the disclosure comprises a VH disclosed herein linked or fused to an IgG4 constant region or stabilized IgG4 constant region (e.g., as discussed above) and the VL is linked to or fused to a kappa light chain constant region.

The present disclosure also provides a protein or antibody which inhibits G-CSF-induced proliferation of a BaF3 cell expressing hG-CSFR with an IC50 of at least about 6nM. For example, the IC50 is 5.9nM or less. In another example, the IC50 is 2nM or less or 1nM or less or 0.7nM or less or 0.6nM or less or 0.5nM or less. In one example, the IC50 is determined by culturing BaF3 cells (e.g. about 2x104 cells) in the presence of about 0.5ng/ml hG-CSF, e.g., for about 48 hours. In one example, the proliferation of the BaF3 cells is determined by measuring 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

The present disclosure also provides a protein or antibody which inhibits G- CSF-induced proliferation of a BaF3 cell expressing hG-CSFR with an IC50 of at least about 10μg/ml. For example, the IC50 is 5μg/ml or less. In another example, the IC50 is 3μg/ml or less or 2μg/ml or less or 1μg/ml or less. In one example, the IC50 is about 0.8ug/ml. In one example, the IC50 is determined by culturing BaF3 cells (e.g. about 1x104 cells) in the presence of about 10ng/ml hG-CSF, e.g., for about 48 hours. In one example, the proliferation of the BaF3 cells is determined by measuring H-thymidine incorporation.

In one example, a protein or antibody of the disclosure binds to a soluble hG-CSFR comprising amino acid 1-311 of SEQ ID NO: 1 expressed as a fusion with an antibody Fc region (hG-CSFR-Fc) with an affinity of at least about 1.5nM. For example, the affinity is at least about 0.5nM or 0.4nM or 0.35nM or 0.33nM. In one example, the affinity of the protein is determined using a biosensor, e.g., by surface plasmon resonance. For example, the hG-CSFR-Fc is immobilized and the affinity of the protein of the disclosure is determined.

In one example, a protein or antibody of the disclosure binds to hG-CSFR expressed on the surface of a cell at an affinity of at least about 1nM, for example, at least about 0.5nM, such as, at least 0.4nM, for example, at least 0.3nM, such as, at least 0.2nM.

In one example, a protein as described herein according to any example is capable of reducing the number of neutrophils in circulation when or if administered to a cynomolgus monkey. For example, the protein reduces the number of neutrophils in circulation when or if administered to a cynomolgus monkey at a dose of between 0.05m/kg-30mg/kg, preferably between 0.1mg/kg-10mg/kg, e.g., administered at a dose of 0.1mg/kg or 1mg/kg or 2mg/kg or 5 mg/kg or 10mg/kg. For example, the protein reduces the number of neutrophils in circulation when or if administered following administration of G-CSF or filgrastim or a PEGylated form thereof, e.g., when or if the protein is administered about 12 hours after administration of G-CSF or filgrastim or a PEGylated form thereof. In one example, the reduction is a 2 fold or 3 fold reduction. In one example, the neutrophils are reduced about 10-24 hours, e.g., about 12 hours following administration.

In one example, a protein or antibody as described herein is isolated and/or recombinant.

In one example, a protein or antibody of the disclosure is conjugated to another compound, for example, a detectable label or a compound that extends the half-life of the protein or antibody, such as polyethylene glycol or an albumin binding protein.

The present disclosure also provides a nucleic acid encoding the protein or antibody of the present disclosure.

In one example, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be in a vector, e.g., a plasmid.

In examples of the disclosure directed to single polypeptide chain proteins, the expression construct may comprise a promoter linked to a nucleic acid encoding that polypeptide chain.

In examples directed to multiple polypeptide chains that form a protein, an expression construct comprises a nucleic acid encoding a polypeptide comprising, e.g., a VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, e.g., a VL operably linked to a promoter.

In another example, the expression construct is a bicistronic expression construct, e.g., comprising the following operably linked components in 5 ' to 3' order: (i) a promoter

(ii) a nucleic acid encoding a first polypeptide;

(iii) an internal ribosome entry site; and

(iv) a nucleic acid encoding a second polypeptide,

wherein the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

The present disclosure also contemplates separate expression constructs one of which encodes a first polypeptide comprising a VH and another of which encodes a second polypeptide comprising a VL. For example, the present disclosure also provides a composition comprising:

(i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

The present disclosure also provides an isolated or recombinant cell expressing a protein of the disclosure.

In one example, the cell comprises the expression construct of the disclosure or:

(i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

Examples of cells of the present disclosure include bacterial cells, yeast cells, insect cells or mammalian cells.

The present disclosure additionally provides methods for producing a protein or antibody of the disclosure. For example, such a method involves maintaining the expression construct(s) of the disclosure under conditions sufficient for the protein to be produced.

