Humanized anti-interleukin 3 receptor alpha chain antibodies

[0001] The invention relates to antibodies directed against the interleukin 3 receptor alpha chain and their use.

BACKGROUND OF THE INVENTION [0002] The functional interleukin 3 receptor is a heterodimer that contains a specific alpha chain (IL3Ra; CD123) and the "common" IL-3 beta chain (β_σ; CD131), which is common to receptors of granulocyte and macrophage colony stimulating factor (GM-CSF) and interleukin 5 (IL-5).

[0003] IL-3Rα is a type I transmembrane protein, with an inferred molecular weight of about 41 kDa, 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 consists of two regions: a N-terminal region about 100 amino acids in length, with a sequence that shows similarity to the equivalent regions of the alpha chains of GM-CSF and IL-5 receptors; and a region closer to the transmembrane domain that contains four conserved cysteine ​​residues and the WSXWS motif, common among other proteins of this family of cytokine receptors.

[0004] The IL-3 binding domain contains cytokine receptor (CRM) motifs with about 200 amino acid residues consisting of two domains folded like Ig. The IL-3Rα extracellular domain is highly glycosylated, with N-glycosylation required for both ligand binding and receptor signaling.

[0005] IL-3Ra expression occurs commonly in the entire hematopoietic system, including hematopoietic progenitor cells, mast cells, erythroid cells, megakaryocytes, basophils, eosinophils, monocytes / macrophages, neutrophils and CD5 B lymphocytes⁺. IL-3Ra is also expressed in non-hematopoietic cells, such as plasmacytoid dendritic cells (pDCs), Leydig cells, endothelial cells and stromal cells.

[0006] IL-3Rα expression also occurs in cells involved in certain disease states, including myelodysplastic syndrome, myeloid leukemia (e.g., acute myeloid leukemia (AML)), malignant lymphoproliferative disorders such as lymphomas, allergies and autoimmune diseases such as lupus, Sjogren's syndrome or scleroderma. Accordingly, anti-IL-3Ra antibodies are desirable for therapeutic applications.

SUMMARY OF THE INVENTION [0007] The invention provides an isolated or recombinant antibody that is capable of specifically binding the interleulin (IL) -3Ra chain, wherein the antibody is humanized and comprises:

(i) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8;

(ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and (iii) a heavy chain constant region containing the amino acid substitution S239D and I332E according to the EU numbering system according to Kabat.

[0008] The invention also provides a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

[0009] The invention also provides a nucleic acid encoding the antibody of the invention.

[0010] The invention also provides a genetic construct comprising a nucleic acid encoding the heavy chain of an antibody of the invention operably linked to a promoter and a nucleic acid encoding a light chain of an antibody of the invention operably linked to a promoter.

[0011] The invention also provides an isolated cell that expresses the antibody of the invention or a genetically modified cell such that it is capable of expressing the antibody of the invention.

[0012] The invention also provides an antibody of the invention or a composition of the invention for use in the treatment of diseases or conditions in mammals.

SUMMARY OF DISCLOSURE [0013] The disclosure is based on the production by the inventors of a humanized antibody that specifically binds to IL-3Ra. After humanization, the inventors have found that reducing the affinity of the antibody for IL3Rα. Accordingly, the inventors have carried out affinity optimization (maturation) to increase the affinity of the antibody for IL-3Rα. It has surprisingly been found that the antibody obtained by affinity optimization contains mutations in the framework regions (FR) of the heavy chain variable region (VH) and light chain variable region (VL) as well as in the light chain CDR1. [0014] The inventors also created forms of this antibody capable of inducing enhanced levels of effector functions.

[0015] The inventors have also found that a particular modification induces an enhancement of effector functions, resulting in an additional desirable property, which is that when a modified antibody is administered to a mammal, the number of natural killer (NK) cells in the mammal initially decreases, but then increases higher than before administration. The inventors have also shown that there is a correlation between the number of NK cells and lysis of target cells, e.g. leukemia cells.

[0016] The invention is generally directed to an immunoglobulin based on a murine antibody capable of specifically binding the IL-3Ra chain.

[0017] In one example, the invention provides an isolated or recombinant immunoglobulin that specifically binds and contains VL determining regions containing the sequence set forth in SEQ ID NO: 8 and the VH CDR shown in SEQ ID NO: 9.

IL-3Ra chain complementarity (CDR) [0018] The invention additionally or alternatively provides an isolated or recombinant antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is capable of specifically binding the IL-3Ra chain and includes VL CDRs comprising the sequence set forth in SEQ ID NO: 8 and the VH CDR shown in SEQ ID NO: 9.

[0019] The invention additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment is capable of specifically binding the IL-3Ra chain and includes V CDRs_L, containing the sequence shown in SEQ ID NO: 8 and the VH CDR shown in SEQ

ID NO: 9.

[0020] In one example, the invention provides an isolated or recombinant immunoglobulin that specifically binds to the IL-3Ra chain and comprises the amino acid sequence according to SEQ ID NO: 2-7, respectively.

[0021] The invention additionally or alternatively provides an isolated or recombinant antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment is capable of specifically binding the IL-3Rα chain and comprises the amino acid sequence set forth in SEQ ID NO: 2-7.

[0022] The invention additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment is capable of specifically binding the IL-3Rα chain and comprises the amino acid sequence set forth in SEQ ID NO: 2-7.

