Humanized anti-interleukin 3 receptor alpha chain antibodies

1 225631/2

HUMANIZED ANTI-INTERLEUKIN 3 RECEPTOR ALPHA CHAIN ANTIBODIES

FIELD

The present disclosure relates to anti-interleukin 3 receptor alpha chain antibodies and uses thereof.

BACKGROUND

The functional interleukin 3 receptor is a heterodimer that comprises a specific alpha chain (IL-3Ra; CD123) and a “common” IL-3 receptor beta chain (βο; CD131) that is shared with the receptors for granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 5 (IL-5). IL-3Ra is a type I transmembrane protein with a deduced Molecular Weight of about 41kDa containing an extracellular domain involved in IL-3 binding, a transmembrane domain and a short cytoplasmic tail of about 50 amino acids. The extracellular domain is composed of two regions: an N-terminal region of about 100 amino acids, the sequence of which exhibits similarity to equivalent regions of the GM-CSF and IL-5 receptor alpha-chains; and a region proximal to the transmembrane domain that contains four conserved cysteine residues and a WSXWS motif, common to other members of this cytokine receptor family.

The IL-3 binding domain comprises about 200 amino acid residue cytokine receptor motifs (CRMs) made up of two Ig-like folding domains. The extracellular domain of IL-3Ra is highly glycosylated, with N-glycosylation necessary for both ligand binding and receptor signaling. IL-3Ra is expressed widely throughout the hematopoietic system including hematopoietic progenitors, mast cells, erythroid cells, megakaryocytes, basophils, eosinophils, monocytes/macrophages, neutrophils and CD5+ B-lymphocytes. Non-hematopoietic cells such as plasmacytoid dendritic cells (pDCs), Leydig cells, endothelial cells and stromal cells also express IL-3Ra. IL-3Ra is also expressed by cells involved in certain disease states including myelodysplastic syndrome, myeloid leukemia (for example, acute myelogenous leukemia (AML)), malignant lymphoproliferative disorders such as lymphoma, allergies and autoimmune disease, such as lupus, Sjogren's syndrome or scleroderma. Accordingly, anti-IL-3Ra antibodies are desirable for therapeutic applications. WO/2009/070844 discloses a method for inhibiting leukemic stem cells expressing IL-3Ra comprising contacting the cells with an antigen binding molecule which binds selectively to IL-3Ra. The method can be used for treatment of a hematologic cancer condition by administration of the antigen binding molecule. The antigen binding molecule may be a chimeric or a humanized antibody. 2 225631/2

SUMMARY

The present disclosure is based on the inventor's production of a humanized antibody that binds specifically to IL-3Ra. Following humanization, the inventor found that the affinity of the antibody for IL-3Ra was reduced. Accordingly, the inventor performed affinity maturation to improve the affinity of the antibody for IL-3Ra. Unpredictably, the affinity matured antibody included mutations in the framework regions (FRs) of the heavy chain variable region (VH) and light chain variable region (VL), as well as in CDR1 of the light chain.

The inventor also produced forms of this antibody capable of inducing enhanced levels of effector function.

The inventor also found that a particular modification to induce enhanced effector function resulted in an additional desirable property in that following administration of the modified antibody to a mammal the number of NK cells in the mammal were initially reduced, however then expanded to levels greater than prior to administration. The inventor has also shown that there is a correlation between the number of NK cells and lysis of target cells, e.g., leukemia cells.

The present disclosure is broadly directed to an immunoglobulin based on a murine antibody capable of specifically binding to an IL-3Ra chain.

In one example, the present disclosure provides an isolated or recombinant immunoglobulin that specifically binds to an IL-3Ra chain and comprises complementarity determining regions (CDRs) of a VL comprising a sequence set forth in SEQ ID NO: 8 and CDRs of a VH set forth in SEQ ID NO: 9.

The present disclosure additionally or alternatively provides an isolated or recombinant antibody or antigen binding fragment thereof, the antibody or antigen binding fragment capable of specifically binding to IL-3Ra chain and comprising CDRs of a VL comprising a sequence set forth in SEQ ID NO: 8 and CDRs of a VH set forth in SEQ ID NO: 9.

The present disclosure additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof, the antibody or antigen binding fragment capable of specifically binding to IL-3Ra chain and comprising CDRs of a VL comprising a sequence set forth in SEQ ID NO: 8 and CDRs of a VH set forth in SEQ ID NO: 9.

In one example, the present disclosure provides an isolated or recombinant immunoglobulin that specifically binds to an IL-3Ra chain and comprises amino acid sequences according to SEQ ID NOs: 2-7, respectively.

The present disclosure additionally or alternatively provides an isolated or recombinant antibody or antigen binding fragment thereof, the antibody or antigen binding fragment capable of specifically binding to IL-3Ra chain and comprising amino acid sequences according to SEQ ID NOs: 2-7.

The present disclosure additionally or alternatively provides an isolated or WO 2012/021934 PCT/AU2011/001056 3 recombinant humanized antibody or antigen binding fragment thereof, the antibody or antigen binding fragment capable of specifically binding to IL-3Ra chain and comprising amino acid sequences according to SEQ ID NOs: 2-7.

For example, the immunoglobulin or antibody comprises: 5 (i) a light chain variable region (Vl) comprising CDRs 1, 2 and 3 as set forth in SEQ ID NOs: 2, 3 and 4, respectively; and (ii) a heavy chain variable region (Vh) comprising CDRs 1, 2 and 3 as set forth in SEQ ID NOs: 5, 6 and 7, respectively.

For example, the immunoglobulin or antibody comprises: 10 (i) a Vl comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 2 (or encoded by a sequence set forth in SEQ ID NO: 14); b) a CDR2 comprising a sequence set forth in SEQ ID NO: 3(or encoded by a sequence set forth in SEQ ID NO: 15); and 15 c) a CDR3 comprising a sequence set forth in SEQ ID NO: 4 (or encoded by a sequence set forth in SEQ ID NO: 16); and (ii) a Vh comprising: d) a CDR1 comprising a sequence set forth in SEQ ID NO: 5 (or encoded by a sequence set forth in SEQ ID NO: 17); 20 e) a CDR2 comprising a sequence set forth in SEQ ID NO: 6 (or encoded by a sequence set forth in SEQ ID NO: 18); and f) a CDR3 comprising a sequence set forth in SEQ ID NO: 7 (or encoded by a sequence set forth in SEQ ID NO: 19).

The present disclosure additionally or alternatively provides an isolated or 25 recombinant humanized antibody or antigen binding fragment thereof, the antibody or antigen binding fragment capable of specifically binding to IL-3Ra chain and comprising a Vl comprising an amino acid sequence according to SEQ ID NO: 8 (or encoded by a sequence set forth in SEQ ID NO: 20) and/or a Vh comprising an amino acid sequence according to SEQ ID NO: 9 (or encoded by a 30 sequence set forth in SEQ ID NO: 21).

The present disclosure additionally or alternatively provides an isolated or

recombinant humanized antibody or antigen binding fragment thereof, the antibody or antigen binding fragment capable of specifically binding to IL-3Ra chain and comprising a Vl comprising an amino acid sequence according to SEQ 35 ID NO: 8 (or encoded by a sequence set forth in SEQ ID NO: 20) and a Vh comprising an amino acid sequence according to SEQ ID NO: 9 (or encoded by a sequence set forth in SEQ ID NO: 21).

Exemplary antigen binding fragments contemplated by the present disclosure include: 40 (i) a domain antibody (dAb); (ii) a Fv; (iii) a scFv or stabilized form thereof (e.g., a disulfide stabilized scFv); WO 2012/021934 PCT/AU2011/001056 4 (iv) a dimeric scFv or stabilized form thereof; (v) a diabody, triabody, tetrabody or higher order multimer; (vi) Fab fragment; (vii) a Fab’ fragment; 5 (viii) a F(ab’) fragment; (ix) a F(ab’)2 fragment; (x) any one of (i)-(ix) fused to a Fc region of an antibody; (xi) any one of (i)-(ix) fused to an antibody or antigen binding fragment thereof that binds to an immune effector cell. 10 In one example, the immunoglobulin or antibody depletes or at least partly eliminates cells to which it binds, e.g., leukemic cells and/or basophils and/or pDCs.

As will be apparent to the skilled artisan from the disclosure herein, exemplary immunoglobulins or antibodies are capable of depleting or at least 15 partly eliminating cells to which it binds without being conjugated to a toxic compound.

In one example, the immunoglobulin or antibody is capable of inducing an effector function, e.g., an effector function that results in killing a cell to which the immunoglobulin or antibody binds. Exemplary effector functions include ADCC, 20 antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).

In one example, the immunoglobulin or antibody is capable of inducing ADCC.

In one example, the immunoglobulin or antibody comprises an antibody Fc 25 region capable of inducing an effector function. For example, the effector function is Fc-mediated effector function. In one example, the Fc region is an

IgGl Fc region or an IgG3 Fc region or a hybrid IgGl/IgG2 Fc region.

In one example, the immunoglobulin or antibody is capable of inducing a similar (e.g., not significantly different or within about 10%) or the same level of 30 effector function as a wild-type human IgGl and/or human IgG3 Fc region.

In one example, the immunoglobulin or antibody is capable of inducing an enhanced level of effector function.

In one example, the level of effector function induced by the immunoglobulin or antibody is enhanced relative to that of the immunoglobulin or 35 antibody when it comprises a wild-type IgGl Fc region.

In one example the immunoglobulin or antibody is afucosylated or comprises a Fc region that is afucosylated.

In another example, the immunoglobulin or antibody has a lower level of fucosylation compared to an immunoglobulin or antibody produced by a human or 40 a CHO cell that has not been altered to reduce the level of fucosylation of proteins. In accordance with this example, a lower level of fucosylation will be understood to mean that in a composition comprising the immunoglobulin or antibody the WO 2012/021934 PCT/AU2011/001056 5 percentage of fucosylated immunoglobulins (e.g., glycosyl groups attached to Asn297 of an antibody comprising fucose) is lower than produced by a human or a CHO cell that has not been altered to reduce the level of fucosylation of proteins.

For example, the immunoglobulin or antibody is an afucosylated 5 humanized antibody comprising a Vl comprising a sequence set forth in SEQ ID NO: 8 (or encoded by a sequence set forth in SEQ ID NO: 20) and a Vh comprising a sequence set forth in SEQ ID NO: 9 (or encoded by a sequence set forth in SEQ ID NO: 21). For example, the immunoglobulin or antibody is an afucosylated humanized antibody comprising a light chain comprising a sequence 10 set forth in SEQ ID NO: 13 (or encoded by a sequence set forth in SEQ ID NO: 23) and a heavy chain comprising a sequence set forth in SEQ ID NO: 10 (or encoded by a sequence set forth in SEQ ID NO: 22).

In one example, the immunoglobulin or antibody is a humanized antibody comprising a light chain comprising a sequence set forth in SEQ ID NO: 13 (or 15 encoded by a sequence set forth in SEQ ID NO: 23) and a heavy chain comprising a sequence set forth in SEQ ID NO: 10 (or encoded by a sequence set forth in SEQ ID NO: 22) expressed by a mammalian cell (e.g., a CHO cell) that does not express detectable levels of (or expresses reduced levels of) a-l,6-fucosyltransferase (FUT8). 20 In one example, the immunoglobulin or antibody comprises an Fc region comprising one or more amino acid sequence substitutions that enhance the effector function induced by the immunoglobulin. For example, the one or more amino acid sequence substitutions increase the affinity of the Fc region for a Fey receptor (FcyR) compared to a Fc region not comprising the substitutions. For 25 example, the one or more amino acid substitutions enhance increase the affinity of the Fc region for a FcyR selected from the group consisting of FcyRI, FcyRIIa, FcyRIIc and FcyRIIIa compared to a Fc region not comprising the substitutions. In one example, the one or more amino acid sequence substitutions are: (i) S239D, A33OL and I332E according to the EU numbering system of Rabat; 30 or (ii) S239D and I332E according to the EU numbering system of Rabat.

For example, the immunoglobulin or antibody is a humanized antibody comprising a Vl comprising a sequence set forth in SEQ ID NO: 8 (or encoded by a sequence set forth in SEQ ID NO: 20) and a VH comprising a sequence set forth 35 in SEQ ID NO: 9 (or encoded by a sequence set forth in SEQ ID NO: 21), wherein the antibody comprises a Fc region comprising one or more amino acid sequence substitutions selected from the group consisting of: (i) S239D, A33OL and I332E according to the EU numbering system of Rabat; and 40 (ii) S239D and 133 2E according to the EU numbering system of Rabat.

In one example, the Fc region comprises a sequence set forth between

residues 234-450 of SEQ ID NO: 11 (comprising the S239D and I332E WO 2012/021934 PCT/AU2011/001056 6 substitutions according to the EU numbering system of Kabat).

In one example, the Fc region comprises a sequence set forth between residues 234-450 of SEQ ID NO: 12 (comprising the S239D, A330L and I332E substitutions according to the EU numbering system of Kabat). 5 In one example, the immunoglobulin or antibody is selected from the group consisting of: (i) an antibody comprising a light chain comprising a sequence set forth in SEQ ID NO: 13 and a heavy chain comprising a sequence set forth in SEQ ID NO: 11; (ii) an antibody comprising a comprising a light chain comprising a sequence 10 set forth in SEQ ID NO: 13 and a heavy chain comprising a sequence set forth in SEQ ID NO: 12.