In one example, a method for producing a protein or antibody of the disclosure comprises culturing the cell of the disclosure under conditions sufficient for the protein or antibody to be produced and, optionally, secreted.

In one example, the method for producing a protein of the disclosure additionally comprises isolating the protein or antibody and, optionally, formulating the protein or antibody into a pharmaceutical composition.

The present disclosure additionally provides a composition comprising a protein or antibody of the disclosure and a pharmaceutically acceptable carrier.

The present disclosure additionally provides a composition comprising:

(i) an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65; and

(ii) (a) an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65; and/or

(b) an antibody comprising one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID NO: 65,

and, optionally, a pharmaceutically acceptable carrier.

The present disclosure also provides a method for treating or preventing a G-CSF-mediated condition in a subject, the method comprising administering the protein, antibody or composition of the disclosure. In this regard, a protein, antibody or composition can be used to prevent a relapse of a condition, and this is considered preventing the condition.

In one example, the G-CSF-mediated condition is an autoimmune disease, an inflammatory disease or cancer. For example, the autoimmune disease or the inflammatory disease is arthritis, multiple sclerosis, pulmonary inflammation or chronic obstructive pulmonary disease.

In one example, the method comprises administering an amount of the protein or antibody sufficient to reduce the number of neutrophils in a subject without inducing neutropenia.

The present disclosure alternatively or additionally provides a method for reducing the number of neutrophils in a subject without inducing neutropenia, the method comprising administering a protein comprising an antigen binding site of an antibody that binds (or specifically binds) to hG-CSFR to the subject. An exemplary

protein is an antibody or comprises an antigen binding domain thereof (e.g., a VH and/or a VL) or is an antigen binding fragment thereof. Exemplary proteins and antibodies are described herein.

In one example, a method described herein comprises administering an amount of the protein or antibody sufficient to reduce the number of neutrophils in a subject without inducing moderate neutropenia.

In one example, a method described herein comprises administering an amount of the protein or antibody sufficient to reduce the number of neutrophils in a subject without inducing severe neutropenia.

In one example, a method described herein comprises administering between about 0.05mg/kg and 30mg/kg of the protein or antibody. For example, the method comprising administering between 0.1mg/kg and 10mg/kg or between 0.2mg/kg and

5mg/kg of the protein or antibody. In one example, the method comprises administering about 0.5-2.0mg/kg of the protein or antibody.

The present disclosure also provides for use of a protein or antibody as described herein in any example in medicine.

The present disclosure also provides for use of a protein or antibody as described herein according to any example in the manufacture of a medicament to treat a G-CSF-mediated condition.

The present disclosure also provides a method for localizing and/or detecting and/or diagnosing and/or prognosing G-CSF-mediated condition associated with a cell expressing G-CSFR, the method comprising detecting in vivo a protein or antibody as described herein bound to the G-CSFR expressing cell, if present, wherein the protein or antibody is conjugated to a detectable tag.

In one example, the method additionally comprises administering the protein to the subject.

The present disclosure also provides a method for detecting G-CSFR or a cell expressing same in a sample, the method comprising contacting the sample with a protein or antibody as described herein according to any example such that a complex forms and detecting the complex, wherein detection of the complex is indicative of G-CSFR or a cell expressing same in the sample.

The present disclosure also provides a method for diagnosing or prognosing a G-CSF-mediated condition, the method comprising performing a method as described herein according to any example to detect G-CSFR or a cell expressing same, wherein detection of the G-CSFR or cell expressing same is diagnostic or prognostic of the condition.

The present disclosure also provides a kit comprising a protein or antibody as described herein according to any example packaged with instructions for use in a method as described herein.

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 - amino acids 25-335 of Homo sapiens G-CSFR (hG-CSFR) with a C-terminal polyhistidine tag