[0023] For example, an immunoglobulin or antibody comprises:

(i) a light chain variable (VL) region comprising CDRs 1, 2 and 3 as shown in SEQ ID NO: 2, 3 and 4, respectively;

and (ii) a heavy chain variable (VH) region comprising CDRs 1, 2 and 3 as shown in SEQ ID NOs 5, 6 and 7, respectively. [0024] For example, an immunoglobulin or antibody comprises:

(i) a VL containing:

a) CDR1 containing the sequence shown in SEQ ID NO: 2 (or encoded by the sequence shown in SEQ ID NO: 14);

b) CDR2 comprising the sequence shown in SEQ ID NO: 3 (or encoded by the sequence shown in SEQ ID NO: 15); and

c) CDR3 comprising the sequence shown in SEQ ID NO: 4 (or encoded by the sequence shown in SEQ ID NO: 16); and (ii) a VH containing:

d) CDR1 comprising the sequence shown in SEQ ID NO: 5 (or encoded by the sequence shown in SEQ ID NO: 17);

e) CDR2 comprising the sequence shown in SEQ ID NO: 6 (or encoded by the sequence shown in SEQ ID NO: 18); and

f) CDR3 comprising the sequence set forth in SEQ ID NO: 7 (or encoded by the sequence shown in SEQ ID NO: 19).

[0025] The invention additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment is capable of specifically binding the IL-3Rα chain and includes V_L, comprising the amino acid sequence of SEQ ID NO: 8 (or encoded by the sequence shown in SEQ ID NO: 20) and / or VH containing the amino acid sequence of SEQ ID NO: 9 (or encoded by the sequence shown in SEQ ID NO: 21).

[0026] The invention additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment is capable of specifically binding the IL-3Ra chain and includes V_L, containing the amino acid sequence according to SEQ ID NO: 8 (or encoded by the sequence shown in SEQ ID NO: 20) and / or VH containing the amino acid sequence according to SEQ ID NO: 9 (or encoded by the sequence shown in SEQ ID NO: 21 ).

[0027] Exemplary antigen binding fragments of the invention include:

(i) antibody domain (dAb);

(ii) Fv;

(iii) scFv or a stabilized form thereof (e.g., scFv stabilized with disulfide bridges);

(iv) dimeric scFv or a stabilized form thereof;

(v) a diabody, tricepus, tetrafile or a higher degree multitool;

(vi) Fab fragment;

(vii) Fab 'fragment;

(viii) fragment F (ab ');

(ix) fragment F (ab ') 2;

(x) any of (i) - (ix) fused to the antibody Fc region;

(xi) any of (i) - (ix) fused to an antibody or antigen-binding fragment thereof that binds to an immune effector cell.

[0028] In one example, an immunoglobulin or antibody lowers or at least partially eliminates cells to which it binds, for example, leukemia cells and / or basophils and / or pDCs.

[0029] It will be apparent to those skilled in the art on the basis of this disclosure that exemplary immunoglobulins or antibodies are capable of lowering or at least partially eliminating the cells to which they bind, without necessarily having to be conjugated to a toxic compound.

[0030] In one example, an immunoglobulin or antibody is capable of inducing an effector function, e.g., an effector function, which results in killing the cell to which the immunoglobulin or antibody binds. Exemplary effector functions include ADCC, antibody dependent cellular phagocytosis (ADCP) and / or complement dependent cytotoxicity (CDC).

[0031] In one example, the immunoglobulin or antibody is capable of inducing ADCC.

[0032] In one example, the immunoglobulin or antibody comprises an antibody Fc region capable of inducing effector function. For example, the effector function is an Fc dependent effector function. In one example, the Fc region is an IgG1 Fc region or an IgG3 Fc region or a hybrid of the IgG1 / IgG2 Fc region.

[0033] In one example, the immunoglobulin or antibody is capable of inducing a similar (e.g., slightly different or within about 10%) or at the same level of effector function as the human IgG1 and / or wild type human IgG3 Fc region.

[0034] In one example, the immunoglobulin or antibody is capable of inducing effector function at an enhanced level.

[0035] In one example, the level of effector function induced by an immunoglobulin or antibody is enhanced relative to that immunoglobulin or antibody when it contains the wild type IgG1 Fc region. [0036] In one example, the immunoglobulin or antibody is non-fucosylated or contains an Fc region that is non-fucosylated.

[0037] In another example, an immunoglobulin or antibody has a lower degree of fucosylation compared to an immunoglobulin or antibody produced by a human cell or CHO cell that has not been altered to reduce the level of protein fucosylation. According to this example, a lower level of fucosylation should be understood to mean that in a composition containing an immunoglobulin or antibody, the percentage of fucosylated immunoglobulins (e.g., glycosyl groups attached to Asn297 fucose containing antibody) is lower than if they were produced by a human cell or CHO cell, which was not changed to reduce the level of protein fucosylation.

[0038] For example, the immunoglobulin or antibody is a non-fucosylated humanized antibody, comprising a VL comprising the sequence set forth in SEQ ID NO: 8 (or encoded by the sequence shown in SEQ ID NO: 20) and a VH containing the sequence shown in SEQ ID NO: 9 ( or encoded by the sequence depicted in SEQ ID NO: 21). For example, an immunoglobulin or antibody is a non-fucosylated humanized antibody comprising a light chain comprising the sequence set forth in SEQ ID NO: 13 (or encoded by the sequence shown in SEQ ID NO: 23) and a heavy chain containing the sequence shown in SEQ ID NO: 10 (or encoded by the sequence depicted in SEQ ID NO: 22).

[0039] In one example, the immunoglobulin or antibody is a humanized antibody comprising a light chain comprising the sequence set forth in SEQ ID NO: 13 (or encoded by the sequence shown in SEQ ID NO: 23) and a heavy chain comprising the sequence shown in SEQ ID NO: 10 (or encoded by the sequence shown in SEQ ID NO: 22) which is expressed in a mammalian cell (e.g., a CHO cell) that is not expressed at detectable levels (or expressed at reduced levels) of α-1,6-fucosyltransferase (FUT8).