As discussed herein above, the inventor has determined that following administration of an antibody described herein, the number of NK cells in circulation in a mammal are initially reduced and then increased compared to the

15 number of NK cells in circulation in the mammal prior to administration. The inventor has also shown that increasing the number of NK cells relative to target cells results in increased efficacy, i.e., a greater number of target cells are killed. This effect is induced by an antibody comprising a constant region or Fc region comprising amino acid substitutions S239D and I332E according to the EU 20 numbering system of Kabat.

Thus, in one example, the present disclosure provides an isolated or recombinant antibody, which is capable of specifically binding to IL-3Ra chain and comprising: (i) a light chain variable region (Vl) comprising CDRs 1, 2 and 3 as set forth in 25 SEQ ID NOs: 2, 3 and 4, respectively; (ii) a heavy chain variable region (VH) comprising CDRs 1, 2 and 3 as set forth in SEQ ID NOs: 5, 6 and 7, respectively; and (iii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat. 30 In one example, the present disclosure provides an isolated or recombinant humanized antibody, the antibody capable of specifically binding to IL-3Ra chain and comprising: (i) a Vl comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 2 (or encoded 35 by a sequence set forth in SEQ ID NO: 14); b) a CDR2 comprising a sequence set forth in SEQ ID NO: 3(or encoded by a sequence set forth in SEQ ID NO: 15); and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 4 (or encoded by a sequence set forth in SEQ ID NO: 16); 40 (ii) a Vh comprising: d) a CDR1 comprising a sequence set forth in SEQ ID NO: 5 (or encoded by a sequence set forth in SEQ ID NO: 17); WO 2012/021934 PCT/AU2011/001056 7 e) a CDR2 comprising a sequence set forth in SEQ ID NO: 6 (or encoded by a sequence set forth in SEQ ID NO: 18); and f) a CDR3 comprising a sequence set forth in SEQ ID NO: 7 (or encoded by a sequence set forth in SEQ ID NO: 19); and 5 (iii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

In one example, the heavy chain constant region is a hybrid of a human IgGl and a human IgG2 constant regions.

In one example, the constant region comprises a sequence set forth between 10 residues 121-450 (inclusive) of SEQ ID NO: 11.

The present disclosure additionally or alternatively provides an isolated or recombinant humanized antibody, the antibody capable of specifically binding to IL-3Ra chain and comprising: (i) a Vl comprising an amino acid sequence according to SEQ ID NO: 8 (or 15 encoded by a sequence set forth in SEQ ID NO: 20) and/or a Vh comprising an amino acid sequence according to SEQ ID NO: 9 (or encoded by a sequence set forth in SEQ ID NO: 21); and (ii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat. 20 In one example, the heavy chain constant region is a hybrid of a human IgGl and a human IgG2 constant regions.

In one example, the constant region comprises a sequence set forth between residues 121-450 (inclusive) of SEQ ID NO: 11.

The present disclosure additionally or alternatively provides an isolated or 25 recombinant humanized antibody, the antibody capable of specifically binding to IL-3Ra chain and comprising a Vl comprising an amino acid sequence according to SEQ ID NO: 8 (or encoded by a sequence set forth in SEQ ID NO: 20), a Vh comprising an amino acid sequence according to SEQ ID NO: 9 (or encoded by a sequence set forth in SEQ ID NO: 21) and a heavy chain constant region 30 comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

In one example, the heavy chain constant region is a hybrid of a human IgGl and a human IgG2 constant regions.

In one example, the constant region comprises a sequence set forth between 35 residues 121-450 (inclusive) of SEQ ID NO: 11

In one example, the present disclosure provides an isolated or recombinant humanized antibody, the antibody capable of specifically binding to IL-3Ra chain and comprising a light chain comprising a sequence set forth in SEQ ID NO: 13 and a heavy chain comprising a sequence set forth in SEQ ID NO: 11. 40 In one example, an immunoglobulin or antibody or antigen binding fragment thereof of the present disclosure neutralizes IL-3 signaling.

In one example, an immunoglobulin or antibody of the present disclosure is WO 2012/021934 PCT/AU2011/001056 8 a naked immunoglobulin or an antibody or antigen binding fragment thereof of the present disclosure is a naked antibody or antigen binding fragment thereof.

In one example, an immunoglobulin or antibody of the present disclosure is a full length antibody. 5 In one example, an immunoglobulin or antibody of the present disclosure binds to IL-3Ra chain with an equilibrium dissociation constant (Kd) of 1x10’8M or less, such as 5xlO’9M or less, for example, 3xlO"9M or less, such as 2.5xlO’9M or less.

In one example, an immunoglobulin or antibody of the present disclosure 10 binds to IL-3Ra chain with a Kd of about 2.2xlO’9M or less. In one example, the Kd is between about 1x10’9M and about 2.5x10"9M, for example is about 2.2x10"9M.

In one example, an immunoglobulin or antibody of the present disclosure binds to IL-3Ra chain with a Kd of about 9xlO"loM or less, for example, about 15 8xlO"loM or less. In one example, the Kd is between about 5xlO’loM and about 9xlO"loM, for example is about 7.8xlO’loM.

The disclosure also includes fragments, variants and derivatives of the immunoglobulin or antibody of the disclosure.

In one example, an immunoglobulin or antibody of the present disclosure is 20 capable of reducing the number of NK cells when administered to a mammal (e.g., a non-human primate, such as a cynomolgus monkey). For example, the immunoglobulin or antibody is capable of reducing the number of NK cells when administered to the mammal by at least about 20%, such as at least about 30% or 40% within 12 hours or 10 hours or 8 hours of administration. For example, the 25 immunoglobulin or antibody is capable of reducing the number of NK cells when administered to the mammal by at least about 50% within 6 hours of administration. In one example, the immunoglobulin or antibody is capable of reducing the number of NK cells when administered at a dose of between O.OOOlmg/kg and 50mg/kg, such as between about 0.0005mg/kg and about 30 40mg/kg, for example, between about O.OOlmg/kg and about 30mg/kg, such as about 0.005mg/kg and about 20mg/kg, such as about O.Olmg/kg and about lOmg/kg. For example, the immunoglobulin or antibody is capable of reducing the number of NK cells when administered to the mammal by at least about 50% within 6 hours of administration when administered at a dose of about O.Olmg/kg 35 or O.lmg/kg or lmg/kg or lOmg/kg. In one example, the dose is about O.Olmg/kg or 0. lmg/kg.

In one example, the number of NK cells in the mammal is increased about 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 22 or 29 or 57 days after administering the immunoglobulin or antibody compared to the

40 number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells is increased at least about 8 or 11 or 17 or 22 or 29 days after administration. For example, the number of NK WO 2012/021934 PCT/AU2011/001056 9 cells in the mammal is increased by about 10% or 20% or 30% or 50% or 60% or 70% or 80% compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells in the mammal is increased by about 20% at least 8 days after 5 administration of the antibody or immunoglobulin at a dose between 0.001 mg/kg and 0.1 mg/kg compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells in the mammal is increased by about 50% at least 11, 17, 22 or 29 days after administration of the antibody or immunoglobulin at a dose between 10 O.OOlmg/kg and O.lmg/kg compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. In one example, the antibody or immunoglobulin is administered at a dose of between O.Olmg/kg and O.lmg/kg. For example, the immunoglobulin or antibody is administered at a dose of O.Olmg/kg or O.lmg/kg. 15 Based on the disclosure herein, it will be apparent to the skilled artisan that the present disclosure provides an immunoglobulin or antibody that, when administered to a mammal, causes an increase in the number of NK cells in the mammal. For example, the immunoglobulin or antibody, when administered to a mammal, causes a reduction in the number of NK cells in the mammal followed 20 by an increase in the number of NK cells. In one example, the number of NK cells is increased or reduced compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody.

In one example, the immunoglobulin or antibody is capable of reducing the number of NK cells in the mammal by at least about 50% within 6 hours of 25 administration when administered at a dose of about O.Olmg/kg or O.lmg/kg and the number of NK cells in the mammal is increased by about 20% at least 8 days after administration of the antibody or immunoglobulin at a dose between O.OOlmg/kg and O.lmg/kg compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. 30 In one example, the disclosure provides a pharmaceutical composition comprising an immunoglobulin or antibody according to the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure also provides an isolated nucleic acid encoding an immunoglobulin or antibody of the present disclosure. 35 Exemplary sequences of nucleic acids are discussed in the context of encoding antibodies or immunoglobulins of the disclosure and are to be taken to apply mutatis mutandis to the present example of the disclosure.

The present disclosure also provides a nucleic acid capable of hybridizing to a nucleic acid of the disclosure under high stringency hybridization conditions. 40 The disclosure also includes fragments, homologs and derivatives of an isolated nucleic acid of the disclosure.

The present disclosure also provides a genetic construct comprising an WO 2012/021934 PCT/AU2011/001056 10 isolated nucleic acid of the disclosure and one or more additional nucleotide sequences, such as a promoter operably linked to the nucleic acid.

In one example, the genetic construct is an expression construct comprising an expression vector and an isolated nucleic acid of the disclosure, wherein said 5 isolated nucleic acid is operably linked to one or more regulatory nucleic acids in said expression vector.

In one example, the genetic construct of the disclosure comprises a nucleic acid encoding a polypeptides (e.g., comprising a VH) operably linked to a promoter and a nucleic acid encoding another polypeptide (e.g., comprising a Vl) 10 operably linked to a promoter.

In another example, the genetic construct is a bicistronic genetic 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; 15 (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide.

For example, the first polypeptide comprises a Vh and the second polypeptide comprises a Vl, or the first polypeptide comprises a Vl and the second polypeptide comprises a Vh. 20 The present disclosure also contemplates separate genetic constructs one of which encodes a first polypeptide (e.g., comprising a Vh) and another of which encodes a second polypeptide (e.g., comprising a Vl). For example, the present disclosure also provides a composition comprising: (i) a first expression construct comprising a nucleic acid encoding a 25 polypeptide (e.g., comprising a Vh) operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a Vl) operably linked to a promoter.

The disclosure also provides a host cell comprising a genetic construct according to the present disclosure. 30 In one example, the present disclosure provides an isolated cell expressing an immunoglobulin or antibody or antigen binding fragment of the disclosure or a recombinant cell genetically-modified to express the immunoglobulin, antibody or antigen binding fragment.

In one example, the cell comprises the genetic construct of the disclosure or: 35 (i) a first genetic construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a Vh) operably linked to a promoter; and (ii) a second genetic construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a Vl) operably linked to a promoter, wherein the first and second polypeptides form an immunoglobulin, antibody or 40 antigen binding fragment of the present disclosure.

The genetic construct can be integrated into the cell or remain episomal. WO 2012/021934 PCT/AU2011/001056 11

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

The present disclosure additionally provides a method for producing an immunoglobulin, antibody or antigen binding fragment of the disclosure, the 5 method comprising maintaining the genetic construct(s) of the disclosure under conditions sufficient for the immunoglobulin, antibody or antigen binding fragment to be produced.

In one example, the method for producing an immunoglobulin, antibody or antigen binding fragment of the disclosure comprises culturing the cell of the 10 disclosure under conditions sufficient for the immunoglobulin, antibody or antigen binding fragment to be produced and, optionally, secreted.

In one example, the method for producing an immunoglobulin, antibody or antigen binding fragment of the disclosure additionally comprises isolating the immunoglobulin, antibody or antigen binding fragment. 15 The present disclosure additionally provides a method of producing a recombinant immunoglobulin or antibody of the disclosure, the method including the steps of: (i) culturing a host cell containing an expression vector according to the disclosure such that the recombinant immunoglobulin or antibody is expressed in 20 said host cell; and (ii) isolating the recombinant immunoglobulin.

In one example, a method for producing an immunoglobulin, antibody or antigen binding fragment of the disclosure additionally comprises formulating the immunoglobulin, antibody or antigen binding fragment with a pharmaceutically 25 acceptable carrier.

The present disclosure also provides a method of prophylactic or therapeutic treatment of a disease or condition in a mammal, the method including the step of administering the immunoglobulin or antibody of the disclosure to the mammal to thereby treat or prevent the disease or condition. 30 In one example, the mammal is a human.

In one example, the mammal is in need of treatment or prophylaxis.

In one example, the mammal in need suffers from the disease or condition.

In one example, the mammal in need is at risk of developing the disease or condition or a relapse thereof. 35 The present disclosure also provides for use of an immunoglobulin, antibody or antigen binding fragment of the disclosure or a composition of the disclosure in medicine.

The present disclosure additionally or alternatively provides for use of an immunoglobulin, antibody or antigen binding fragment of the disclosure in the 40 manufacture of a medicament for the treatment of a disease or condition in a mammal.

The present disclosure also provides an immunoglobulin, antibody or WO 2012/021934 PCT/AU2011/001056 12 antigen binding fragment of the disclosure for use in the treatment of a disease or condition in a mammal.

In one example, the disease or condition is an IL-3Ra-mediated disease or condition. 5 In one example, the disease or condition is myelodysplastic syndrome.

In one example, the disease or condition is cancer, such as a hematologic cancer, for example, leukemia, such as an acute leukemia (e.g., acute myelogenous leukemia) or a chronic leukemia (e.g., chronic myelomonocytic leukemia).

In one example, the disease or condition is an IL-3Ra-associated cancer, e.g., 10 leukemia, i.e., the cancer (or leukemia) is characterized by cancer (or leukemia) cells expressing fL-3Ra.

In another example, the cancer is a malignant lymphoproliferative disorder such as lymphoma.

In one example, the disease or condition is an autoimmune condition or an 15 inflammatory condition. For example, the condition is lupus, e.g., systemic lupus erythrematosus, Sjogren’s syndrome or scleroderma (e.g., systemic sceroderma).