SEQ ID NO: 2 - VH of C1.2

SEQ ID NO: 3 - VL of C1.2

SEQ ID NO: 4 - VH of C1.2G

SEQ ID NO: 5 - VL of C1.2G

SEQ ID NO: 6 - HCDR1 of C1.2

SEQ ID NO: 7 - HCDR2 of C1.2

SEQ ID NO: 8 - HCDR3 of C1.2

SEQ ID NO: 9 - LCDR1 of C1.2

SEQ ID NO: 10 - LCDR2 of C1.2

SEQ ID NO: 11 - LCDR3 of C1.2

SEQ ID NO . 12 - consensus sequence of HCDR3 of C1.2

SEQ ID NO: 13 - consensus sequence of HCDR3 of C1.2

SEQ ID NO: 14 - VL of antibody 987

SEQ ID NO: 15 - VH of antibody 987

SEQ ID NO: 16 - VL of antibody 95

SEQ ID NO: 17 - VL of antibody 79

SEQ ID NO: 18 - VL of antibody 83

SEQ ID NO: 19 - VL of antibody 1003

SEQ ID NO: 20 - VH of antibody 1003

SEQ ID NO: 21 - VL of antibody 44

SEQ ID NO: 22 - VL of antibody 97

SEQ ID NO: 23 - VL of antibody 986

SEQ ID NO: 24 - VH of antibody 986

SEQ ID NO: 25 - VL of antibody 56

SEQ ID NO: 26 - VL of antibody 77

SEQ ID NO: 27 - VL of antibody 54

SEQ ID NO. 28 - VH of antibody 54

SEQ ID NO: 29 - VL of antibody 802

SEQ ID NO: 30 - VL of antibody 967

SEQ ID NO: 31 - VH of antibody 967

SEQ ID NO: 32 - VL of antibody 989

SEQ ID NO: 33 - VH of antibody 989

SEQ ID NO: 34 - VL of antibody 63

SEQ ID NO: 35 - VL of antibody 1002

SEQ ID NO: 36 - VH of antibody 1002

SEQ ID NO: 37 - VL of antibody 994

SEQ ID NO: 38 - VH of antibody 994

SEQ ID NO: 39 - VL of antibody 969

SEQ ID NO: 40 - VH of antibody 969

SEQ ID NO: 41 - VL of antibody 1000

SEQ ID NO: 42 - VH of antibody 1000

SEQ ID NO: 43 - VL of antibody 94

SEQ ID NO: 44 - VL of antibody 975

SEQ ID NO: 45 - VH of antibody 975

SEQ ID NO: 46 - VL of antibody 75

SEQ ID NO: 47 - VL of antibody 814

SEQ ID NO: 48 - VL of antibody 973

SEQ ID NO: 49 - VH of antibody 973

SEQ ID NO: 50 - VL of antibody 977

SEQ ID NO: 51 - VH of antibody 977

SEQ ID NO: 52 - VL of antibody 984

SEQ ID NO: 53 - VH of antibody 984

SEQ ID NO: 54 - VL of antibody 61

SEQ ID NO: 55 - VH of antibody 852

SEQ ID NO: 56 - VL of antibody 996

SEQ ID NO: 57 - VH of antibody 996

SEQ ID NO: 58 - VL of antibody 43

SEQ ID NO: 59 - VH of antibody 43

SEQ ID NO: 60 - VL of antibody 999

SEQ ID NO: 61 - VH of antibody 999

SEQ ID NO: 62 - VL of antibody 870

SEQ ID NO: 63 - VL of antibody 877

SEQ ID NO: 64 - Heavy chain of C1.2G with stabilized IgG4 constant region SEQ ID NO: 65 - Light chain of C1.2G with kappa constant region

SEQ ID NO: 66 - sequence of exemplary h-GCSFR

SEQ ID NO: 67 - polypeptide comprising Ig and CRH domains of Macaca ƒascicularis G-CSFR (cynoG-CSFR) with a C-terminal polyhistidine tag

SEQ ID NO: 68 - Heavy chain of C1.2G with stabilized IgG4 constant region and lacking C-terminal lysine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation showing inhibition of G-CSF-mediated proliferation of BaF3 cells by increasing concentrations of various anti-G-CSFR antibodies. The relative IC50 values for each antibody were; 10. 1μg/mL for mAb711 , 37.4μg/ml for mAb 774, 0.8μg/mL for C1.2G and was not determinable for mAb 744.

Figure 2 A is a graphical representation showing the relative binding of C1.2G and mAb744 to a series of alanine point mutants of SEQ ID NO: 1 compared to their binding to SEQ ID NO: 1 (positions of mutations are indicated with reference to SEQ ID NO: 1). The fold decrease in KD of the antibody for the mutant receptor compared to SEQ ID NO: 1 is depicted.

Figure 2B is a graphical representation showing the relative binding of C1.2G, mAb744 and mAb774 to a series of alanine point mutants of SEQ ID NO: 1 compared to their binding to SEQ ID NO: 1 (positions of mutations are indicated with reference to SEQ ID NO: 1). The fold decrease in KD of the antibody for the mutant receptor compared to SEQ ID NO: 1 is depicted.

Figure 3 is a graphical representation showing results of an assay in which pegylated G-CSF was administered to cynomolgus monkeys and one day later C1.2 was administered. The number of neutrophils per blood was assessed.

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohisto chemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook ex al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences oƒ Proteins oƒ Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Selected Definitions