[0040] In one example, the immunoglobulin or antibody comprises an Fc region that contains one or more substitutions in the amino acid sequence that enhance the immunoglobulin-induced effector function. For example, one or more substitutions in the amino acid sequence increase the affinity of the Fc region for the Fcy receptor (Fc? R), compared to the non-substitution Fc region. For example, one or more substitutions in the amino acid sequence increase the affinity of the Fc region for Fc? R selected from the group consisting of Fc? RI, FcYRIIa, Fc? RIIc and FcYRIIIa, as compared to the non-substitutable Fc region. In one example, one or more substitutions in the amino acid sequence are:

(i) S239D, A330L and I332E, according to the EU Kabat numbering system; or (ii) S239D and I332E, according to the EU Kabat numbering system.

[0041] For example, an immunoglobulin or antibody is a humanized antibody comprising a VL comprising the sequence shown in SEQ ID NO: 8 (or encoded by the sequence shown in SEQ ID NO: 20) and a VH comprising the sequence shown by SEQ ID NO: 9 (or encoded by SEQ ID NO: 21), wherein the antibody comprises an Fc region comprising one or more substitutions in the amino acid sequence selected from the group consisting of:

(i) S239D, A330L and I332E, according to the EU Kabat numbering system; and (ii) S239D and I332E, according to the EU Kabat numbering system. [0042] In one example, the Fc region comprises a sequence comprising residues 234-450 in SEQ ID NO: 11 (including substitutions S239D and I332E, according to the EU Kabat numbering system).

[0043] In one example, the Fc region comprises a sequence comprising residues 234-450 in SEQ ID NO: 12 (including substitutions S239D, A330L and I332E, according to the EU Kabat numbering system).

[0044] In one example, the immunoglobulin or antibody is selected from the group consisting of:

(i) an antibody comprising a light chain comprising the sequence depicted in SEQ ID NO: 13 and a heavy chain comprising the sequence depicted in SEQ ID NO: 11;

(ii) an antibody comprising a light chain comprising the sequence depicted in SEQ ID NO: 13 and a heavy chain comprising the sequence depicted in

SEQ ID NO: 12.

[0045] As discussed above in this specification, the inventors have found that after administration of the antibody described herein, the number of NK cells in the bloodstream of a mammal initially decreases and then increases compared to the number of NK cells in the bloodstream of a mammal prior to administration. The inventors have also shown that the increased number of NK cells relative to the target cells results in increased efficacy, i.e. a greater number of target cells are killed. This effect is induced by an antibody containing a constant region or Fc region, including the amino acid substitutions S239D and I332E, according to the EU Kabat numbering system.

[0046] Thus, in one example, the invention provides an isolated or recombinant antibody that is capable of binding specifically to the IL-3Rα chain and comprises:

(i) a light chain variable (VL) region comprising CDRs 1, 2 and 3 as shown in SEQ ID NO: 2, 3 and 4, respectively;

(ii) a heavy chain variable (VH) region comprising CDRs 1, 2 and 3 as shown in SEQ ID NOs: 5, 6 and 7, respectively; and (iii) a heavy chain constant region containing the amino acid substitutions S239D and I332E according to the EU Kabat numbering system.

[0047] In one example, the invention provides an isolated or recombinant humanized antibody, wherein the antibody is capable of specifically binding the IL-3Rα chain and comprises:

(i) VL including:

a) CDR1, containing the sequence shown in SEQ

ID NO: 2 (or encoded by the sequence shown in SEQ ID NO: 14);

b) CDR2, containing the sequence shown in SEQ

ID NO: 3 (or encoded by the sequence shown in SEQ ID NO: 15); and

c) CDR3, containing the sequence shown in SEQ

ID NO: 4 (or encoded by the sequence shown in SEQ ID NO: 16);

(ii) VH including:

d) CDR1, containing the sequence shown in SEQ

ID NO: 5 (or encoded by the sequence shown in SEQ ID NO: 17);

e) CDR2, containing the sequence shown in SEQ

ID NO: 6 (or encoded by the sequence depicted in SEQ ID NO: 18); and

f) CDR3, comprising the sequence shown in SEQ ID NO: 7 (or encoded by the sequence shown in SEQ ID NO: 19); and (iii) a heavy chain constant region containing the amino acid substitutions S239D and I332E according to the EU Kabat numbering system.

[0048] In one example, the heavy chain constant region is a hybrid of human IgG1 and human IgG2 constant regions. [0049] In one example, the constant region comprises the sequence represented by residues 121-450 (inclusive) of SEQ ID NO: 11. [0050] The invention additionally or alternatively provides an isolated or recombinant humanized antibody, wherein the antibody is capable of specifically binding the IL chain -3Rα and contains:

(i) VL, comprising the amino acid sequence according to SEQ

ID NO: 8 (or encoded by the sequence shown in SEQ ID NO: 20) and / or VH, containing the amino acid sequence according to SEQ ID NO: 9 (or encoded by the sequence shown in SEQ ID NO: 21); and (ii) a heavy chain constant region containing the amino acid substitutions S239D and I332E according to the EU Kabat numbering system.

[0051] In one example, the heavy chain constant region is a hybrid of human IgG1 and human IgG2 constant regions. [0052] In one example, the constant region comprises the sequence represented by residues 121-450 (inclusive) of SEQ ID NO: 11.

[0053] The invention additionally or alternatively provides an isolated or recombinant humanized antibody, wherein the antibody is capable of binding specifically to IL-3Ra chain and includes V_L, containing the amino acid sequence according to SEQ ID NO: 8 (or encoded by the sequence shown in SEQ ID NO: 20), VH containing the amino acid sequence according to SEQ ID NO: 9 (or encoded by the sequence shown in SEQ ID NO: 21), and heavy chain constant region containing the amino acid substitutions S239D and I332E according to the EU Kabat numbering system.