In one example, the method comprises administering an effective amount of the immunoglobulin, such as a therapeutically effective amount of the 20 immunoglobulin, antibody or antigen binding fragment.

In one example, the method comprises administering between about O.OOOlmg/kg and 50mg/kg of immunoglobulin, antibody or antigen binding fragment to the mammal. For example, the method comprises administering between about 0.0005mg/kg to about 40mg/kg. For example, the method 25 comprises administering between about 0.0005mg/kg to about 30mg/kg. For example, the method comprises administering between about 0.001 mg/kg to about 20mg/kg. For example, the method comprises administering between about O.OOlmg/kg to about lOmg/kg. For example, the method comprises administering between about O.Olmg/kg to about 5mg/kg. For example, the 30 method comprises administering between about O.OOlmg/kg to about lmg/kg.

In one example, the method comprises administering between about 0. lmg/kg to about lOmg/kg of the immunoglobulin, antibody or antigen binding fragment. For example, the method comprises administering between about 0. lmg/kg to about 5mg/kg. For example, the method comprises administering 35 between about 0. lmg/kg to about lmg/kg.

In one example, the method comprises administering between about lOmg/kg to about 30mg/kg of the immunoglobulin, antibody or antigen binding fragment. For example, the method comprises administering between about 20mg/kg to about 30mg/kg. 40 In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of O.Olmg/kg.

In one example, the immunoglobulin, antibody or antigen binding WO 2012/021934 PCT/AU2011/001056 13 antibody or antigen binding antibody or antigen binding fragment is administered at a dose of 0.1 mg/kg.

In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of 1 mg/kg.

In one example, the immunoglobulin, 5 fragment is administered at a dose of lOmg/kg.

In one example, the immunoglobulin, fragment is administered at a dose of 30mg/kg.

In one example, the immunoglobulin or antibody is administered to the mammal a plurality of times. In one example, the period between 10 administrations is at least about 7 days, such as at least about 8 days, for example, at least about 9 days or 10 days. In one example, the period between administrations is at least about 11 days. In another example, the period between administrations is at least about 15 days, such as at least about 16 days, for example, at least about 18 days or 20 days. In one example, the period between 15 administrations is at least about 22 days. In another example, the period between administrations is at least about 25 days, such as at least about 30 days, for example, at least about 40 days or 45 days. In one example, the period between administrations is at least about 57 or 60 days.

For example, the immunoglobulin, antibody or antigen binding domain is 20 administered at a dose of between 0.000lmg/kg and 5mg/kg, such as between 0.0005mg/kg and 5mg/kg, for example, between O.OOlmg/kg and 5mg/'kg, and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 17 days or 21 days or 22 days or 28 days or 29 days or 30 days or 1 calendar month. For example, the immunoglobulin, antibody or 25 antigen binding domain is administered at a dose of between O.Olmg/kg and 5mg/kg and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days. For example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of between O.Olmg/kg and 2mg/kg and the period between administrations is at least about 7 30 days or 8 days or 9 days or 10 days or 11 days or 14 days. For example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of between O.Olmg/kg and lmg/kg and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days. In some examples, the period between administrations is at least about 7 days and less than 35 about 22 days, such as at least about 11 days or 15 days and less than about 20 days, for example at least about 13 days and less than about 18 days.

In one example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of O.Olmg/kg and the period between administrations is 6 days or 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 15 days. 40 In one example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of 0. lmg/kg and the period between administrations is 6 days or 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 15 days. WO 2012/021934 PCT/AU20U/001056 14

In one example, the immunoglobulin or antibody is administered at a dose of 1 mg/kg and the period between administrations is 6 days or 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 15 days or 20 days or 21 days or 22 days. 5 For example, the immunoglobulin or antibody is administered at a dose of between 6mg/kg and 50mg/kg and the period between administrations is at least about 15 days. For example, the immunoglobulin or antibody is administered at a dose of between lOmg/kg and 30mg/kg and the period between administrations is at least about 14 or 15 days. For example, the immunoglobulin or antibody is 10 administered at a dose of between 20mg/kg and 30mg/kg and the period between administrations is at least about 14 orl5 days. In some examples, the period between administrations is at least about 20 days and less than about 70 days, such as at least about 21 or 22 days and less than about 65 days, for example at least about 25 days and less than about 57 days. 15 In one example, the immunoglobulin or antibody is administered at a dose of lOmg/kg and the period between administrations is 14 days or 15 days or 21 days or 22 days or 30 days or 48 days or 50 days or 56 days or 57 days or 60 days.

In one example, the immunoglobulin or antibody is administered at a dose of 30mg/kg and the period between administrations is 14 days or 15 days or 21 20 days or 22 days or 30 days or 48 days or 50 days or 56 days or 57 days or 60 days.

As discussed above, the inventor has found that following administration of an immunoglobulin or antibody of the disclosure the number of NK cells is increased above the number present before administration within about 8 days. The inventor has also shown that increased numbers of NK cells increase the 25 efficacy of an antibody or immunoglobulin of the disclosure in inducing death of target cells. Thus, once the number of NK cells is increased, a further dose of the antibody or immunoglobulin can be administered to induce a therapeutic/prophylactic effect. For example, an immunoglobulin, antibody or antigen binding fragment of the disclosure is administered a plurality of times at a 30 dose of between about O.OOlmg/kg and about lmg/kg with a period between administrations of at least about 7 days, such as, at least about 8 days, for example, at least about 11 days, such as at least about 14 days, for example, at least about 17 days, such as at least about 21 days, for example, at least about 22 days, for example, 28 days or 29 days or one calendar month or 56 or 57 or 60 days. In 35 one example, the period between doses is about 7 days. In one example, the period between doses is about 8 days. In one example, the period between doses is about 11 days. In one example, the period between doses is about 14 days. In one example, the period between doses is about 17 days. In one example, the period between doses is about 21 days. In one example, the period between 40 doses is about 22 days. In one example, the period between doses is about 28 days. In one example, the period between doses is about 29 days. In one example, the dose is between about 0.0 lmg/kg and about 0.1 mg/kg, such as a dose WO 2012/021934 PCT/AU2011/001056 15 of about O.Olmg/kg or O.lmg/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA is a graphical representation showing the number of NK cells in 5 a sample from a non-human primate subject at various time points (as indicated) following administration of antibody CSL362X2. The number of cells is represented as a percentage of the number of cells prior to administering the antibody. Dosages of the antibody are indicated.

Figure IB is a graphical representation showing the number of NK cells in a 10 sample from a non-human primate subject at various time points (as indicated) following administration of antibody CSL362B. The number of cells is represented as a percentage of the number of cells prior to administering the antibody. Dosages of the antibody are indicated.

Figure 1C is a graphical representation showing the number of NK cells in 15 a sample from a non-human primate subject at various time points (as indicated) following administration of a chimeric antibody (designated CSL360) comprising a human IgGl constant domain and the variable region of antibody 7G3. The number of cells is represented as a percentage of the number of cells prior to administering the antibody. Dosages of the antibody are indicated. 20 Figure 2A is a graphical representation showing the percentage of TF-1 cells lysed in the presence of the indicated cell population and antibody CSL362X1 at various concentrations (as indicated on the X axis).

Figure 2B is a graphical representation showing the percentage of AML cells in the presence of various numbers of NK cells and antibody CSL362X1. 25 The ratio of effector cells (NK cells; E) to target cells (leukemia cells; T) is indicated on the X axis. Results generated suing cells from two patients are depicted.

DETAILED DESCRIPTION 30 Key to Sequence Listing SEQ ID NO: 1 - amino acid sequence of IL-3 Ra chain SEQ ID NO: 2 - amino acid sequence of LCDR1 of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 3 - amino acid sequence of LCDR2 of humanized anti- IL-3Ra chain 35 antibody CSL362 and modified forms thereof SEQ ID NO: 4 - amino acid sequence of LCDR3 of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 5 - amino acid sequence of HCDR1 of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof 40 SEQ ID NO: 6 - amino acid sequence of HCDR2 of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 7 - amino acid sequence of HCDR3 of humanized anti- IL-3Ra WO 2012/021934 PCT/AU2011/001056 16 chain antibody CSL362 and modified forms thereof SEQ ID NO: 8 - amino acid sequence of light chain variable region of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 9 - amino acid sequence of heavy chain variable region of 5 humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof

SEQ ID NO: 10 - amino acid sequence of heavy chain of humanized anti- IL-3Ra chain antibody CSL362 and CSL362B SEQ ID NO: 11 - amino acid sequence of heavy chain of humanized anti- IL-3Ra chain antibody CSL362X1 10 SEQ ID NO: 12 - amino acid sequence of heavy chain of humanized anti- IL-3Ra chain antibody CSL362X2 SEQ ID NO: 13 - amino acid sequence of light chain of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 14 - nucleotide sequence encoding a LCDR1 of humanized anti- 15 IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 15 - nucleotide sequence encoding a LCDR2 of humanized anti-IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 16 - nucleotide sequence encoding a LCDR3 of humanized anti-IL-3Ra chain antibody CSL362 and modified forms thereof 20 SEQ ID NO: 17 - nucleotide sequence encoding a HCDR1 of humanized anti-IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 18 - nucleotide sequence encoding a HCDR2 of humanized anti-IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 19 - nucleotide sequence encoding a HCDR3 of humanized anti- 25 IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 20 - nucleotide sequence encoding a light chain variable region of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof SEQ ID NO: 21 - nucleotide sequence encoding a heavy chain variable region of humanized anti- IL-3Ra chain antibody CSL362 and modified forms thereof

30 SEQ ID NO: 22 - nucleotide sequence encoding a heavy chain of humanized anti-IL-3Ra chain antibody CSL362 and CSL362B SEQ ID NO: 23 - nucleotide sequence encoding a light chain of humanized anti-IL-3Ra chain antibody CSL362 and modified forms thereof 35 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 40 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. WO 2012/021934 PCT/AU2011/001056 17

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 5 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. 10 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 15 one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, 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

20 and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et 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 25 Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and L.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 30 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 maybe further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, 35 National Institutes of Health, Bethesda, Md., 1987 and 1991.

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 40 “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 WO 2012/021934 PCT/AU2011/001056 18 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 5 directly from that source.

Selected Definitions

As used herein, the term “immunoglobulin” includes any antigen-binding protein product of the immunoglobulin gene complex, including immunoglobulin 10 isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Exemplary immunoglobulins are antibodies. Exemplary immunoglobulins are monoclonal. Included in the term “immunoglobulin” are any immunoglobulins that are appropriately de-immunized to thereby reduce or eliminate an immune response by a mammal to an immunoglobulin that has been administered to the 15 mammal. In the case of treatment of humans, suitable immunoglobulins include chimeric, humanized or human immunoglobulins. Also included within the term “immunoglobulin” are modified, mutagenized, chimeric and/or humanized immunoglobulins that comprise altered or variant amino acid residues, sequences or glycosylation, whether naturally occurring or produced by human intervention 20 (e.g. by recombinant DNA technology). Exemplary proteins include an Fc receptor binding portion. For example, proteins encompassed by the term “immunoglobulin” include domain antibodies, camelid antibodies and antibodies from cartilaginous fish (i.e., immunoglobulin new antigen receptors (IgNARs)). Generally, camelid antibodies and IgNARs comprise a Vh, however lack a Vl and 25 are often referred to as heavy chain immunoglobulins. Other “immunoglobulins” include T cell receptors and other immunoglobulin-like domain containing proteins that are capable of binding to an antigen, e.g., by virtue of an antigen binding site comprising a variable region.

The skilled artisan will be aware that an “antibody” is generally considered 30 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 or constant fragment or fragment crystallizable (Fc). A Vh and a Vl interact to form a Fv comprising an antigen 35 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., IgGb IgG2, IgG3, IgGzi, IgAi and IgA2) or subclass. The term “antibody” encompasses 40 humanized antibodies.

The term “humanized antibody” shall be understood to refer to an antibody comprising a human-like variable region including CDRs from an antibody from a WO 2012/021934 PCT/AU2011/001056 19 non-human species (e.g., mouse) grafted onto or inserted into FRs from a human antibody (this type of antibody is also referred to a “CDR-grafted antibody”). Humanized antibodies also include antibodies in which one or more residues of the human antibody are modified by one or more amino acid substitutions and/or 5 one or more FR residues of the human protein are replaced by corresponding non-human residues. As exemplified herein, humanized antibodies may also comprise residues which are found in neither the human antibody or in the non-human antibody. Any additional regions of the antibody (e.g., Fc region) are generally human. Humanization can be performed using a method known in the 10 art, e.g., US5225539, US6054297, US7566771 or US5585089. The term “humanized antibody” also encompasses a super-humanized protein, e.g., as described in US7732578.

The terms “full-length antibody”, “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as 15 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. In some cases, the intact antibody may be capable of inducing one or more effector 20 functions.

The term “naked antibody” refers to an antibody that is not conjugated to another compound, e.g., a toxic compound or radiolabel.