For the purposes of nomenclature only and not limitation an exemplary sequence of a human G-CSFR is set out in NCBI Reference Sequence: NP_000751.1 (and set out in SEQ ID NO: 66). The sequence of cynomolgus monkey G-CSFR can be determined using sequences provided herein and/or in publically available databases and/or determined using standard techniques (e.g., as described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) Reference to human G-CSFR may be abbreviated to hG-CSFR and reference to cynomolgus monkey G-CSFR may be abbreviated to cynoG-CSFR. Reference to soluble G-CSFR refers to polypeptides comprising the ligand binding region of G-CSFR. The Ig and CRH domains of the G-CSFR are involved in ligand binding and receptor dimerization (Layton et al., J. Biol Chem., 272: 29735-29741, 1997 and Fukunaga et al, EMBO J. 10: 2855-2865, 1991). Soluble forms of G-CSFR comprising these portions of the receptor have been used in various studies of the receptor and mutation of the free cysteines at positions 78, 163, and 228 of the receptor assists in expression and isolation of the soluble receptor polypeptide (Mine et al., Biochem., 43: 2458-2464 2004) without affecting ligand binding. In the present studies soluble forms of the receptor comprising amino acids 25-335 of hG-CSFR with mutations C78A, C163S and C228S were used (e.g. SEQ ID NO: l) and the corresponding segment of cynoG-CSFR with the cysteine mutations was used (e.g., SEQ ID NO 67) for studies on the cynomolgus monkey receptor. Various point mutations of the soluble receptor of SEQ ID NO: 1 and SEQ ID NO: 67 have also been utilized. Reference to hG-CSFR-Fc means the polypeptide of SEQ ID NO: l wherein the C-terminal polyhistidine tag has been replaced with an Fc sequence (e.g., a polypeptide comprising amino acids 1 -311 of SEQ ID NO: 1 fused to an Fc). cynoG-CSFR-Fc means the corresponding segment of cynoG-CSFR with an Fc sequence attached to its C-terminus (e.g., a

polypeptide comprising amino acids 1-311 of SEQ ID NO: 67 fused to an Fc). The inventors have shown that antibodies and proteins comprising antigen binding sites thereof (e.g., Fab) bind to wild type hG-CSF polypeptides and to these mutant proteins with highly similar affinity. Accordingly, studies using the mutant proteins are a model of studies using hG-CSFR and/or cynoG-CSFR.

Reference herein to G-CSF includes native forms of G-CSF, mutant forms thereof, e.g., filgrastim and pegylated forms of G-CSF or filgrastim. This term also encompasses mutant forms of G-CSF retaining activity to bind to G-CSFR (e.g., hG-CSFR) and induce signaling.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally-associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By "substantially purified" is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

The term "recombinant" shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally-occurring within a subject's body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

As used herein, the term "antigen binding site" shall be taken to mean a structure formed by a protein that is capable of binding or specifically binding to an antigen. The antigen binding site need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain. For example, in a Fv produced from two different polypeptide chains the antigen binding site is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always in the one or more of the CDRs in each variable region. In some examples, an antigen binding site is a VH or a VL or a Fv.

The skilled artisan will be aware that an "antibody" is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a VL and a polypeptide comprising a VH. An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A VH and a VL interact to form a Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a κ light chain or a λ light chain and a heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The term "antibody" also encompasses humanized antibodies, primatized antibodies, human antibodies and chimeric antibodies.

The terms "full-length antibody," "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

As used herein, "variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. In the case of a protein derived from an IgNAR, the protein may lack a CDR2. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

As used herein, the term "complementarity determining regions" (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences oƒ Proteins oƒ Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems in the performance of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and/or Al-Lazikani et al., J Mol Biol 273: 927-948, 1997; the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol, 27: 55-77, 2003; or the AHO numbering system of Honnegher and Plükthun J. Mol. Biol, 309: 657-670, 2001. For example, according to the numbering system of Kabat, VH framework regions (FRs) and CDRs are positioned as follows: residues 1-30 (FR1 ), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above. In one example, reference herein to a CDR (or a FR) is in respect of those regions according to the Kabat numbering system.

"Framework regions" (FRs) are those variable region residues other than the CDR residues.

As used herein, the term "Fv" shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. The VH and the VL which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding sites which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means. A "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A "Fab2" fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A "single chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

As used herein, the term "binds" in reference to the interaction of a protein or an antigen binding site thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the protein, will reduce the amount of labeled "A" bound to the antibody.

As used herein, the term "specifically binds" or "binds specifically" shall be taken to mean that a protein of the disclosure reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a protein binds to G-CSFR (e.g., hG-CSFR) with materially greater affinity (e.g., 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other cytokine receptor or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). In an example of the present disclosure, a protein that

"specifically binds" to one form of hG-CSFR or a polypeptide comprising a region thereof (e.g., the ligand binding domain of hG-GCSFR) or a polypeptide comprising amino acids 1-311 of SEQ ID NO: 1 with an affinity at least 20 fold or 40 fold or 60 fold or 80 fold or 100 fold or 150 fold or 200 fold greater than it does to a mutant form of hG-CSFR or a polypeptide comprising a region thereof (e.g., a mutant form of the ligand binding domain of h-GCSFR) or a mutant form of SEQ ID NO: 1 comprising an alanine substituted for the native arginine at position 287. Additional exemplary changes to SEQ ID NO: 1 and their effect on binding are described herein. Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

As used herein, the term "does not detectably bind" shall be understood to mean that a protein, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or 8% or 6% or 5% above background. The background can be the level of binding signal detected in the absence of the protein and/or in the presence of a negative control protein (e.g., an isotype control antibody) and/or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the protein is immobilized and contacted with an antigen.