[0054] In one example, the heavy chain constant region is a hybrid of human IgG1 and human IgG2 constant regions. [0055] In one example, the constant region comprises the sequence set forth between residues 121-450 (inclusive) of SEQ ID NO:

11.

[0056] In one example, the invention provides an isolated or recombinant humanized antibody, wherein the antibody is capable of binding specifically to the IL-3Rα chain and comprises a light chain comprising the sequence set forth in SEQ ID NO: 13, and a heavy chain comprising the sequence shown in SEQ ID NO: 11.

[0057] In one example, an immunoglobulin or antibody or antigen binding fragment thereof of the invention abolishes IL-3 signaling.

[0058] In one example, the immunoglobulin or antibody of the invention is a naked immunoglobulin or antibody, or the antigen binding fragment thereof of the invention is a naked antibody or antigen binding fragment thereof. [0059] In one example, the immunoglobulin or antibody of the invention is a full length antibody.

[0060] In one example, an immunoglobulin or antibody of the invention binds to the IL-3Rα chain with a dissociation equilibrium constant (KD) of 1 x 10^-8 M or less, such as 5 x 10^-9 M or less, for example, 3 x 10^-9 M or less, such as 2.5 x 10^-9 M or less.

[0061] In one example, an immunoglobulin or antibody of the invention binds to an IL-3Rα chain with a KD of about 2.2 x 10^-9 M or less. In one example, the KD is between about 1 x 10^-9 M and about 2.5 x 10^-9 M, for example, is about 2.2 x 10-9 M.

[0062] In one example, an immunoglobulin or antibody of the invention binds to an IL-3Rα chain with a KD of about 9 x 10^-10 M or less, for example, about 8 x 10^-10 M or less. In one example, the KD is from about 5 x 100^-10 M to about 9x10^-10 M, for example, is about 7.8 x 10^-10 M.

[0063] The disclosure also includes fragments, variants and derivatives of an immunoglobulin or antibody of the invention. [0064] In one example, the immunoglobulin or antibody of the invention when administered to a mammal (e.g., a non-human primate, such as a macaque) is capable of reducing the number of NK cells. For example, an immunoglobulin or antibody is capable of reducing the number of NK cells when administered to a mammal by at least about 20%, such as at least about 30% or 40% within 12 hours or 10 hours or 8 hours after administration . For example, an immunoglobulin or antibody when administered to a mammal is capable of reducing NK cell numbers by at least about 50% within 6 hours of administration. In one example, an immunoglobulin or antibody is able to reduce the number of NK cells when administered at a dose of 0.0001 mg / kg to 50 mg / kg, such as from about 0.0005 mg / kg to about 40 mg / kg, for example from about 0.001 mg / kg to about 30 mg / kg, such as for example from about 0.005 mg / kg to about 20 mg / kg, such as for example from about 0.01 mg / kg to about 10 mg / kg. For example, an immunoglobulin or antibody when administered to a mammal is able to reduce NK cell numbers by at least about 50% within 6 hours of administration when administered at a dose of about 0.01 mg / kg or 0.1 mg / kg or 1 mg / kg or 10 mg / kg. In one example, the dose is about 0.01 mg / kg or 0.1 mg / kg. [0065] In one example, the number of NK cells in a mammal increases 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 administration of an immunoglobulin or antibody, compared to the number of NK cells in a mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells increases at least about 8 or 11 or 17 or 22 or 29 days after administration. For example, the number of NK cells in a mammal increases by about 10% or 20% or 30% or 50% or 60% or 70% or 80%, compared to the number of NK cells in a mammal prior to administration of an immunoglobulin or antibody. For example, the number of NK cells in a mammal increases by about 20% at least 8 days after administration of the antibody or immunoglobulin at a dose of 0.001 mg / kg to 0.1 mg / kg, compared to the number of NK cells in the mammal prior to administration of the immunoglobulin. or antibodies. For example, the number of NK cells in a mammal increases by about 50% at least 11, 17, 22 or 29 days after administration of the antibody or immunoglobulin at a dose of 0.001 mg / kg to 0.1 mg / kg, compared to the number NK cells in a mammal prior to administration of the immunoglobulin or antibody. In one example, the antibody or immunoglobulin is administered at a dose of 0.01 mg / kg to 0.1 mg / kg. For example, the immunoglobulin or antibody is administered at a dose of 0.01 mg / kg or 0.1 mg / kg.

[0066] Based on this disclosure, it will be apparent to those skilled in the art that the invention 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, an immunoglobulin or antibody when administered to a mammal results in a decrease in the number of NK cells in the mammal, followed by an increase in the number of NK cells. In one example, the number of NK cells is increased or decreased, compared to the number of NK cells in a mammal prior to administration of an immunoglobulin or antibody.

[0067] In one example, the immunoglobulin or antibody is capable of reducing the number of NK cells in a mammal by at least about 50% within 6 hours after administration when administered at a dose of about 0.01 mg / kg or 0.1 mg / kg, and the number of NK cells in a mammal increases by about 20%, at least 8 days after administration of the antibody or immunoglobulin at a dose of 0.001 mg / kg to 0.1 mg / kg, compared to the number of NK cells in the mammal before administration of immunoglobulin or antibody.

[0068] In one example, the disclosure provides a pharmaceutical composition comprising the immunoglobulin or antibody of this disclosure and a pharmaceutically acceptable carrier, diluent or excipient.

[0069] This disclosure also provides an isolated nucleic acid encoding an immunoglobulin or antibody according to this disclosure.