An “antigen binding fragment” of an antibody comprises the antigen binding domain and/or the variable region of the intact antibody. Examples of 25 antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

In the context of the present disclosure, “effector functions” refer to those biological activities mediated by cells or proteins that bind to the Fc region (a 30 native sequence Fc region or amino acid sequence variant Fc region) of an antibody that result in killing of a cell. Examples of effector functions induced by antibodies or immunoglobulins include: complement dependent cytotoxicity; antibody-dependent-cell-mediated cytotoxicity (ADCC); antibody-dependent-cell-phagocytosis (ADCP); and B-cell activation. In the 35 context of the present disclosure, the term or “effector function induced by an antibody” or like term is used interchangeably with “effector function of an immunoglobulin” or “effector function of an antibody” or like term and each provides literal support for the other. “Antibody-dependent-cell-mediated cytotoxicity” or “ADCC” refers to a 40 form of cytotoxicity in which secreted Ig bound onto Fc receptors (“FcRs”) present on certain cytotoxic cells (e.g., natural killer (“NK”) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an WO 2012/021934 PCT/AU2011/001056 20 antigen-bearing target-cell and subsequently kill the target-cell with cytotoxins. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (“PBMC”) and NK cells. 5 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, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally 10 FR4) together with three CDRs. 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 major contributors to specific 15 antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”. According to the 20 numbering system of Kabat, Vh 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). 25 “Framework regions” (hereinafter FR) are those variable domain residues other than the CDR residues.

The term “constant region” as used herein, refers to a portion of heavy chain or light chain of an antibody other than the variable region. In a heavy chain, the constant region generally comprises a plurality of constant domains and 30 a hinge region, e.g., a IgG constant region comprises the following linked components, a constant heavy (Ch)1, a linker, a Ch2 and a Ch3. In a heavy chain, a constant region comprises a Fc. In a light chain, a constant region generally comprise one constant domain (a ClI).

The term “fragment crystalizable” or “Fc” or “Fc region” or “Fc portion” 35 (which can be used interchangeably herein) refers to a region of an antibody comprising at least one constant domain and which is generally (though not necessarily) glycosylated and which is capable of binding to one or more Fc receptors and/or components of the complement cascade. The heavy chain constant region can be selected from any of the five isotypes: α, δ, ε, γ, or μ. 40 Furthermore, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, proteins with desired effector function WO 2012/021934 PCT/AU2011/001056 21 can be produced. Exemplary heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2) and gamma 3 (IgG3), or hybrids thereof.

A “constant domain” is a domain in an antibody the sequence of which is highly similar in antibodies/antibodies of the same type, e.g., IgG or IgM or IgE. A 5 constant region of an antibody generally comprises a plurality of constant domains, e.g., the constant region of γ, a or δ heavy chain comprises two constant domains.

As used herein, the term “specifically binds” shall be taken to mean an immunoglobulin or antibody by which is meant that the binding interaction 10 between an immunoglobulin or antibody and IL-3Ra chain is dependent on the presence of the antigenic determinant or epitope of an IL-3R.cc chain bound by the immunoglobulin or antibody. Accordingly, the immunoglobulin or antibody preferentially binds or recognizes an IL-3Ra chain antigenic determinant or epitope even when present in a mixture of other molecules or organisms. In one 15 example, the immunoglobulin or antibody reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with IL-3Ra or cell expressing same than it does with alternative antigens or cells. It is also understood by reading this definition that, for example, an immunoglobulin or antibody specifically binds to IL-3Ra may or may not specifically bind to a second 20 antigen. As such, “specific binding” does not necessarily require exclusive binding or non-detectable binding of another antigen. The term “specifically binds” is used interchangeably with “selectively binds” herein. Generally, reference herein to binding means specific binding, and each term shall be understood to provide explicit support for the other term. In one example, “specific binding” to with 25 IL-3Ra or cell expressing same, means that the immunoglobulin or antibody binds to the with IL-3Ra or cell expressing same with an equilibrium constant (RD) of lOOnM or less, such as 50nM or less, for example 20nM or less, such as, InM or less, e.g., 0.8nM or less.

The term “EU numbering system of Rabat” will be understood to mean the 30 numbering of an antibody heavy chain is according to the EU index as taught in Rabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgGl EU antibody.

As used herein, the term “IL-3Ra-mediated condition” will be understood 35 to mean a condition associated with or caused by excessive IL-3Ra expression and/or an excessive number of IL-3Ra expressing cells in a mammal, such as cancer cells (e.g., leukemic cells) and/or immune cells (e.g., piasmacytoid dendritic cells).

As used herein, the term “myelodysplastic syndrome” or “MDS” will be 40 understood to refer to a diverse collection of hematological medical conditions that involve ineffective production (or dysplasia) of the myeloid class of blood cells. Subjects with MDS often develop severe anemia and can require frequent WO 2012/021934 PCT/AU2011/001056 22 blood transfusions. In many cases, as MDS progresses the subject develops cytopenias (low blood counts) due to progressive bone marrow failure. In about one third of patients with MDS, the disease transforms into acute myelogenous leukemia (AML). The MDS can be diagnosed or classified to various systems, 5 including the French-American-British Classification System (Bennett et al., Br. J. Haematol. 33: 451^458, 1976), The International Prognostic Scoring System (Greenberg et al., Blood 89: 2079-88, 1997) or a system published by the World Health Organization.

As used herein, the term “treatment” refers to clinical intervention 10 designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disease are mitigated or 15 eliminated.

As used herein, the term “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to or at risk of developing the disease or disease relapse but has not yet been diagnosed with the disease or the relapse. 20 As used herein, a mammal “at risk” of developing a disease or condition or relapse thereof or relapsing may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment according to the present disclosure. “At risk” denotes that a mammal has one or more risk factors, which are measurable 25 parameters that correlate with development of the disease or condition, as known in the art and/or described herein.

An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more 30 administrations. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect treatment of a disease or condition as hereinbefore described. The effective amount may vary according to the disease or condition to be treated and also according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the 35 mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period. 40 A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disorder (e.g., SLE). A therapeutically effective amount herein may vary according to factors such as 23 225631/2 the disease state, age, sex, and weight of the patient, and the ability of the immunoglobulin or antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the immunoglobulin or antibody are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in mammals prior to or at an earlier stage of disease, a prophylactically effective amount may be less than a therapeutically effective amount.

Reference herein to “the number of NK cells in the mammal” will be understood to include the number of NK cells in a sample from a mammal and not to require determining the total number of NK cells in a mammal. This number can be expressed as, for example, cells per mL or dL or as a percentage of the number of cells from a sample taken at a different point in time from the mammal.

For the purposes of nomenclature only and not limitation, the amino acid sequence of an IL-3Ra chain is taught in Gene ID Accession Number 3563 and/or in SEQ ID NO: 1.

The “mammal” treated according to the present disclosure may be a mammal, such as a non-human primate or a human. In one example, the mammal is a human.

The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Sequence identity may be determined by alignment of compared sequences using computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

The disclosure also provides derivatives of anti-IL-3Ra immunoglobulins, antibodies or antigen binding fragments of the disclosure. As used herein, “derivative” proteins have been altered, for example by post-translational modification (e.g phosphorylation, acetylation etc), modification of glycosylation (e.g. adding, removing or altering glycosylation) and/or inclusion of additional amino acid sequences as would be understood in the art. WO 2012/021934 PCT/AU2011/001056 10 15 20 25 30 35 40 24

The term “nucleic acid” as used herein designates single-or double-stranded DNA and RNA capable of encoding an immunoglobulin, antibody or antigen binding fragment of the disclosure or a polypeptide component thereof. DNA includes genomic DNA and cDNA. RNA includes mRNA and cRNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto. “Hybridize and Hybridization ” is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purines and pyrimidines as are well known in the art.

In this regard, it will be appreciated that modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine) may also engage in base pairing. “Stringency” as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences. “High stringency conditions” designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

Reference herein to high stringency conditions include and encompass :- (i) from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C; (ii) 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65°C, and (a) 0.1 x SSC, 0.1% SDS; or (b) 0.5% BSA, ImM EDTA, 40 mMNaHP04 (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C for about one hour; and (iii) 0.2 x SSC, 0.1% SDS for washing at or above 68°C for about 20 minutes.

In general, washing is carried out at Tm = 69.3 + 0.41 (G + C) % -12°C. In general, the Tm of a duplex DNA decreases by about 1°C with every increase of 1% in the number of mismatched bases.

Notwithstanding the above, stringent conditions are known in the art, such as described in Chapters 2.9 and 2.10 of. Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY John Wiley & Sons, Inc. 1995-2009 WO 2012/021934 PCT/AU2011/001056 25

An “expression vector” may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

Immunoglobulins 5 Suitably, an immunoglobulin or antibody of the disclosure selectively binds IL-3Ra chain, by which is meant that the binding interaction between the immunoglobulin or antibody and IL-3Ra chain is dependent on the presence of the antigenic determinant or epitope of an IL-3RCC chain bound by the immunoglobulin. Accordingly, the immunoglobulin or antibody preferentially 10 binds or recognizes an IL-3RCC chain antigenic determinant or epitope even when present in a mixture of other molecules or organisms.

Antibodies

In one example, an immunoglobulin as described herein according to any 15 example is an antibody, e.g., comprising CDRs and/or one or more variable regions described herein.

Suitably, the immunoglobulin is a humanized, chimeric or human antibody that comprises light and heavy chain CDR 1, 2 and 3 amino acid sequences according to SEQ ID NOs: 2-7 respectively. In one example, the antibody 20 comprise a heavy chain variable region amino acid sequence according to SEQ ID NO: 9 and a light chain variable region amino acid sequence according to SEQ ID NO: 8. In one example, the antibody comprises a heavy chain amino acid sequence according to SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 and a light chain amino acid sequence according to SEQ ID NO: 13. 25 In one example, the antibody is a recombinant antibody. Methods for producing antibodies comprising CDRs and/or variable regions described herein will be apparent to the skilled artisan based on the disclosure herein and/or documents referred to herein.

In one example, an antibody of the disclosure comprises a Vh and/or a Vl 30 comprising CDRs as set forth herein and FRs from a human antibody. Optionally, the FRs comprise one or more amino acid substitutions, e.g., 2 or more or 3 or more or 4 or more of 5 or more or 10 or more or 15 or more. In one example, the FRs comprise no more than 30 amino acid substitutions, e.g., no more than 20 amino acid substitutions compared to the human FRs. 35 In one example, an antibody of the disclosure comprises a Vh and/or a Vl comprising CDRs as set forth herein and FRs from a non-human primate antibody, i.e., the antibody is Synhumanized, e.g., as described in W02007/019620.

In one example, an antibody of the disclosure is a composite antibody, e.g., as described in W02006/082406. 40 One exemplary antibody of the disclosure is a humanized antibody, e.g., as defined herein. Exemplary humanized antibodies are described herein. In one example, the humanized antibody has been affinity matured. In one example, the WO 2012/021934 PCT/AU2011/001056 26 humanized antibody comprises: (i) a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 2 (or encoded by a sequence set forth in SEQ ID NO: 14); 5 b) a CDR2 comprising a sequence set forth in SEQ ID NO: 3(or encoded by a sequence set forth in SEQ ID NO: 15); and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 4 (or encoded by a sequence set forth in SEQ ID NO: 16); and (ii) a Vh comprising: 10 d) a CDR1 comprising a sequence set forth in SEQ ID NO: 5 (or encoded by a sequence set forth in SEQ ID NO: 17); e) a CDR2 comprising a sequence set forth in SEQ ID NO: 6 (or encoded by a sequence set forth in SEQ ID NO: 18); and f) a CDR3 comprising a sequence set forth in SEQ ID NO: 7 (or 15 encoded by a sequence set forth in SEQ ID NO: 19).

Those skilled in the art will appreciate that immunoglobulins or antibodies of the disclosure may include modifications that introduce sequences not naturally found in humans, for example to increase affinity or effector function.

The disclosure also provides variants of the immunoglobulin or antibody of 20 the disclosure. Suitably, variants have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the amino acid sequences set forth in SEQ ID NOS:2-13.

For example, it is understood in the art that some amino acids may be substituted or deleted without adversely or significantly affecting the IL-3Ra 25 specificity and/or effector function of the immunoglobulin or antibody (e.g. conservative substitutions).

Derivatives of antibodies or immunoglobulins contemplated by the disclosure include, but are not limited to, modification to amino acid side chains, incorporation of unnatural amino acids and/or their derivatives during peptide or 30 polypeptide synthesis and the use of crosslinkers.

Exemplary antibodies and antigen binding fragments thereof of the present disclosure are described in Table 1. WO 2012/021934 PCT/AU2011/001056 27

Table 1: Exemplary antibodies and antigen binding fragments thereof

Name Heavy Chain SEQ ID NO: Light Chain SEQ ID NO: CSL362 Fv 91 81 CSL362 10 13 CSL362B 102 13 CSL362X1 11 13 CSL362X2 12 13 1 Variable region sequence only. 2 Heavy chain constant region is afucosylated. 5 Antibody Fragments

Single-Domain Antibodies

In some examples, an antigen binding fragment of an antibody of the disclosure is or comprises a single-domain antibody (which is used interchangeably with the term “domain antibody” or “dAb”). A single-domain 10 antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody.

Diabodies, Triabodies, Tetrabodies

In some examples, an antigen binding fragment of the disclosure is or 15 comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in W098/044001 and/or W094/007921.

For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure Vl-X-Vh or Vh-X-Vl, wherein X is a linker comprising insufficient residues to permit the Vh and Vl in a 20 single polypeptide chain to associate (or form an Fv) or is absent, and wherein the Vh of one polypeptide chain binds to a Vl of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The Vl and Vh can be the same in each polypeptide chain or the Vl and Vh can be different in each polypeptide chain so as to form a 25 bispecific diabody (i.e., comprising two Fvs having different specificity). A diabody, triabody, tetrabody, etc capable of inducing effector activity can be produced using an antigen binding domain capable of binding to IL-3Ra and an antigen binding domain capable of binding to a cell surface molecule on an immune cell, e.g., a T cell (e.g., CD3). 30

Single Chain Fv (scFv) Fragments

The skilled artisan will be aware that scFvs comprise Vh and Vl regions in a single polypeptide chain and a polypeptide linker between the Vh and Vl which enables the scFv to form the desired structure for antigen binding (i.e., for the Vh 35 and Vl of the single polypeptide chain to associate with one another to form a Fv). WO 2012/021934 PCT/AU2011/001056 28

For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3 being one of the more favored linkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR 5 of Vl and the cysteine residues linked by a disulfide bond to yield a stable Fv.