As used herein, the term "does not significantly bind" shall be understood to mean that the level of binding of a protein of the disclosure to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the protein and/or in the presence of a negative control protein (e.g., an isotype control antibody) and/or the level of binding detected in the presence of a negative control polypeptide. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the protein is immobilized and contacted with an antigen.

As used herein, phrases referring to "reduced binding" or "binding being at a lower level" in relation to an antigen will be understood to mean that an antibody binds to an antigen (e.g., an alanine point mutant of SEQ ID NO: 1 at any one of positions 287, 237, 198, 172, 171 or 111) with an affinity at least about 20 fold or 40 fold or 60 fold less than a control epitope or antigen (e.g. SEQ ID NO: 1). For example, a protein of the present disclosure can bind to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 237 at a level 20 fold or 40 fold or 60 fold less than it binds to a polypeptide of SEQ ID NO: 1. Preferably, the protein binds at a level 20 fold less, more preferably 40 fold less, still more preferably 60 fold less.

A protein or antibody may be considered to "preferentially bind" to a polypeptide if it binds that polypeptide with a dissociation constant (KD) that is less than the protein's or antibody's KD for another polypeptide. In one example, a protein or antibody is considered to preferentially bind to a polypeptide if it binds the polypeptide with an affinity (i.e., KD) that is at least about 20 fold or 40 fold or 60 fold or 80 fold or 100 fold or 120 fold or 140 fold or 160 fold more than the protein's or antibody's KD for another polypeptide.

As used herein, the term "similar affinity" will be understood to mean that a protein of the present disclosure binds to two antigens (e.g., the ligand binding domain of G-CSFR from humans and from cynomolgus monkeys) with affinities that are within about 5 fold or less of one another, e.g., within about 4, 3, 2, or 1 fold of one another, such as, within about 0.5 fold of one another or the levels of binding are substantially identical, e.g.. when the affinity is assessed by immobilizing the two antigens (e.g., the ligand binding domain of G-CSFR or extracellular domains from humans and from cynomolgus monkeys) and contacting the immobilized proteins with a protein of the disclosure.

For the purposes of clarification and as will be apparent to the skilled artisan based on the exemplified subject matter herein, reference to "affinity" in this specification is a reference to KD of a protein or antibody.

For the purposes of clarification and as will be apparent to the skilled artisan based on the description herein, reference to an "affinity of at least about" will be understood to mean that the affinity (or KD) is equal to the recited value or higher (i.e., the value recited as the affinity is lower), i.e., an affinity of 2nM is greater than an affinity of 3nM. Stated another way, this term could be "an affinity of X or less", wherein X is a value recited herein.

An "IC50 of at least about" will be understood to mean that the IC50 is equal to the recited value or greater (i.e., the value recited as the IC50 is lower), i.e., an IC50 of 2nM is greater than an IC50 of 3nM. Stated another way, this term could be "an IC50 of X or less", wherein X is a value recited herein.

As used herein, the term "epitope" (syn. "antigenic determinant") shall be understood to mean a region of hG-CSFR to which a protein comprising an antigen binding site of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and/or 5-10 or 2-5 or 1-3 amino acids outside of this region. In some examples, the epitope comprises a series of discontinuous amino acids that are positioned close to one another when hG-

CSFR is folded, i.e., a "conformational epitope". For example, a conformational epitope comprises amino acids in one or more or two or more or all of the regions corresponding to 111-115, 170-176, 218-234 and/or 286-300 of SEQ ID NO: 1. The skilled artisan will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three dimensional structural characteristics, and/or specific charge characteristics.

The term "competitively inhibits" shall be understood to mean that a protein of the disclosure (or an antigen binding site thereof) reduces or prevents binding of a recited antibody or protein to G-CSFR, e.g., to hG-CSFR. This may be due to the protein (or antigen binding site) and antibody binding to the same or an overlapping epitope. It will be apparent from the foregoing that the protein need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably, the protein reduces binding of the antibody by at least about 30%, more preferably by at least about 50%, more preferably, by at least about 70%, still more preferably by at least about 75%, even more preferably, by at least about 80% or 85% and even more preferably, by at least about 90%. Methods for determining competitive inhibition of binding are known in the art and/or described herein. For example, the antibody is exposed to G-CSFR either in the presence or absence of the protein. If less antibody binds in the presence of the protein than in the absence of the protein, the protein is considered to competitively inhibit binding of the antibody. In one example, the competitive inhibition is not due to steric hindrance.

CLAIMS:

1. A protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, and wherein the protein inhibits growth of colony forming units - granulocytes (CFU-G) from CD34+ bone marrow cells grown in the presence of G-CSF with an IC50 of at least about 0.2nM.