[0070] Exemplary nucleic acid sequences are discussed in the context of encoding the antibodies or immunoglobulins of the disclosure and should be considered suitable for use mutatis mutandis in accordance with this example of the disclosure.

[0071] This disclosure also provides a nucleic acid capable of hybridizing to the nucleic acid of the disclosure under stringent hybridization conditions.

[0072] The disclosure also includes fragments, homologues and derivatives of the isolated nucleic acid of the disclosure.

[0073] This disclosure is also provided by a genetic construct comprising the isolated nucleic acid of the disclosure and one or more additional nucleotide sequences, such as a promoter operably linked to a nucleic acid.

[0074] In one example, the genetic construct is an expression construct, comprising an expression vector and an isolated nucleic acid of the disclosure, wherein the isolated nucleic acid is operably linked to one or more regulatory nucleic acids present in the expression vector.

[0075] In one example, the genetic construct of the disclosure comprises a nucleic acid encoding polypeptides (e.g., comprising VH), operably linked to a promoter and a nucleic acid encoding another polypeptide (e.g., comprising VL), operably linked to a promoter.

[0076] In another example, the genetic construct is a bicistronic genetic construct, e.g. containing, in the 5 'to 3' direction, the following components operably linked:

(i) a promoter (ii) a nucleic acid encoding the first polypeptide;

(iii) an internal ribosome binding site; and (iv) a nucleic acid encoding a second polypeptide.

[0077] For example, the first polypeptide comprises VH and the second polypeptide comprises VL, or the first polypeptide comprises VL, and the second polypeptide comprises VH.

[0078] The disclosure also contemplates separate genetic constructs, one of which encodes the first polypeptide (e.g., comprising VH), the other encodes the second polypeptide (e.g., comprising VL). For example, this disclosure also provides a composition comprising:

antibodies, disclosures, genetically (i) a first expression construct comprising a promoter-linked nucleic acid encoding a polypeptide (e.g., comprising VH); and (ii) a second expression construct comprising a promoter-linked nucleic acid encoding a polypeptide (e.g., comprising VL).

[0079] The disclosure also provides a host cell comprising the genetic construct of the disclosure.

[0080] In one example, the invention provides an isolated cell that expresses an immunoglobulin or antigen-binding fragment according to, or a recombinant cell modified to be capable of expressing an immunoglobulin, antibody or antigen-binding fragment.

[0081] In one example, the cell comprises the genetic construct of the disclosure or:

(i) a first genetic construct comprising a nucleic acid encoding a polypeptide (e.g. comprising VH) operably linked to a promoter; and (ii) a second genetic construct comprising a promoter-linked nucleic acid encoding a polypeptide (e.g., comprising VL), wherein the first and second polypeptides form an immunoglobulin, antibody or antigen binding fragment of the disclosure.

[0082] The genetic construct may be integrated into the cell genome or remain episomal DNA.

[0083] Examples of the cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0084] The disclosure further provides a method of producing an immunoglobulin, antibody or antigen binding fragment of the disclosure that comprises holding the genetic construct (s) of the disclosure under conditions suitable for producing the immunoglobulin, antibody or antigen binding fragment.

[0085] In one example, the method of producing an immunoglobulin, antibody or antigen binding fragment of the disclosure comprises culturing the cell of the disclosure under conditions suitable for the production and, optionally, secretion of the immunoglobulin, antibody or antigen binding fragment.

[0086] In one example, the method of producing an immunoglobulin, antibody or antigen binding fragment of the disclosure further includes isolating the immunoglobulin, antibody or antigen binding fragment.

[0087] The disclosure further provides a method of producing a recombinant immunoglobulin or antibody of the disclosure, the method comprising the steps of:

(i) culturing the host cell containing the expression vector of the disclosure so that said host cell expresses a recombinant immunoglobulin or antibody; and (ii) isolating the recombinant immunoglobulin.

[0088] In one example, the method of producing an immunoglobulin, antibody or antigen binding fragment according to the disclosure further comprises forming an immunoglobulin preparation, antibody or antigen binding fragment with a pharmaceutically acceptable carrier.

[0089] The disclosure also provides a method for the prophylactic or therapeutic treatment of a disease or condition in a mammal, the method comprising the step of administering to the mammal an immunoglobulin or antibody of the disclosure to thereby treat or prevent the disease or condition.

[0090] In one example, the mammal is a human.

[0091] In one example, the mammal needs treatment or prevention.

[0092] In one example, a needy mammal suffers from a disease or condition.

[0093] In one example, in the case of a mammal in need, there is a risk of developing the disease or condition or recurrence.

[0094] The disclosure also provides an immunoglobulin, antibody or antigen binding fragment of the disclosure, or a composition of the disclosure for use in medicine. [0095] The invention additionally or alternatively also provides an immunoglobulin, antibody or antigen binding fragment according to the disclosure for use in the manufacture of a medicament for treating a disease or condition in a mammal.

[0096] The disclosure also provides an immunoglobulin, antibody or antigen binding fragment of the disclosure for treating a disease or condition in a mammal.

[0097] In one example, the disease or condition is IL-3Rα mediated disease or condition. [0098] In one example, the disease or condition is myelodysplastic syndrome.

[0099] In one example, the disease or condition is a cancer, such as a hematological cancer, for example, leukemia such as acute leukemia (e.g. acute myelogenous leukemia) or chronic leukemia (e.g. chronic myelomonocytic leukemia).

[0100] In one example, the disease or condition is an IL-3Ra-related cancer, e.g., leukemia, i.e., the tumor (or leukemia) is characterized by tumor (leukemia) cells that express IL-3Ra.

[0101] In another example, the cancer is malignant lymphoproliferative disorder, such as lymphoma.