Alternatively, or in addition, the present disclosure encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to 10 permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

The present disclosure also contemplates a dimeric scFv capable of inducing effector activity. For example, one scFv binds to IL-3Ra and comprises CDRs and/or variable regions described herein and another scFv binds to a cell 15 surface molecule on an immune cell, e.g., a T cell (e.g., CD3 or CD19). In one example, the dimeric protein is a combination of a dAb and a scFv. Examples of bispecific antibody fragments capable of inducing effector function are described, for example, in US7235641. 20 Other Antibodies and Antibody Fragments

The present disclosure also contemplates other antibodies and antibody fragments, such as: (i) “key and hole” bispecific proteins as described in US5,731,168; (ii) heteroconjugate proteins, e.g., as described in US4,676,980; 25 (iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4,676,980; and (iv) Fab3 (e.g., as described in EP19930302894).

Constant Regions 30 The present disclosure encompasses immunoglobulins and antibodies and antigen binding fragments comprising a constant region of an antibody and/or a Fc region of an antibody.

Sequences of constant regions and/or Fc regions useful for producing the immunoglobulins, antibodies or antigen binding fragments of the present 35 disclosure may be obtained from a number of different sources. In some examples, the constant region, Fc or portion thereof of the immunoglobulin, antibody or antigen binding fragment is derived from a human antibody. The constant region, Fc or portion thereof may be derived from any antibody class, including IgA, IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgGl, IgG2, IgG3 and 40 IgG4. In one example, the constant region or Fc is human isotype IgGl or human isotype IgG2 or human isotype IgG3 or a hybrid of any of the foregoing. WO 2012/021934 PCT/AU2011/001056 29

In one example, the constant region or Fc region is capable of inducing an effector function. For example, the constant region or Fc region is a human IgGI or IgG3 Fc region. In another example, the constant region or Fc region is a hybrid of an IgGI and an IgG2 constant region or Fc region or a hybrid of an IgGI 5 and an IgG3 constant region or Fc region or a hybrid of an IgG2 and an IgG3 constant region or Fc region. Exemplary hybrids of human IgGI and IgG2 constant region or Fc regions are described in Chappel et al., Proc. Natl Acad. Sci. USA, 88: 9036-9040, 1991.

Methods for determining whether or not a Fc region can induce effector 10 function will be apparent to the skilled artisan and/or described herein.

Effector Function

Suitably, an anti-IL-3Ra immunoglobulin, antibody or antigen binding fragment of the disclosure has or displays an effector function that facilitates or 15 enables at least partial depletion, substantial depletion or elimination of IL-3Ra+ cells. Such an effector function may be enhanced binding affinity to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell mediated phagocytosis (ADCP) and/or complement dependent cytotoxicity (CDC). 20 As will be apparent to the skilled artisan based on the description herein, some examples of the present disclosure include an immunoglobulin, antibody or antigen binding fragment capable of inducing effector function.

For the IgG class of antibodies, these effector functions are governed by engagement of the Fc region with a family of receptors referred to as the Fey 25 receptors (FcyRs) which are expressed on a variety of immune cells and/or with complement, e.g., Clq. Formation of the Fc/FcyR complex recruits these cells to sites of bound antigen, typically resulting in signaling and subsequent immune responses. Methods for optimizing the binding affinity of the FcyRs to the antibody Fc region in order to enhance the effector functions, in particular to alter 30 the ADCC and/or CDC activity relative to the “parent” Fc region, are known to persons skilled in the art. These methods can include modification of the Fc region of the antibody to enhance its interaction with relevant Fc receptors and increase its potential to facilitate ADCC and ADCP. Enhancements in ADCC activity have also been described following the modification of the 35 oligosaccharide covalently attached to IgGI antibodies at the conserved Asn297 in the Fc region.

In this regard, it will be appreciated that in some non-limiting examples, enhancing effector function such as ADCC may be achieved by modification of an immunoglobulin or antibody which has a normally glycosylated wild-type 40 constant domain, including alteration or removal of glycosylation (see for example WOOO/61739) and/or amino acid sequence mutations (see for example W02008036688). WO 2012/021934 PCT/AU2011/001056 30

In one example, the immunoglobulin, antibody or antigen binding fragment binds to IL-3Ra in such a manner that it is capable of inducing an effector function, such as, ADCC.

In one example, the immunoglobulin, antibody or antigen binding fragment 5 binds to an epitope within IL-3Ra that permits it to induce an effector function, such as ADCC.

In another example, the immunoglobulin, antibody or antigen binding fragment is capable of binding to IL-3Ra on a cell in a mammal to thereby induce an effector function, such as ADCC. 10 For example, the immunoglobulin, antibody or antigen binding fragment remains bound to IF-3Ra on the surface of a cell for a time sufficient to induce an effector function, such as ADCC. For example, the immunoglobulin or antibody is not internalized too quickly to permit ADCC to be induced.

Alternatively, or in addition, the immunoglobulin, antibody or antigen 15 binding fragment is bound to the IL-3Ra on the surface of the cell in a manner permitting an immune effector cell to bind to a constant region or Fc region in the immunoglobulin, antibody or antigen binding fragment and induce an effector function, such as ADCC. For example, the Fc region of the immunoglobulin, antibody or antigen binding fragment is exposed in such a manner when the 20 immunoglobulin, antibody or antigen binding fragment is bound to the IF-3Ra that is capable of interacting with a Fc receptor (e.g., a FcyR) on an immune effector cell. In the context of the present disclosure, the term “immune effector cell” shall be understood to mean any cell that expresses a Fc receptor and that is capable of killing a cell to which it is bound by ADCC or ADCP. In one 25 example, the immune effector cell is a NK cell.

Each of the above paragraphs relating to effector functions of an immunoglobulin, antibody or antigen binding fragment shall be taken to apply mutatis mutandis to inducing CDC. For example, the immunoglobulin, antibody or antigen binding fragment is bound to the IL-3Ra on the surface of the cell in a 30 manner permitting complement component Clq to bind to a constant region or Fc region in the immunoglobulin, antibody or antigen binding fragment and induce CDC.

In one example, the immunoglobulin, antibody or antigen binding fragment is capable of inducing an enhanced level of effector function. 35 In one example, the level of effector function induced by the constant region or Fc region is enhanced relative to a wild-type constant region or Fc region of an IgGl antibody or a wild-type constant region or Fc region of an IgG3 antibody.

In another example, the constant region or Fc region is modified to increase 40 the level of effector function it is capable of inducing compared to the constant region or Fc region without the modification. Such modifications can be at the WO 2012/021934 PCT/AU2011/001056 10 15 20 25 30 35 40 31 amino acid level and/or the secondary structural level and/or the tertiary structural level and/or to the glycosylation of the constant region or Fc region.

The skilled addressee will appreciate that greater effector function may be manifested in any of a number of ways, for example as a greater level of effect, a more sustained effect or a faster rate of effect.

For example, the anti-IL-3Ra immunoglobulin, antibody or antigen binding fragment has or displays an effector function that includes antibody-dependent cell-mediated cytotoxicity (ADCC).

In one example, the constant region or Fc region comprises one or more amino acid modifications that increase its ability to induce enhanced effector function. In one example, the constant region or Fc region binds with greater affinity to one or more FcyRs. In one example, the constant region or Fc region has an affinity for an FcyR that is more than 1-fold greater than that of a wild-type constant region or Fc region or more than 5-fold greater than that of a wild-type constant region or Fc region or between 5-fold and 300-fold greater than that of a wild-type constant region or Fc region. In one example, the constant region or Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, numbered according to the EU index of Kabat. In one example, the constant region or Fc region comprise at least one amino acid substitution selected from the group consisting of: P230A, E233D, F234E, F234Y, F234I, F235D, F235S, F235Y, F235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243F, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276F, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, F328M, F328I, F328Q, F328D, L328V, F328T, A330Y, A33OL, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y, numbered according to the EU index of Kabat. In one example, the constant region or Fc region comprises amino acid substitutions selected from the group consisting of V264I, F243L/V264I, F328M, I332E, F328M/I332E, V264FI332E, S298A/I332E, S239E/I332E, S239Q/I332E, S239E, A330Y, I332D, F328FI332E, F328Q/I332E^ V264T, V240I, V266I, S239D, S239D/I332D, S239D/I332E, S239D/I332H S239D/I332Q, S239E/I332D, S239E/I332N, S239E/I332Q, S239N/I332D^ S239N/I332E, S239Q/I332D, A330Y/I332E, V264FA330Y/I332E, A330F/I332E^ V264FA330F/I332E, F234E, F234Y, L234I, F235D, F235S, F235Y, L235I S239T, V240M, V264Y, A330I, N325T, F328D/I332E, F328V/I332E, F328T/I332E, L328TI332E, S239E/V264TI332E, S239Q/V264FI332E^ S23 9 E/V2641/A3 3 0Y/I3 32E, S23 9D/A3 3 ΟΥ/B 32E, S23 9D/A3 3OF/I3 32E, S239N/A33 0L/I332E, S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264PS298A/I332E, S239D/V264FA330F/I332E, S239D/I332E/A330I P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R, K274F, K274Y, S239N/A330Y/I332E, V264FS298A/I332E, S239D/V264FI332E, WO 2012/021934 PCT/AU2011/001056 32 F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240FV266I, S239D/A330Y/I332E/L234I, S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I, S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, and 5 S239D/A330Y/I332E/K326T, numbered according to the EU index of Kabat.

In another example, the constant region or Fc region binds to FcyRIIIa more efficiently than to FcyRIIb. For example, the constant region or Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 234, 235, 239, 240, 264, 296, 330, and 1332, numbered 10 according to the EU index of Kabat. In one example, the constant region or Fc region comprises at least one amino acid substitution selected from the group consisting of: L234Y, L234I, L235I, S239D, S239E, S239N, S239Q, V240A, V240M, V264I, V264Y, Y296Q, A33OL, A330Y, A330I, I332D, and I332E, numbered according to the EU index of Kabat. For example, the constant region 15 or Fc region comprises amino acid substitutions selected from the group consisting of: I332E, V264FI332E, S239E/I332E, S239Q/I332E, Y296Q, A33OL, A33OY, I332D, S239D, S239D/I332E, A330Y/I332E, V264FA330Y/I332E, A330L/I332E, V264FA330L/I332E, L234Y, L234I, L235I, V240A, V240M, V264Y, A330I, S239D/A330L/I332E, S239D/S298A/I332E, 20 S239N/S298A/I332E, S239D/V264FI332E, S239D/V264FS298A/I332E, and S239D/V264FA330L/I332E, numbered according to the EU index of Kabat.

In a further example, the constant region or Fc region induces ADCC at a level greater than that mediated by a wild-type constant region or Fc region. For example, the constant region or Fc region induces ADCC at a level that is more 25 than 5-fold or between 5-fold and 1000-fold greater than that induced by a wild-type constant region or Fc region. In one example, the constant region or Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, numbered 30 according to the EU index of Kabat. In one example, the constant region or Fc region comprises at least one amino acid substitutions selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, 35 F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A33OL, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y, numbered according to the EU index of Kabat. In one example, the constant region or Fc region comprises amino acid substitutions selected from the group consisting of: V264I, 40 F243L/V264I, L328M, I332E, L328M/I332E, V264FI332E, S298A/I332E, S239E/I332E, S239Q/I332E, S239E, A330Y, I332D, L328FI332E, L328Q/I332E, V264T, V240I, V266I, S239D, S239D/I332D, S239D/I332E, S239D/I332N, WO 2012/021934 PCT/AU2011/001056 10 15 20 25 30 35 40 33 S239D/I332Q, S239E/I332D, S239E/I332N, S239E/I332Q, S239N/I332D, S239N/I332E, S239Q/I332D, A330Y/I332E, V264FA330Y/I332E, A330L/I332E, V264PA330L/I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D/I332E, L328V/I332E, L328T/I332E, L328TI332E, S239E/V264TI332E, S239Q/V264EI332E, S23 9 E/V2641/A3 3 0Y/I3 32E, S23 9D/A3 3 ΟΥ/B 32E, S23 9D/A3 3OL/I3 32E, S239N/A33 0L/I332E, S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264PS298A/I332E, S239D/V264FA330L/I332E, S239D/I332E/A330I, P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240FV266I, S239D/A330Y/I332E/L234I, S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I, S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, and S239N/A330Y/I332E, V264FS298A/I332E, S239D/V264FI332E, S239D/A330Y/I332E/K326T, numbered according to the EU index of Kabat.

In one example, the constant region or Fc region comprises the following amino acid substitutions S239D/I332E, numbered according to the EU index of Kabat. This constant region or Fc region has about 14 fold increase in affinity for FcyPllla compared to a wild-type constant region or Fc region and about 3.3 increased ability to induce ADCC compared to a wild-type constant region or Fc region. In one example, the constant region comprises a sequence set forth between residues 121-450 (inclusive) of SEQ ID NO: 11. In one example, the Fc region comprises a sequence set forth between residues 234-450 of SEQ ID NO: 11.