2. A protein comprising an antigen binding site of an antibody, wherein the antigen binding site binds to both human and cynomolgus monkey granulocyte-colony stimulating factor receptor (G-CSFR) with a similar affinity and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling.

3. A protein comprising an antigen binding site of an antibody wherein the antigen binding site binds specifically to the same epitope in human granulocyte-colony stimulating factor receptor (hG-CSFR) as that bound by C1.2 (comprising a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5).

4. A protein comprising an antigen binding site of an antibody, wherein (i) the protein competitively inhibits binding of C1.2 (comprising a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5) to human granulocyte-colony stimulating factor receptor (hG-CSFR); (ii) the protein neutralizes granulocyte colony stimulating factor (G-CSF) signaling; and (iii) the level of binding of the protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1;

(b) the histidine at position 237 of SEQ ID NO: 1;

(c) the methionine at position 198 of SEQ ID NO: 1;

(d) the tyrosine at position 172 of SEQ ID NO: 1;

(e) the leucine at position 171 of SEQ ID NO: 1 ; or

(f) the leucine at position 1 11 of SEQ ID NO: 1 :

is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.

5. The protein of claim 4, wherein the antigen binding site does not detectably bind to the polypeptide comprising the alanine substitution position 287 of SEQ ID NO: 1.

6. A protein comprising an antigen binding site of an antibody, wherein (i) the protein binds to human granulocyte colony stimulating factor receptor (hG-CSFR); (ii) the protein neutralizes granulocyte colony stimulating factor (G-CSF) signaling; and (iii) the level of binding of the protein to a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO: 1;

(b) the histidine at position 237 of SEQ ID NO: 1 ;

(c) the methionine at position 198 of SEQ ID NO: 1;

(d) the tyrosine at position 172 of SEQ ID NO: 1;

(e) the leucine at position 171 of SEQ ID NO: 1 ; or

(f) the leucine at position 1 11 of SEQ ID NO: 1 :

is lower than the level of binding of the protein to a polypeptide of SEQ ID NO: 1.

7. The protein of any one of claims 4 to 6, wherein the polypeptide to which the protein binds at a reduced level additionally comprises one or more of the following:

(i) an alanine at a position 111 of SEQ ID NO: 1 ;

(ii) an alanine at a position 171 of SEQ ID NO: 1 ;

(iii) an alanine at a position 172 of SEQ ID NO: 1 ; and

(iv) an alanine at a position 198 of SEQ ID NO: 1.

8. The protein of any one of claims 1 to 7, wherein the antigen binding site cross-reacts with:

(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ ID NO: 1; and/or

(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of SEQ ID NO: 1.

9. The protein of any one of claims 1 to 8, which competitively inhibits the binding of C1.2 (comprising a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G (comprising a VH comprising a sequence set forth in SEQ ID NO: 4 and a VL comprising a sequence set forth in SEQ ID NO: 5) to one or more of:

(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the lysine at position 167 of SEQ ID NO: 1; and/or

(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted for the histidine at position 168 of SEQ ID NO: 1.

10. A protein that binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the protein comprising at least one of:

(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 6, a CDR2 comprising a sequence set forth in SEQ ID NO: 7 and a CDR3 comprising a sequence at least about 55% identity to the sequence set forth in SEQ ID NO: 8;

(ii) a VH comprising a sequence at least about 80% identical to a sequence set forth in SEQ ID NO: 2 and/or 4;

(iii) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 9, a CDR2 comprising a sequence set forth in SEQ ID NO: 10 and a CDR3 comprising a sequence at least about 33% identity to the sequence set forth in SEQ ID NO: 11; and (iv) a VL comprising a sequence at least about 80% identical to a sequence set forth in SEQ ID NO: 3 and/or 5.

11. The protein of claim 10, the VH comprises a CDR3 comprising a sequence LGELGX1X2X3X4 (SEQ ID NO: 12), wherein:

X1 is an amino acid selected from the group consisting of tryptophan, glutamine, methionine, serine, phenylalanine, glutamic acid and histidine;

X2 is an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, serine, glycine and isoleucine;

X3 is an amino acid selected from the group consisting of aspartic acid, methionine, glutamine, serine, leucine, valine, arginine and histidine; and

X4 is an amino acid selected from the group consisting of proline, glutamic acid, alanine, leucine, phenylalanine, tyrosine, threonine, asparagine, aspartic acid, serine, glycine, arginine, and lysine.

12. The protein of claim 11, wherein:

X1 is a neutral amino acid;

X2 is phenylalanine, tyrosine, methionine or serine;

X3 is aspartic acid, methionine, glutamine, serine or leucine; and

X4 is proline, glutamic acid, alanine, leucine, phenylalanine or tyrosine.