[0102] In one example, the disease or condition is an autoimmune or inflammatory condition. For example, the condition is lupus, e.g., systemic lupus erythematosus, Sjogren's syndrome, or scleroderma (e.g., systemic scleroderma).

[0103] In one example, the method comprises administering an effective amount of an immunoglobulin, such as a therapeutically effective amount of an immunoglobulin, antibody or antigen binding fragment.

[0104] In one example, the method comprises administering to the mammal from about 0.0001 mg / kg to 50 mg / kg of immunoglobulin, antibody or antigen binding fragment. For example, the method involves administration from about 0.0005 mg / kg to about 40 mg / kg. For example, the method involves administration from about 0.0005 mg / kg to about 30 mg / kg. For example, the method includes administration from about 0.001 mg / kg to about 20 mg / kg. For example, the method involves administration from about 0.001 mg / kg to about 10 mg / kg. For example, the method includes administration from about 0.01 mg / kg to about 5 mg / kg. For example, the method involves administration from about 0.001 mg / kg to about 1 mg / kg. [0105] In one example, the method comprises administering from about 0.1 mg / kg to about 10 mg / kg of an immunoglobulin, antibody or antigen binding fragment. For example, the method includes administration from about 0.1 mg / kg to about 5 mg / kg. For example, the method involves administration from about 0.1 mg / kg to about 1 mg / kg.

[0106] In one example, the method comprises administering from about 10 mg / kg to about 30 mg / kg of an immunoglobulin, antibody or antigen binding fragment. For example, the method involves administration from about 20 mg / kg to about 30 mg / kg.

[0107] In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of 0.01 mg / kg.

[0108] In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of 0.1 mg / kg.

[0109] In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of 1 mg / kg.

[0110] In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of 10 mg / kg.

[0111] In one example, the immunoglobulin, antibody or antigen binding fragment is administered at a dose of 30 mg / kg.

[0112] In one example, the immunoglobulin or antibody is administered to the mammal repeatedly. In one example, the interval between doses 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 interval between doses is at least about 11 days. In another example, the interval between doses 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 interval between doses is at least about 22 days. In another example, the interval between doses 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 interval between doses is at least about 57 or 60 days.

[0113] For example, an immunoglobulin, antibody or antigen binding domain is administered at a dose of 0.0001 mg / kg to 5 mg / kg, such as between 0.0005 mg / kg to 5 mg / kg,

<td>on</td><td>example, in between</td><td>0.001 mg / kg</td><td>down</td><td> 5</td><td>mg / kg, a</td><td>period</td>

<td colspan="2">between doses is</td><td>at least</td><td colspan="2">about</td><td>7 days or 8</td><td>days,</td>

<td>or</td><td>9 days or 10 days,</td><td>or 11 days,</td><td>or</td><td> 14</td><td>days, or 17</td><td>days,</td>

<td>or</td><td>21 days or 22 days</td><td>or 28 days,</td><td>or</td><td> 29</td><td>days, or 30</td><td>days,</td>

or 1 calendar month. For example, an immunoglobulin, antibody or antigen binding domain is administered in a dose of 0.01 mg / kg to 5 mg / kg, and the interval between doses 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 0.01 mg / kg to 2 mg / kg, and the interval between doses is at least about 7 days or 8 days, or 9 days, or 10 days. or 11 days or 14 days. For example, an immunoglobulin, antibody or antigen binding domain is administered at a dose of 0.01 mg / kg to 1 mg / kg, and the interval between doses 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 interval between doses is at least about 7 days and less than 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. days.

[0114] In one example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of 0.01 mg / kg, and the interval between doses is 6 days or 7 days, or 8 days, or 9 days, or 10 days, or 11 days, or 14 days, or 15 days.

[0115] In one example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of 0.1 mg / kg, and the interval between doses is 6 days or 7 days, or 8 days, or 9 days, or 10 days, or 11 days, or 14 days, or 15 days.

[0116] In one example, the immunoglobulin or antibody is administered at a dose of 1 mg / kg, and the interval between doses 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.

[0117] For example, the immunoglobulin or antibody is administered at a dose of between 6 mg / kg to 50 mg / kg, and the interval between doses is at least about 15 days. For example, the immunoglobulin or antibody is administered at a dose of between 10 mg / kg to 30 mg / kg, and the interval between doses is at least about 14 or 15 days. For example, the immunoglobulin or antibody is administered at a dose of between 20 mg / kg to 30 mg / kg, and the interval between doses is at least about 14 or 15 days. In some examples, the interval between doses 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 days.

[0118] In one example, the immunoglobulin or antibody is administered at a dose of 10 mg / kg, and the interval between doses 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.

[0119] In one example, the immunoglobulin or antibody is administered at a dose of 30 mg / kg, and the interval between doses 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.

[0120] As discussed above, the inventors have found that within about 8 days after administration of the immunoglobulin or antibody of the disclosure, the number of NK cells increases above the number of cells present prior to administration. The inventors have also shown that an increased number of NK cells increases the effectiveness of an antibody or immunoglobulin, according to the disclosure, to induce target cell death. Thus, after increasing the number of NK cells, another dose of antibody or immunoglobulin may be given to induce a therapeutic / prophylactic effect. For example, an immunoglobulin, antibody or antigen-binding fragment according to the disclosure is administered multiple times at a dose of from about 0.001 mg / kg to about 1 mg / kg, with a period between doses of at least about 7 days, such as at least about 8 days, e.g., at least about 11 days, such as at least about 14 days, e.g., at least about 17 days, e.g., at least about 21 days, e.g., at least about 22 days, e.g, days or 29 days or one calendar month or 56 or 57 or 60 days. In one example, the interval between doses is about 7 days. In one example, the interval between doses is about 8 days. In one example, the interval between doses is about 11 days. In one example, the interval between doses is about 14 days. In one example, the interval between doses is about 17 days. In one example, the interval between doses is about 21 days. In one example, the interval between doses is about 22 days. In one example, the interval between doses is about 28 days. In one example, the interval between doses is about 29 days. In one example, the dose is from about 0.01 mg / kg to about 0.1 mg / kg, such as for example the dose is about 0.01 mg / kg or 0.1 mg / kg.