In one example, the constant region or Fc region comprises the following amino acid substitutions S239D/A330L/I332E, numbered according to the EU index of Kabat. This constant region or Fc region has about 138 fold increase in affinity for FcyRIHa compared to a wild-type constant region or Fc region and about 323 increased ability to induce ADCC compared to a wild-type constant region or Fc region. In one example, the constant region comprises a sequence set forth between residues 121-450 (inclusive) of SEQ ID NO: 12. In one example, the Fc region comprises a sequence set forth between residues 234-450 of SEQ ID NO: 12.

Additional amino acid substitutions that increase ability of a Fc region to induce effector function are known in the art and/or described, for example, in US6737056 or US7317091.

In one example, the glycosylation of the constant region or Fc region is altered to increase its ability to induce enhanced effector function. In this regard, native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the Ch2 domain of the constant region or Fc region. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), WO 2012/021934 PCT/AU2011/001056 34 galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some examples, constant regions or Fc regions according to the present disclosure comprise a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region, i.e., the 5 Fc region is “afucosylated”. Such variants may have an improved ability to induce ADCC. Methods for producing afucosylated antibodies include, expressing the immunoglobulin or antibody in a cell line incapable of expressing a-l,6-fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et al., Biotechnol. Bioengineer., 87: 614-622, 2004), expressing the immunoglobulin or 10 antibody in cells expressing a small interfering RNA against FUT8 (e.g., as described in Mori et al., Biotechnol. Bioengineer., 88: 901-908, 2004), expressing the immunoglobulin or antibody in cells incapable of expressing guanosine diphosphate (GDP)-mannose 4,6-dehydratase (GMD) (e.g., as described in Kanda et al., J. Biotechnol., 130: 300-310, 2007). The present disclosure also 15 contemplates the use of immunoglobulins having a reduced level of fucosylation, e.g., produced using a cell line modified to express β—(l,4)-A-acetylglucosaminyltransferase ΠΙ (GnT-III) (e.g., as described in Umanaet al., Nat. Biotechnol., 17: 176-180, 1999).

In one example, an immunoglobulin or antibody according to the present 20 disclosure is afucosylated. For example, the immunoglobulin or antibody is produced in a cell (e.g., a mammalian cell, such as a CHO cell) that does not express FUT8.

Other methods include the use of cell lines which inherently produce antibodies capable of inducing enhanced Fc-mediated effector function (e.g. duck 25 embryonic derived stem cells for the production of viral vaccines, W02008/129058; Recombinant protein production in avian EBX® cells, WO 2008/142124).

Immunoglobulins useful in the methods of the present disclosure also include those with bisected oligosaccharides, e.g., in which a biantennary 30 oligosaccharide attached to the constant region or Fc region is bisected by GlcNAc. Such immunoglobulins may have reduced fucosylation and/or improved ADCC function. Examples of such immunoglobulins are described, e.g., in US6602684 and US20050123546.

Immunoglobulins with at least one galactose residue in the oligosaccharide 35 attached to the constant region or Fc region are also contemplated. Such immunoglobulins may have improved CDC function. Such immunoglobulins are described, e.g., in WO1997/30087 and WO1999/22764.

Non-limiting examples of immunoglobulins that induce enhanced levels of ADCC activity include: 40 (i) an antibody comprising a heavy chain comprising a sequence set forth in SEQ ID NO: 11 and a light chain comprising a sequence set forth in SEQ ID NO: 13; WO 2012/021934 PCT/AU2011/001056 35 (ii) an antibody comprising a heavy chain comprising a sequence set forth in SEQ ID NO: 12 and a light chain comprising a sequence set forth in SEQ ID NO: 13;and (iii) an antibody comprising a heavy chain comprising a sequence set forth in 5 SEQ ID NO: 10 and a light chain comprising a sequence set forth in SEQ ID NO: 13, wherein the heavy chain constant region is afucosylated.

An advantageous antibody of the disclosure that induces enhanced levels of ADCC activity comprises a heavy chain comprising a sequence set forth in SEQ ID NO: 11 and a light chain comprising a sequence set forth in SEQ ID NO: 10 13. As discussed herein, after administering this antibody to a mammal (e.g., at least 7 or 8 or 11 or 17 or 22 or 29 days after administration) the number of NK cells in a mammal is increased compared to one or more of: (i) the number of NK cells in the mammal prior art administration of the antibody; 15 (ii) the number of NK cells in a mammal administered an antibody comprising a heavy chain comprising a sequence set forth in SEQ ID NO: 10 and a light chain comprising a sequence set forth in SEQ ID NO: 13, wherein the heavy chain constant region is afucosylated (e.g., as assessed on the same day after administration); and 20 (iii) the number of NK cells in a mammal administered an antibody that binds specifically to IL-3Ra chain and has a human IgGl constant region (e.g., as assessed on the same day after administration).

Methods for determining the ability of an immunoglobulin or antibody to induce effector function and known in the art and/or described in more detail 25 herein.

Additional Modifications

The present disclosure also contemplates additional modifications to an immunoglobulin. 30 For example, the immunoglobulin or antibody comprises one or more amino acid substitutions that increase the half-life of the immunoglobulin. For example, the immunoglobulin or antibody comprises a constant region or Fc region comprising one or more amino acid substitutions that increase the affinity of the constant region or Fc region for the neonatal Fc region (FcRn). For 35 example, the constant region or Fc region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in an endosome. In one example, the constant region or Fc region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which facilitates the re-release of constant region or Fc into blood following cellular recycling. These amino acid 40 substitutions are useful for extending the half life of an immunoglobulin, by reducing clearance from the blood. WO 2012/021934 PCT/AU2011/001056 36

Exemplary amino acid substitutions include T250Q and/or M428L according to the EU numbering system of Kabat. Additional or alternative amino acid substitutions are described, for example, inUS20070135620. 5 Nucleic Acids

Another example of the disclosure provides an isolated nucleic acid that encodes an immunoglobulin or antibody of the disclosure, inclusive of fragments, variants and derivatives of the immunoglobulin or antibody.

Certain examples of nucleic acids comprise a nucleotide sequence set forth 10 in one or more of SEQ ID NOS: 14-19, which respectively encode CDRs 1-6 of an immunoglobulin or antibody of the disclosure.

Other examples of nucleic acids comprise a nucleotide sequence set forth in one or more of SEQ ID NOS: 20-23.

The disclosure also contemplates nucleic acid homologs that encode a 15 variant of an immunoglobulin or antibody of the disclosure, as hereinbefore described.

Exemplary, nucleic acid homologs share at least 80% or 85%, such as at least 90% or 95% or 99% nucleotide sequence identity with an isolated nucleic acid that encodes any one of SEQ ID NOS:2-13. Suitably, the nucleic acid 20 homolog does not encode a protein comprising a murine variable region capable of binding specifically to IL-3Ra..

Nucleic acid homologs may hybridize to isolated nucleic acids of the disclosure under high stringency conditions. 25 Protein Production

Recombinant Expression

In one example, an immunoglobulin or antibody described herein according to any example is recombinant.

By way of example only, a recombinant immunoglobulin or antibody of the 30 disclosure may be produced by a method including the steps of: (i) preparing an expression construct which comprises an isolated nucleic acid of disclosure, operably linked to one or more regulatory nucleotide sequences (e.g., a promoter); (ii) transfecting or transforming a suitable host cell with the 35 expression construct; (iii) expressing a recombinant immunoglobulin or antibody in the host cell; and (iv) isolating the recombinant immunoglobulin or antibody from the host cell. 40 In the case of a recombinant immunoglobulin, nucleic acid encoding same can be cloned into expression vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian WO 2012/021934 PCT/AU2011/001056 37 COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce immunoglobulin or antibody protein.

Exemplary cells used for expressing an immunoglobulin or antibody are 5 CHO cells, myeloma cells or HEK cells. The cell may further comprise one or more genetic mutations and/or deletions that facilitate expression of a modified immunoglobulin or antibody. One non-limiting example is a deletion of a gene encoding an enzyme required for fucosylation of an expressed immunoglobulin or antibody. For example, the deleted gene encodes FUT8. A commercially 10 available source of FUT8-deleted CHO cells is Biowa (Potelligent™ cells). For example, the cells used for expression of an afucosylated immunoglobulin or antibody are FUT8-deleted CHO cells, such as, Biowa’s Potelligent™ cells.

Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et al., (editors), Current Protocols in 15 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). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also 20 known in the art. SeeUS4816567 orUS5530101.

Poliowing isolation, the nucleic acid is inserted operably linked to a promoter in an genetic construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells. Thus, another example of the disclosure provides a genetic construct that comprises an 25 isolated nucleic acid of the disclosure and one or more additional nucleotide sequences. Suitably, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other 30 host cells, for manipulation by recombinant DNA technology and/or for expression of the nucleic acid or an encoded immunoglobulin or antibody of the disclosure. Lor the purposes of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises the nucleic acid of the disclosure operably linked to one or more additional sequences in an 35 expression vector, such as a promoter or a regulatory sequence.

Typically, a regulatory nucleotide sequence may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. 40 As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription WO 2012/021934 PCT/AU2011/001056 38 initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term 5 “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid. 10 As used herein, the term “operably linked to” means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal 15 sequence, a sequence encoding an immunoglobulin or antibody (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of an immunoglobulin. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, 20 penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-a promoter 25 (EF1), small nuclear RNA promoters (Ula and Ulb), α-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, β-actin promoter; hybrid regulatory element comprising a CMV enhancer/ β-actin promoter or an immunoglobulin or antibody promoter or active fragment thereof. Examples of useful mammalian host cell 30 lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as for example 35 a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TPF1 promoter.

Means for introducing the isolated nucleic acid or expression construct 40 comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, WO 2012/021934 PCT/AU2011/001056 39 transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus 5 Inc., WI, USA) amongst others.

The host cells used to produce the immunoglobulin or antibody may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium 10 ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

Isolation of Proteins

Methods for purifying an immunoglobulin or antibody are known in the art 15 and/or described herein.

Where an immunoglobulin or antibody is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be 20 included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The immunoglobulin or antibody prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography 25 (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example in WO99/57134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988).

The skilled artisan will also be aware that an immunoglobulin or antibody 30 can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting immunoglobulin or antibody is then purified using methods known in the art, such as, affinity purification. For 35 example, an immunoglobulin or antibody comprising a hexa-his tag is purified by contacting a sample comprising the immunoglobulin or antibody with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound immunoglobulin, and subsequently eluting the bound immunoglobulin. 40 Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

In one example, the immunoglobulin or antibody also has a protease WO 2012/021934 PCT/AU2011/001056 40 cleavage site, such as for Factor Xa or Thrombin, which allow the relevant protease to partially digest the immunoglobulin or antibody and thereby liberate the immunoglobulin or antibody from the tag. The liberated antibody or immunoglobulin can then be isolated from the fusion partner by subsequent 5 chromatographic separation.

Assaying Activity of an Immunoglobulin

Immunoglobulins or antibodies of the disclosure are readily screened for biological activity, e.g., as described below. 10

Binding Assays

One form of such an assay is an antigen binding assay, e.g., as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such a method generally involves labeling the immunoglobulin 15 or antibody and contacting it with immobilized antigen. Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound protein is detected. Of course, the immunoglobulin or antibody can be immobilized and the antigen labeled. Panning-type assays, e.g., as described or exemplified herein can also be used. 20

Determining Neutralization

In some examples of the present disclosure, an immunoglobulin or antibody is capable of neutralizing IL-3 signaling.

Various assays are known in the art for assessing the ability of an 25 immunoglobulin to neutralize signaling of a ligand through a receptor.

In one example, the immunoglobulin or antibody reduces or prevents IL-3 binding to the 3Ra chain and/or a heterodimer of IL-3 Ra chain and IL-3RP chain. These assays can be performed as a competitive binding assay using labeled IL-3 and/or labeled immunoglobulin. For example, labeled IL-3Ra or an extracellular 30 region thereof fused to an Fc region of an antibody or a cell expressing IL-3R immobilized and labeled IL-3 is then contacted to the immobilized receptor or cell in the presence or absence of a test immunoglobulin or antibody and the amount of bound label detected. A reduction in the amount of bound label in the presence of the antibody or immunoglobulin compared to in the absence of the 35 protein indicates that the immunoglobulin or antibody reduces or prevents binding of IL-3 to IL-3R. By testing multiple concentrations of the immunoglobulin or antibody an IC50 is determined, i.e., a concentration of the protein that reduces the amount of IL-3 that binds to IL-3R, or an EC50 can be determined, i.e., a concentration of the protein that achieves 50% of the maximum inhibition of 40 binding of IL-3 to IL-3R achieved by the immunoglobulin or antibody

In another example, the immunoglobulin or antibody reduces or prevents IL-3-mediated histamine release from basophils. For example, low density WO 2012/021934 PCT/AU2011/001056 41 leukocytes comprising basophils are incubated with IgE, IL-3 and various concentrations of the immunoglobulin or antibody. Control cells do not comprise immunoglobulin (positive control) or IL-3 (negative control). The level of released histamine is then assessed using a standard technique, e.g., RIA. 5 An immunoglobulin or antibody that reduces the level of histamine release to a level less than the positive control is considered to neutralize IL-3 signaling. In one example, the level of reduction is correlated with immunoglobulin or antibody concentration. An exemplary method for assessing IL-3-mediated histamine release is described, for example, in Lopez et al., J. Cell. Physiol., 145: 69, 1990. 10 In a further example, the immunoglobulin or antibody reduces or prevents IL-3-mediated proliferation of leukemic cell line TF-1. For example, TF-1 cells are cultured without IL-3 or GM-CSF for a time sufficient for them to stop proliferating (e.g., 24-48 hours). Cells are then cultured in the presence of IL-3 and various concentrations of the immunoglobulin or antibody. Control cells are 15 not contacted with the immunoglobulin or antibody (positive control) or IL-3 (negative control). Cell proliferation is then assessed using a standard technique, e.g., 3H-thymidine incorporation. An immunoglobulin or antibody that reduces or prevents cell proliferation in the presence of IL-3 to a level less than the positive control is considered to neutralize IL-3 signaling. 20 Another assay for assessing IL-3 signaling neutralization comprises determining whether or not the immunoglobulin or antibody reduces or prevents IL-3-mediated effects on endothelial cells. For example, human umbilical vein endothelial cells (HUVECs) are cultured in the presence of IL-3 (optionally, with IFN-γ) and various concentrations of the immunoglobulin or antibody. The 25 amount of secreted IL-6 is then assessed, e.g., using an enzyme linked immunosorbent assay (ELISA). Control cultures do not comprise immunoglobulin or antibody (positive control) or IL-3 (negative control). An immunoglobulin or antibody that reduces or prevents IL-6 production in the presence of IL-3 to a level less than the positive control is considered to neutralize 30 IL-3 signaling.