13. The protein of claim 10, wherein the VL comprising a CDR3 comprising a sequence X1X2X3X4X5X6X7X8X9 (SEQ ID NO: 13), wherein:

X1 is an amino acid selected from the group consisting of glutamine, glutamic acid, histidine, alanine and serine;

X2 is an amino acid selected from the group consisting of glutamine, valine, phenylalanine, asparagine and glutamic acid;

X3 is an amino acid selected from the group consisting of serine and glycine;

X4 is an amino acid selected from the group consisting of tryptophan, methionine, phenylalanine, tyrosine, isoleucine and leucine;

X5 is an amino acid selected from the group consisting of glutamic acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine, alanine, arganine, histidine, tyrosine, lysine and threonine;

X6 is an amino acid selected from the group consisting of tyrosine, methionine, isoleucine and threonine;

X7 is an amino acid selected from the group consisting of proline, alanine, histidine, glycine and lysine;

X8 is an amino acid selected from the group consisting of leucine, glutamine, methionine, alanine, phenylalanine, isoleucine, lysine, histidine and glycine;

X9 is an amino acid selected from the group consisting of threonine, phenylalanine, tyrosine, methionine, lysine, serine, histidine, proline, tryptophan, isoleucine, glutamine, glycine and valine.

14. The protein of claim 13, wherein:

X1 is a hydrophobic amino acid, such as glutamine or glutamic acid;

X2 is glutamine;

X3 is an amino acid selected from the group consisting of serine and glycine;

X4 is an amino acid selected from the group consisting of tryptophan, methionine, phenylalanine and tyrosine;

X5 is an amino acid selected from the group consisting of glutamic acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine and alanine;

X6 is an amino acid selected from the group consisting of tyrosine, threonine and methionine;

X7 is an amino acid selected from the group consisting of proline, alanine, histidine and glycine;

X8 is an amino acid selected from the group consisting of leucine, glutamine, methionine, alanine, phenylalanine and isoleucine;

X9 is an amino acid selected from the group consisting of threonine, phenylalanine, tyrosine, methionine, lysine, serine, histidine and phenylalanine.

15. A protein that binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the protein comprising at least one variable region of an antibody selected from the group consisting of:

(i) a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 2;

(ii) a light chain variable region (VL) comprising an amino acid sequence set forth in

SEQ ID NO: 3;

(iii) a VH comprising ; an amino acid sequence set forth in SEQ ID NO: 4;

(iv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 5;

(v) a VL comprising an amino acid sequence set forth in SEQ ID NO: 14;

(vi) a VH comprising ; an amino acid sequence set forth in SEQ ID NO: 15,

(vii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 16;

(viii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 17;

(ix) a VL comprising an amino acid sequence set forth in SEQ ID NO: 18;

(x) a VL comprising an amino acid sequence set forth in SEQ ID NO: 19;

(xi) a VH comprising ; an amino acid sequence set forth in SEQ ID NO: 20,

(xii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 21;

(xiii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 22;

(xiv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 23;

(XV) a VH comprising ; an amino acid sequence set forth in SEQ ID NO: 24,

(xvi) a VL comprising an amino acid sequence set forth in SEQ ID NO: 25;

(xvii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 26;

(xviii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 27;

(xix) a VH comprising ; an amino acid sequence set forth in SEQ ID NO: 28,

(XX) a VL comprising an amino acid sequence set forth in SEQ ID NO: 29;

(xxi) a VL comprising an amino acid sequence set forth in SEQ ID NO: 30;

(xxii) a VH comprising ; an amino acid sequence set forth in SEQ ID NO: 31 ,

(xxiii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 32;

(xxiv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 33;

(xxv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 34;

(xxvi) a VL comprising an amino acid sequence set forth in SEQ ID NO: 35;

(xxvii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 36;

(xxviii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 37;

(xxix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 38;

(xxx) a VL comprising an amino acid sequence set forth in SEQ ID NO: 39;

(xxxi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 40;

(xxxii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 41 ;

(xxxiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 42;

(xxxiv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 43;

(xxxv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 44;

(xxxvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 45;

(xxxvii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 46;

(xxxviii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 47;

(xxix) a VL comprising an amino acid sequence set forth in SEQ ID NO: 48

(xl) a VH comprising an amino acid sequence set forth in SEQ ID NO: 49

(xli) a VL comprising an amino acid sequence set forth in SEQ ID NO: 50

(xlii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 51

(xliii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 52

(xliv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 53

(xlv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 54

(xlvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 55

(xlvii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 56

(xlviii)a VH comprising an amino acid sequence set forth in SEQ ID NO: 57

(xlix) a VL comprising an amino acid sequence set forth in SEQ ID NO: 58

(l) a VH comprising an amino acid sequence set forth in SEQ ID NO: 59

(li) a VL comprising an amino acid sequence set forth in SEQ ID NO: 60

(lii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 61

(liii) a VL comprising an amino acid sequence set forth in SEQ ID NO: 62; and (liv) a VL comprising an amino acid sequence set forth in SEQ ID NO: 63.