BRIEF DESCRIPTION OF THE DRAWING [0121]

Fig. 1A is a graphic representation showing the number of NK cells in a sample from a non-human primate at various time points (as indicated) after administration of the CSL362X2 antibody. The number of cells is represented as a percentage of the number of cells prior to administration of the antibody. Dosage of the antibody is indicated.

Fig. 1B is a graphic representation showing the number of NK cells in a sample from a non-human primate at various time points (as indicated) after administration of the CSL362B antibody. The number of cells is represented as a percentage of the number of cells prior to administration of the antibody. Dosage of the antibody is indicated.

Fig. 1C is a graphical representation showing the number of NK cells in a sample from a non-human primate individual at various time points (as indicated) after administration of a chimeric antibody (designated CSL360) containing the human IgG1 constant domain and the variable region of the 7G3 antibody. The number of cells is represented as a percentage of the number of cells prior to administration of the antibody. Dosage of the antibody is indicated.

Fig. 2A is a graphical representation showing the percentage of lysed k TF-1 cells in the presence of the indicated cell population and CSL362X1 antibody at various concentrations (as indicated on the X axis).

Fig. 2B is a graphical representation showing the percentage of AML cells in the presence of different numbers of NK cells and CSL362X1 antibody. The ratio of effector cells (NK cells; E) to target cells (leukemia cells; T) is indicated on the X axis. The results obtained using cells from two patients are presented. DETAILED DESCRIPTION

Key to the sequence list [0122]

SEQ ID NO: 1 - amino acid sequence of the IL-3Ra chain

SEQ ID NO: 2 - the amino acid sequence of the LCDR1 humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 3 - the amino acid sequence of the LCDR2 humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 4 - the amino acid sequence of the LCDR3 humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 5 - the amino acid sequence of HCDR1 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 6 - the amino acid sequence of HCDR2 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 7 - the amino acid sequence of HCDR3 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 8 - amino acid sequence of the light chain variable region of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 9 - amino acid sequence of the heavy chain variable region of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 10 - amino acid sequence of the humanized antibody CSL362 and CSL362B heavy chain directed against the IL-3Ra chain,

SEQ ID NO: 11 - amino acid sequence of the humanized CSL362X1 antibody directed against the IL-3Ra chain,

SEQ ID NO: 12 - amino acid sequence of the humanized CSL362X2 antibody directed against the IL-3Ra chain,

SEQ ID NO: 13 - amino acid sequence of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 14 - nucleotide sequence coding for LCDR1 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 15 - nucleotide sequence coding for LCDR2 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 16 - nucleotide sequence coding for LCDR3 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 17 - nucleotide sequence coding for HCDR1 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 18 - nucleotide sequence coding for HCDR2 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 19 - nucleotide sequence coding for HCDR3 of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 20 - nucleotide sequence coding the light chain variable region of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 21 - nucleotide sequence coding the heavy chain variable region of the humanized CSL362 antibody directed against the IL-3Ra chain, and its modified forms

SEQ ID NO: 22 - nucleotide sequence coding for the heavy chain of the humanized CSL362 and CSL362B antibodies directed against the IL-3Ra chain,

SEQ ID NO: 23 - nucleotide sequence coding for the heavy chain of the humanized CSL362 antibody directed against the IL-3Rα chain, and its modified forms

General information [0123] In the specification, unless expressly stated otherwise or the context does not indicate otherwise, references to one stage, composition, group of stages or group of compositions should be considered to include one or more (i.e. one or more) of these stages, substance compositions, stage groups or substance composition groups.

[0124] It will be apparent to those skilled in the art that changes and modifications other than those described in detail are possible with the invention. It is understood that the disclosure includes all such changes and modifications. The disclosure also includes all steps, properties, compositions and compounds referred to or indicated in this specification, individually or collectively, and all combinations without exception, or any two or more of said steps or properties.

[0125] The scope of the disclosure is not limited by the specific examples described herein, which are intended to be illustrative only. Functionally equivalent products, compositions and methods are obviously within the scope of this invention.

[0126] In the specification, any example of the invention should be considered suitable for use mutatis mutandis in accordance with any other example of the disclosure, unless expressly indicated otherwise.

[0127] Unless otherwise defined, it is to be understood that all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art (e.g., in the field of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry and biochemistry).

[0128] Unless otherwise indicated, techniques for recombinant proteins, cell cultures, and immunological techniques used in the disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and WileyInterscience (1988, including all updates to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors) Current Protocols in Immunology, John Wiley &amp; Sons (including all updates to date).

[0129] The description and definitions of the variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof in the description can be further clarified using the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991.

[0130] The term "and / or", e.g. "X and / or Y" should be understood to mean either "X and Y" or "X or Y" and it should be understood that both these meanings and each of them are equally important.

[0131] Throughout this description, the word "contain", or variants thereof, such as "includes" or "containing", should be understood to mean the inclusion of a particular element, whole or stage, or group of elements, whole or stages, but not exclusion any other element, whole or stage, or group of elements, whole or stages.

[0132] As used herein, the term "derived from" should be understood to indicate that a given item can be obtained from a given source, although not necessarily directly from that source.