Other methods for assessing neutralization of IL-3 signaling are contemplated by the present disclosure.

Determining Effector Function 35 Methods for assessing ADCC activity are known in the art.

In one example, the level of ADCC activity is assessed using a 51Cr release assay, a europium release assay or a 35S release assay. In each of these assays, cells expressing IL-3Ra are cultured with one or more of the recited compounds for a time and under conditions sufficient for the compound to be taken up by the 40 cell. In the case of a 35S release assay, cells expressing IL-3Ra can be cultured with 35S-labeled methionine and/or cysteine for a time sufficient for the labeled amino acids to be incorporated into newly synthesized proteins. Cells are then WO 2012/021934 PCT/AU2011/001056 42 cultured in the presence or absence of the immunoglobulin or antibody and in the presence of immune effector cells, e.g., peripheral blood mononuclear cells (PBMC) and/or NK cells. The amount of 51Cr, europium and/or 35S in cell culture medium is then detected, and an increase in the presence of the 5 immunoglobulin or antibody compared to in the absence of immunoglobulin indicates that the immunoglobulin has effector function. Exemplary publications disclosing assays for assessing the level of ADCC induced by an immunoglobulin include Hellstrom, et al. Proc. Natl Acad. Sci. USA 83:7059-7063, 1986 and Bruggemann, et al., J. Exp. Med. 166:1351-1361, 1987. 10 Other assays for assessing the level of ADCC induced by an immunoglobulin or antibody include ACTFM nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. CA, USA) or CytoTox 96® non-radioactive cytotoxicity assay (Promega, WI, USA).

Alternatively, or additionally, effector function of an immunoglobulin or 15 antibody is assessed by determining its affinity for one or more FcyRs, e.g., as described in US7317091.

Clq binding assays may also be carried out to confirm that the immunoglobulin or antibody is able to bind Clq and may induce CDC. To assess complement activation, a CDC assay may be performed (see, for example, 20 Gazzano-Santoro et al, J. Immunol. Methods 202: 163, 1996.

Determining NK cell Numbers

As discussed herein, immunoglobulins and/or antibodies of the disclosure can affect the number of NK cells in a mammal. Methods for assessing the 25 number of NK cells in a mammal will be apparent to the skilled artisan.

In one example, following administration of an immunoglobulin or antibody of the disclosure to a mammal (e.g., a non-human mammal, such as a non-human primate, e.g., a cynomolgus monkey) a sample of blood (or serum) is obtained and the number of NK cells assessed using fluorescence activated cell 30 sorting (FACS). NK cells can be detected based on expression of CD16 and/or CD56 and/or lack of expression (or low levels of expression) of CD20 and/or CD3.

The percentage change in the number of NK cells can be determined by comparing to the number of NK cells in a sample obtained earlier (e.g., prior to administration of the antibody or immunoglobulin. 35

Determining Affinity

Optionally, the dissociation constant (Kd) or association constant (Ka) or equilibrium constant (Kd) of a protein for IL-3Ra or an epitope thereof is determined. These constants for an immunoglobulin or antibody is, in one 40 example, measured by a radiolabeled or fluorescently-labeled IL-3Ra-binding assay. This assay equilibrates the protein with a minimal concentration of labeled IL-3Ra (or a soluble form thereof, e.g., comprising an extracellular region of WO 2012/021934 PCT/AU2011/001056 43 IL-3Ra fused to an Fc region) in the presence of a titration series of unlabeled IL-3Ra. Following washing to remove unbound IL-3Ra, the amount of label is determined.

Affinity measurements can be determined by standard methodology for 5 antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka Curr. Opin. Biotechnol 11: :54, 2000; Englebienne Analyst. 123: 1599, 1998), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art.

In one example, the constants are measured by using surface plasmon 10 resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized IL-3Ra or a region thereof. Exemplary SPR methods are described in US7229619.

Assessing Therapeutic Efficacy 15 In Vitro Assays

Various in vitro assays are available to assess the ability of an immunoglobulin or antibody to treat a disease or condition described herein.

For example, an immunoglobulin or antibody is assessed for its ability to kill a cell, e.g., a cancer cell, such as leukemic cell, using a method described 20 herein.

In another example, immune cells, e.g., pDCs and/or basophils or cell populations comprising same (e.g., PBMC) are cultured in the presence or absence of an immunoglobulin or antibody and an inducer of those cells that occurs in a disease or condition (e.g., CpG oligonucleotides and/or immune complexes). 25 The efficacy of the immunoglobulin or antibody in treating the disease or condition is then assessed, e.g., by determining the level of IFNa secreted into cell culture medium using an ELISA. Alternatively or in addition the level of histamine secretion or IL-4, IL-6 and/or IL-13 secretion is assessed. A reduction in the level of any of these cytokines compared to in the absence of 30 immunoglobulin or antibody (or in the presence of an isotype control immunoglobulin or antibody) indicates that the immunoglobulin or antibody is suitable for treating the disease or condition. Alternatively, or in addition, the level of cell death is assessed. An increase in cell death is indicative of an immunoglobulin or antibody suitable for treating the disease or condition. 35

In Vivo Assays

In one example, the efficacy of an immunoglobulin to treat a disease or condition is assessed using an in vivo assay.

In one example, a xenotransplantation model of a cancer is used to assess 40 therapeutic efficacy. For example, NOD/SCID mice are irradiated and optionally treated with anti-CD 122 antibody to deplete NK cells. Human leukemic cells (e.g., acute myeloid leukemia cells) and mouse or human bone marrow stem cells WO 2012/021934 PCT/AU2011/001056 44 are administered to the mice. Following cell engraftment, a test immunoglobulin or antibody is administered to the mice and the level of leukemic cells in circulation and/or bone marrow and/or lymph nodes is assessed. A reduction in the number of leukemic cells in circulation and/or bone marrow and/or lymph 5 nodes in the presence of the antibody or immunoglobulin compared to in the absence of the antibody or immunoglobulin indicates therapeutic efficacy.

In another example, the immunoglobulin or antibody is administered to a non-human animal (e.g., a non-human primate) and the number/level of immune cells, e.g., pDCs and/or basophils, in circulation is assessed. An 10 immunoglobulin or antibody that reduces the number/level of immune cells, e.g., pDCs and/or basophils compared to prior to administration and/or in a control mammal to which the immunoglobulin or antibody has not been administered is considered suitable for treating the disease or condition.

In another example, the level of a cytokine, such as IFNa is detected in the 15 circulation of a mammal, e.g., using an ELISA. An immunoglobulin or antibody that reduces the level of the cytokine compared to the level prior to administration and/or in a control mammal to which the immunoglobulin or antibody has not been administered is considered suitable for treating the disease or condition. Since cytokines such as IFNa are considered to play a role in some 20 diseases/conditions, e.g., lupus, an immunoglobulin or antibody that reduces IFNa production is considered to be suitable for treating such conditions.

Compositions

Suitably, in compositions or methods for administration of the anti-IL-3Ra 25 immunoglobulin or antibody to a mammal, the immunoglobulin or antibody is combined with a pharmaceutically acceptable carrier, diluent and/or excipient, as is understood in the art. Accordingly, one example of the present disclosure provides a pharmaceutical composition comprising the immunoglobulin or antibody of the disclosure combined with a pharmaceutically acceptable carrier, 30 diluent and/or excipient. In another example, the disclosure provides a kit comprising a pharmaceutically acceptable carrier, diluent and/or excipient suitable for combining or mixing with the immunoglobulin or antibody prior to administration to the mammal. In this example, the kit may further comprise instructions for use. 35 In general terms, by “carrier, diluent or excipient” is meant a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating or lubricant that may be safely administered to any mammal, e.g., a human. Depending upon the particular route of administration, a variety of acceptable carriers, diluents or excipients, known in the art may be 40 used, as for example described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).

By way of example only, the carriers, diluents or excipients may be WO 2012/021934 PCT/AU2011/001056 45 selected from a group including sugars (e.g. sucrose, maltose, trehalose, glucose), starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, oils inclusive of vegetable oils, synthetic oils and synthetic mono- or di-glycerides, lower alcohols, polyols, alginic acid, phosphate buffered solutions, lubricants such 5 as sodium or magnesium stearate, isotonic saline and pyrogen-free water. For example, the carrier, diluent or excipient is compatible with, or suitable for, parenteral administration. Parenteral administration includes any route of administration that is not through the alimentary canal. Non-limiting examples of parenteral administration include injection, infusion and the like. By way of 10 example, administration by injection includes intravenous, intra-arterial, intramuscular and subcutaneous injection. Also contemplated is delivery by a depot or slow-release formulation which may be delivered intradermally, intramuscularly and subcutaneously, for example. 15 Combination Therapies

In one example, the immunoglobulin or antibody of the disclosure is administered in combination with another compound or therapeutic treatment useful for treating a disease or condition.

In one example, the immunoglobulin or antibody is administered prior to, 20 e.g., one month or one fortnight or one week prior to radiation therapy, e.g., for the treatment of cancer, such as a hematologic cancer, such as leukemia.

In one example, the other compound is a chemotherapy compound, such as caboplatin, cisplatin, cyclophosphamide, docetaxal, doxorubicin, erlotinib, etoposide, fluorouracil, irinotecan, methotrexate, paclitaxel, topotecan, vincristine 25 or vinblastine. In one example, the chemotherapy compound is selected from the group consisting of methotrexate, 1-asparaginase, vincristine, doxorubicin, danorubicin, cytarabine, idarubicin, mitoxantrone, cyclophosphamide, fludarabine, chlorambucil and combinations thereof.

In one example, the other compound is a chemotherapy compound used in 30 the treatment of acute leukemia, such as, a compound selected from the group consisting of methotrexate, 1-asparaginase, vincristine, doxorubicin, danorubicin, cytarabine, idarubicin, mitoxantrone and combinations thereof.

In one example, the other compound is a chemotherapy compound used in the treatment of acute lymphoblastic leukemia, such as, a compound selected from 35 the group consisting of methotrexate, 1-asparaginase, vincristine, doxorubicin, danorubicin and combinations thereof.

In a further example, the other compound is a chemotherapy compound such as azacytidine.

In one example, the other compound is a biologic useful for treating a 40 cancer, e.g., rituximab, trastuzumab, bevacizumab, alemtuzumab, panitumumab, or cetuximab

In one example, the other compound is an anti-inflammatory compound. WO 2012/021934 PCT/AU2011/001056 46

Alternatively, or additionally, the other compound is an immunosuppressant. Alternatively, or additionally, the other compound is a corticosteroid, such as prednisone and/or prednisolone. Alternatively, or additionally, the other compound is an antimalarial compound, such as hydroxychloroquine or 5 chloroquinine. Alternatively, or additionally, the other compound is methotrexate. Alternatively, or additionally, the other compound is azathioprine.

Alternatively, or additionally, the other compound is cyclophosphamide. Alternatively, or additionally, the other compound is mycophenolate mofetil. Alternatively, or additionally, the other compound is an anti-CD20 antibody (e.g., 10 rituximab or ofatumumab). Alternatively, or additionally, the other compound is an anti-CD22 antibody (e.g., epratuzumab). Alternatively, or additionally, the other compound is an anti-TNF antibody (e.g., infliximab or adalimumab or golimumab). Alternatively, or additionally, the other compound is a CTFA-4 antagonist (e.g., abatacept, CTFA4-Ig). Alternatively, or additionally, the other 15 compound is an anti-IL-6 antibody. Alternatively, or additionally, the other compound is a BFys antagonist, such as an anti-BLys antibody (e.g., belimumab).

Dosages and Timing of Administration

For the prevention or treatment of a disease or condition or relapse 20 thereof, the appropriate dosage of an active agent (i.e., an immunoglobulin or antibody of the disclosure), will depend on the type of disease to be treated, the severity and course of the disease, whether the immunoglobulin or antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the immunoglobulin, and the discretion of the 25 attending physician. The particular dosage regimen, i.e., dose, timing, and repetition, will depend on the particular individual and that individual's medical history as assessed by a physician. Typically, a clinician will administer an immunoglobulin until a dosage is reached that achieves the desired result.

Methods of the present disclosure are useful for treating, ameliorating or 30 preventing the symptoms of diseases or conditions in a mammal, or for improving the prognosis of a mammal. Methods of the present disclosure are also useful for delaying development of or preventing lupus in an individual at risk of developing lupus or a relapse thereof.