16. The protein of any one of claims 1 to 15 comprising at least a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL bind to form a Fv comprising an antigen binding domain.

17. The protein of claim 16, wherein the VH and the VL are in a single polypeptide chain.

18. The protein of claim 17, which is:

(i) a single chain Fv fragment (scFv);

(ii) a dimeric scFv (di-scFv); or

(iii) at least one of (i) and/or (ii) linked to a constant region of an antibody, a Fc or a heavy chain constant domain (CH) 2 and/or CH3.

19. The protein of claim 16, wherein the VL and VH are in separate polypeptide chains.

20. The protein of claim 19, which is:

(i) a diabody;

(ii) a triabody;

(iii) a tetrabody;

(iv) a Fab;

(v) a F(ab')2;

(vi) a Fv; or

(vii) one of (i) to (vi) linked to a constant region of an antibody, a Fc or a heavy chain constant domain (CH) 2 and/or CH3.

21. The protein of claim 19, which is an antibody.

22. The protein of any one of claims 1 to 21 which is chimeric, de-immunized, humanized, human or primatized.

23. An antibody to that binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the antibody comprising:

(i) a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 3;

(ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 5;

(iii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 14;

(iv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 16;

(v) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a comprising an amino acid sequence set forth in SEQ ID NO: 17;

(vi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 18;

(vii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 20 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 19;

(viii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 21 ;

(ix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 22;

(x) a VH comprising an amino acid sequence set forth in SEQ ID NO: 24 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 23;

(ix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 25;

(x) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 26;

(xi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 28 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 27;

(xii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 29;

(xiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 31 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 30;

(xiv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 33 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 32;

(xv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 34;

(xvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 36 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 35;

(xvii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 37;

(xviii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 40 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 39;

(xix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 42 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 41 ;

(xx) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 43;

(xxi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 45 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 44;

(xxii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 46;

(xxiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 47;

(xxiv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 49 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 48;

(xxv) a VH comprising an amino acid sequence set forth in SEQ ID NO: 51 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 50;

(xxvi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 53 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 52;

(xxvii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 54;

(xxviii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 55 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 5;

(xxix) a VH comprising an amino acid sequence set forth in SEQ ID NO: 57 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 56;

(xxx) a VH comprising an amino acid sequence set forth in SEQ ID NO: 59 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 58;

(xxxi) a VH comprising an amino acid sequence set forth in SEQ ID NO: 61 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 60;

(xxxii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 62;

(xxxiii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 63; and

(xxxix) a VH comprising three CDRs of a VH set forth in any one or more of (i) to (xxxiii) and a VL comprising three CDRs of a VL set forth in any one or more of (i) to (xxxiii).

24. An antibody to that binds to human granulocyte-colony stimulating factor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulating factor (G-CSF) signaling, the antibody comprising:

(i) a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 3; or

(ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 5.

25. An antibody comprising:

(i) a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 or 68 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65; or

(ii) one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID NO: 65.

26. The protein or antibody of any one of claims 1 to 25, which inhibits G-CSF-induced proliferation of a BaF3 cell expressing hG-CSFR with an IC50 of at least about 0.5nM.

27. The protein or antibody of any one of claims 1 to 26, which binds to a polypeptide comprising amino acids 1 to 31 1 of SEQ ID NO: 1 expressed as a fusion with an antibody Fc region with an affinity of at least about 0.5nM, wherein affinity is determined in an assay in which the polypeptide immobilized and the protein or antibody contacted to the immobilized polypeptide.

28. The protein or antibody of any one of claims 1 to 27, which binds to hG-CSFR expressed on the surface of a cell at an affinity of at least about 1nM.

29. The protein of any one of claims 1 to 28 conjugated to another compound.

30. A composition comprising the protein or antibody of any one of claims 1 to 29 and a pharmaceutically acceptable carrier.

31. A composition comprising:

(i) an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65; and

(ii) (a) an antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 65 and/or

(b) an antibody comprising one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 64 and one heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 68 and two light chains comprising an amino acid sequence set forth in SEQ ID NO: 65,

and, optionally, a pharmaceutically acceptable carrier.

32. A method for treating or preventing a granulocyte-colony stimulating factor (G-CSF)-mediated condition in a subject, the method comprising administering the protein or antibody of any one of claims 1 to 29 or the composition of claim 30 or 31 to the subject.

33. The method of claim 32, wherein the G-CSF-mediated condition is an autoimmune disease, an inflammatory disease or cancer.

34. The method of claim 33, wherein the autoimmune disease or the inflammatory disease is arthritis, multiple sclerosis, pulmonary inflammation or chronic obstructive pulmonary disease.

35. The method of any one of claims 32 to 34 comprising administering an amount of the protein or antibody sufficient to reduce the number of neutrophils in a subject without inducing neutropenia.

36. The method of claim 35, wherein the amount of the protein is between about 0.05mg/kg and 30mg/kg.