For administration of the immunoglobulins or antibodies described herein, 35 normal dosage amounts may vary from about lOng/kg up to about lOOmg/kg of an individual's body weight or more per day. Exemplary dosages and ranges thereof are described herein. For repeated administrations over several days or longer, depending on the severity of the disease or disorder to be treated, the treatment can be sustained until a desired suppression of symptoms is achieved. 40 In some examples, the immunoglobulin or antibody is administered at an initial (or loading) dose of between about lmg/kg to about 30mg/kg, such as from about lmg/kg to about lOmg/kg, or about 2mg/kg or about 3mg/kg or 4mg/kg or WO 2012/021934 PCT/AU2011/001056 47 5mg/kg. The immunoglobulin or antibody can then be administered at a maintenance dose of between about O.OOOlmg/kg to about lmg/kg, such as from about 0.0005mg/kg to about lmg/kg, for example, from about O.OOlmg/kg to about lmg/kg, such as about O.Olmg/kg to about lmg/kg, for example from about 5 O.Olmg/kg to about 0.lmg/kg, such as about 0.02mg/kg or 0.03mg/kg or 0.04mg/kg or 0.05mgkg. The maintenance doses may be administered every 7-30 days, such as, every 10-15 days, for example, every 10 or 11 or 12 or 13 or 14 or 15 days.

In some examples, the immunoglobulin or antibody is administered at a 10 dose of between about O.OOOlmg/kg to about 50mg/kg, such as between about 0.0005mg/kg to about 50mgzkg, for example, between about O.OOlmg/kg to about 40mg/kg, for example, between about 0.005mg/kg to about 30mg/kg, such as between about O.Olmg/kg to about 20mg/kg. For example, the immunoglobulin is administered at a dose of between about O.Olmg/kg to about lOmg/kg, such as 15 from about O.Olmg/kg to about lmg/kg, such as about 0.02mg/kg or 0.03mg/kg or 0.04mg/kg or 0.05mg/kg or 0.06mg/kg or 0.07mg/kg or 0.08mg/kg or 0.09mg/kg or 0.lmg/kg or 0.2mgzkg or 0.3mg/kg or 0.4mg/kg or 0.5mg/kg or 0.6mg/kg or 0.7mg/kg or 0.8mg/kg or 0.9mg/kg (e.g., without a higher loading dose). In some examples, numerous doses are administered, e.g., every 7-30 days, such as, 20 every 10-22 days, for example, every 10-15 days, for example, every 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days. For example, the immunoglobulin or antibody is administered every 7 days or every 14 days or every 21 days.

In some examples, the immunoglobulin or antibody is administered at a 25 dose of between about lmg/kg to about 30mg/kg, such as from about lmg/kg to about lOmg/kg, or about 2mg/kg or about 3mg/kg or 4mg/'kg or 5mg/kg, or such as from about lOmg/kg to 30mg/kg, such as about lOmg/kg or 15 mg/kg or 20mg/kg or 25mg/kg (e.g., without a lower maintenance dose). In some examples, numerous doses are administered, e.g., every 10-70 days, such as every 30 14-70 days, such as, every 14-60 days, for example, every 14-50 days, such as every 14-40 days, or every 14-30 days. For example the doses are administered every 14 or 21 or 25 or 28 or 35 or 40 or 42 or 49 or 50 or 55 or 57 or 63 or 70 days. For example, the immunoglobulin or antibody is administered every 21 days or every 28 days or every 35 days or every 42 days or every 49 days or every 35 56 days.

In some examples, the immunoglobulin or antibody causes or is associated with a reduction of NK cells in a mammal following administration, e.g., within about 6 hours of administration. In some examples, a further dose of the immunoglobulin or antibody is administered when the number of NK cells in a 40 mammal returns to within 20% or 10% or 5% or 1% of the number of NK cells in the mammal prior to administration. In some examples, a further dose of the immunoglobulin or antibody is administered when the number of NK cells in a WO 2012/021934 PCT/AU2011/001056 48 mammal exceeds the number of NK cells in the mammal prior to administration by at least about 5% or 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80%. The number of NK cells can be assessed in a mammal to determine when to administer the further dose of the immunoglobulin or antibody. Alternatively, 5 the time to administer a further dose of the immunoglobulin or antibody is determined by previous analysis of a population or a model organism (e.g., a non-human primate, such as a cynomolgus monkey). For example, the further dose of the immunoglobulin is administered about 7 days or 8 days or 14 days or 17 days or 21 days or 22 days or 28 days or 29 days following a previous dose. 10 In some examples, at the time of commencing therapy, the mammal is administered the immunoglobulin or antibody on no more than 7 consecutive days or 6 consecutive days or 5 consecutive days or 4 consecutive days.

In the case of a mammal that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, 15 increasing doses may be administered.

In another example, for mammals experiencing an adverse reaction, the initial (or loading) dose may be split over numerous days in one week or over numerous consecutive days.

Dosages for a particular immunoglobulin or antibody may be determined 20 empirically in mammals that have been given one or more administrations of the immunoglobulin. To assess efficacy of an immunoglobulin, a clinical symptom of a disease or condition can be monitored.

Administration of an immunoglobulin according to the methods of the present disclosure can be continuous or intermittent, depending, for example, on 25 the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an immunoglobulin or antibody may be essentially continuous over a preselected period of time or may be in a series of spaced doses, e.g., either during or after development of a condition. 30 The present disclosure includes the following non-limiting examples.

Non-Limiting Examples

Example 1: Humanized antibodies A humanized antibody that binds specifically to human IL-3 Rot chain that 35 acts as an antagonist of IL-3 activity was produced. The humanized antibody was produced by grafting CDR sequences of an antagonistic murine antibody (7G3) onto human variable framework germline sequences selected on the basis of the canonical structure of the donor and acceptor CDRs in accordance with the procedure of Tan et al., (J Immunol. 169, 1119-1125, 2002); sometimes referred to 40 as “superhumanization”. This work was conducted using the antibody in a scFv format. This approach then compares the CDR residues of the donor antibody with those of the variable framework germline acceptor sequences, and selects as WO 2012/021934 PCT/AU2011/001056 49 the acceptor sequence the one with highest correlation of CDR residues. However, in the present case a heavy chain acceptor sequence with a lower level of CDR correlation was selected. The resulting humanized antibody contained entirely human variable framework sequence as a result of the humanization 5 process, however the affinity for IL-3Ra was decreased compared with the parental murine antibody.

Affinity optimization was employed using a ribosome display-based mutagenesis process (Kopsidas et al., BMC Biotechnol. 7, 18, 2007) conducted using the antibody in a scFv format in an effort to increase the binding affinity of 10 the humanized antibody. An affinity optimized scFv was produced that when converted to an IgGi format exhibited slightly improved IL-3Ra binding affinity to that of the parent murine monoclonal antibody. Unpredictably, the affinity optimization process resulted in mutations in the framework of the Vh and Vl, as well as in CDR1 of the light chain. So the set of CDR sequences of the 15 humanized affinity optimized antibody differs from those of the parent murine monoclonal antibody. There affinity optimized antibody is referred to herein as CSL362.

Fc engineered derivatives of the humanized affinity optimized antibody comprising the light and heavy chain variable regions of the antibody and a hybrid 20 IgGi/IgG2 constant domain with the three amino acid substitutions S239D/A330L/I332E (referred to herein as CSL362X2) or with the two amino acid substitutions S239D/I332E (referred to herein as CSL362X1) were produced by expression of an appropriate vector in CHO-S cells; the positions of the identified mutations are based on EU numbering system. 25 An afucosylated version of humanized affinity optimized antibody with a human IgGi constant domain was generated by expression of an appropriate vector in FUT8-knock-out CHO cells from Biowa (Potelligent® Cells) (referred to herein as CSL362B). 30 Example 2: Binding Affinity Determination

Full-Length mAb Kinetics'.

To analyze full-length antibodies, surface piasmon resonance (Biacore) assays were performed in a capture format where a chemically immobilized anti 35 human or anti mouse Fc specific antibody (Anti human Goat anti Human IgG (gamma) mouse adsorbed (Invitrogen, Cat No. Hl0500) or anti mouse Fc specific antibody (Jackson Immuno Research Labs inc. Cat No. 515-005-071) were chemically immobilized on a CM-5 sensor surface using standard amine coupling chemistry) and used to capture the mAb from solution. Soluble human IL-3Ra 40 was then injected over captured antibody at various concentrations. Responses were subtracted from those from a reference flow cell in which antibody was not captured, but otherwise treated identically. The reference subtracted responses WO 2012/021934 PCT/AU2011/001056 50 were then subtracted from the responses from a blank injection.

The final corrected responses were fitted using non-linear regression to a model describing 1:1 kinetics, including a term for mass transport limitation. The Rmax value was fitted locally, to account for slight deviations in the level of 5 antibody captured. Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) were determined.

Antibodies were captured at 0.3pg/ml for 180 seconds

Soluble IL-3Ra was injected for 10 minutes, and dissociation was monitored for 30 minutes. 10 Soluble IL-3Ra was injected at 0, 0.62, 1.25, 2.5, 5, 10, 20 and 40nM, with 2.5 and 5 nM in duplicate

Regeneration was performed after each cycle with a 90 second injection of lOOmM H3PO4

The assay was conducted at 25°C 15 scFv Kinetics:

To analyze scFvs, soluble human IL-3Ra was chemically immobilized on a CM-5 sensor surface using standard amine coupling chemistry, and scFvs were injected at various concentrations. Responses were subtracted from those from a 20 reference flow cell in which IL-3Ra was not immobilized, but otherwise treated identically. The reference subtracted responses were then subtracted from the responses from a blank injection.

The final corrected responses were fitted using non-linear regression to a model describing 1:1 kinetics, including a term for mass transport limitation. 25 The Rmax value was fitted globally, and association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) were determined. scFvs were injected for 10 minutes, and dissociation was monitored for 20 minutes. scFvs were injected at 0, 0.62, 1.25, 2.5, 5, 10, 20 and 40nM, with lOnM in 30 duplicate

Regeneration was performed after each cycle with a 30 second injection of lOOmM H3PO4, 1M NaCl and a 15 second injection of 50mM NaOH.

The assay was conducted at 25°C 35 Antibody and scFv affinities are tabulated below in Table 2:

Table 2: Affinity dissociation constants of antibodies and scFvs_

Antibody KD (M) parental murine mAb (7G3) ~9.2xlO"10 chimeric form of parental murine mAb) (human IgGi) -l.OxlO"9 Superhumanized mAb (IgGi) ~lxl0"8 WO 2012/021934 PCT/AU2011/001056 51

Antibody KD(M) Superhumanized mAb scFv -1.4x1 O'8 CSL362 scFv -2.2x1 O'9 CSL362 (IgGi) ~7.8xlO"10 CSL362B ~4.3xlO"10

Example 3: NK Cell Levels Following Administration of Humanized or Chimeric

Antibodies

Naive monkeys (non-human primates; NHPs) were administered a single 5 dose of CSL362B or CSL362X1 via intravenous infusion. In a separate study naive monkeys were administered repeat doses (weekly x 4) of a chimeric antibody comprising the variable regions of the murine antibody used to produce the humanized antibody (7G3) and a human IgGi constant region). Peripheral blood was collected at various time points and analysis of NHP NK cells 10 performed by flow cytometry.

As shown in Figure IA administration of CSL362X1 resulted in an initial depletion of NK cells, e.g., about 6 hours following administration. At dosages of O.Olmg/kg and O.lmg/kg, this level had exceeded the level observed prior to administration by about 8 days after administration and remained elevated until at 15 least 22 or 29 days after administration. An increase in the number of NK cells was also observed at the 1 mg/kg dosage.

In contrast to the results described in the previous paragraph, administration of CSL362B resulted in depletion of NK cells, however the NK cell numbers did not subsequently exceed the numbers prior to administration 20 (Figure IB).

Repeat administration of the chimeric antibody did not substantially change the number of NK cells detected in circulation (Figure 1C).

Example 4: Enhanced ADCC of Humanized Antibodies in the Presence of NK 25 Cells

Human PBMCs or NK cells were isolated and incubated with TF-1 cells in the presence of various concentrations of CSL362X1. Effector Cells (E; PBMC) and Target cells (T; TF-1 cells) were combined to achieve a ratio of 50:1 (E:T Ratio) or in the case where purified NK cells were used as effectors the ratio was 30 20:1. Cell lysis was measured using a LDH CytoTox 96 Non-Radioactive

Cytotoxicity kit (Promega).

Specific Lysis was determined by the following calculation:

Specific Lysis = [Sample Lysis - Spontaneous Lysis]/[Maximal Lysis -Spontaneous Lysis] x 100%. 35 Maximal Lysis was evaluated by addition of Extran™ to a final concentration of 0.75% (v/v). Spontaneous Lysis was that which occurred in wells with cells alone (no Ab). WO 2012/021934 PCT/AU2011/001056 52

As shown in Figure 2A, lysis of TF-1 cells occurred in the presence of PBMCs and in the presence of NK cells, liowc\'cr was substantially reduced in PBMCs from which NK cells were removed.

In a separate experiment, leukemic cells from two different AML patients 5 were used as target cells. In this assay, a single concentration of antibody (10pg/mL) was used and purified NK cells were added to generate various E:T ratios. Figure 2B shows that the higher the number of NK cells to target cells (peripheral blood blasts from two different AML patients) (i.e., the E:T ratio), the greater the specific lysis of the target cells by the humanized antibody. 10