Selected definitions [0133] As used herein, the term "immunoglobulin"

includes any antigen-binding protein product of an immunoglobulin gene complex, including IgA, IgD, IgM, IgG and IgE isotype immunoglobulins and antigen binding fragments thereof. Examples of immunoglobulins are antibodies. Exemplary immunoglobulins are monoclonal. The term "immunoglobulin" includes any immunoglobulins that have been appropriately deprived of immunogenicity to thereby reduce or eliminate a mammal's immune response to an immunoglobulin that is administered to a mammal. For human treatment, suitable immunoglobulins include chimeric, humanized or human immunoglobulins. The term "immunoglobulin" also includes mutagenized, chimeric and / or modified humanized immunoglobulins that contain altered or variant amino acid residues, sequences or glycosylations, regardless of whether they occur naturally or as a result of human intervention (e.g. by recombinant DNA techniques) . Exemplary proteins include the Fc receptor binding portion. For example, proteins containing the term "immunoglobulin" include antibody domains, camelid antibodies, and cartilaginous fish antibodies (ie, immunoglobulins with new antigen receptors (IgNARs)). In general, camelid antibodies and IgNARs contain VH but are devoid of VL and are often referred to as heavy chain immunoglobulins. Other "immunoglobulins" T cell receptors and other immunoglobulin-like domains containing proteins that are capable of binding to an antigen, eg, by antigen binding sites containing a variable region.

[0134] A person skilled in the art will know that an "antibody" is generally considered a protein that contains a variable region composed of a plurality of polypeptide chains, e.g., a VL-containing polypeptide and a VH-containing polypeptide. The antibody also generally contains constant domains, some of which may be located in the constant region or constant fragment or crystallizable fragment (Fc). VH and VL interact with each other to form an Fv containing an antigen binding region that is capable of specifically binding one or more closely related antigens. Generally, the mammalian light chain is either the κ light chain or the λ light chain, and the mammalian heavy chain is an α, δ, ε, γ, or μ chain. Antibodies can be of different types (e.g. IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses. The term "antibody" includes humanized antibodies.

[0135] The term "humanized antibody" should be understood to refer to an antibody that contains a human-like variable region, including CDRs from a non-human antibody (e.g., mouse) transplanted or inserted into FR (framework regions) from a human antibody (this type of antibody is also referred to as "CDR-grafted antibody"). Humanized antibodies also include antibodies in which one or more residues of a human antibody are modified by one or more amino acid substitutions, and / or one or more residues of the framework regions of a human protein, replaced with corresponding non-human residues. As exemplified herein, humanized antibodies may also contain residues that are not found in either human antibodies or non-human antibodies. Any additional regions of the antibody (e.g., the Fc region) are generally human. Humanization can be carried out using a method known in the art, e.g. from US5225539, US6054297, US7566771 or US5585089. The term "humanized antibody" also includes superhumanized proteins, e.g., as described in US7732578.

[0136] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to designate the antibody in its substantially intact form, as opposed to the antigen-binding fragment of the antibody. In particular, whole antibodies include those containing heavy and light chains, including the Fc region. The constant domains may be wild type constant domain sequences (e.g. human wild-type constant domain sequences) or variants of their amino acid sequences. In some cases, an intact antibody may be capable of inducing one or more effector functions.

[0137] The term "naked antibody" refers to an antibody that is not conjugated to another compound, e.g., a toxic compound or a radioactive label. [0138] An "antigen binding fragment" of an antibody comprises the antigen binding domain and / or the variable region of an intact antibody. Examples of 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.

[0139] In the context of the invention, "effector functions" refer to those biological activities that occur through cells or proteins that bind to the Fc region (native Fc sequence region or variant Fc amino acid sequence region) of an antibody, resulting in cell death. Examples of effector functions induced by antibodies or immunoglobulins include: complement dependent cytotoxicity; antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); and activation of B cells. In the context of this invention, the term "antibody-induced effector function" or the like is used interchangeably with the term "immunoglobulin effector function" or "antibody effector function" or the like, and each is a literal support for another.

[0140] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulin binds to Fc receptors ("FcR") found on some cytotoxic cells (e.g., Natural Killer ("NK cells "), neutrophils and macrophages), allowing these cytotoxic effector cells to specifically bind to the antigen-carrying target cell, followed by killing the target cell with cytotoxins. To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells ("PBMCs") and NK cells.

[0141] As used herein, "variable region" refers to portions of an antibody light and / or heavy chain, as defined herein, that are capable of binding specifically to an antigen and, for example, include a CDR sequence; i.e., CDR1, CDR2, and CDR3, and framework regions (FR). For example, the variable region contains three or four FRs (e.g. FR1, FR2, FR3 and, optionally, FR4) together with three CDRs. VH refers to the heavy chain variable region. VL refers to the light chain variable region.

[0142] As used herein, the term "complementarity determining regions" (syn. CDRs; i.e. CDR1, CDR2, and CDR3) refer to antibody variable region amino acid residues whose presence plays a critical role in specific antigen binding. Each variable region usually has three CDR regions, referred to as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to CDRs and

FR was defined in accordance with Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health,

Bethesda, Md., 1987 and 1991 (also referred to in this description as the "Kabat numbering system." According to the Kabat numbering system, FR and CDR VH are located 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 Kabat numbering system, FR and CDR VL are located in 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).

[0143] "Framework regions" (hereinafter FR) are those variable domain residues that are different from CDR residues.

[0144] The term "constant region" as used herein refers to portions of an antibody heavy chain or light chain that are other than a variable region. In the heavy chain, the constant region generally contains multiple constant domains and the hinge region, e.g., the IgG constant region contains the following bound components, heavy chain constant domain (CH) 1, linker, CH2 and CH3. In the heavy chain, the constant region contains Fc. In the light chain, the constant region generally contains one constant domain (CL1).

[0145] The term "crystallizable fragment" or "Fc" or "Fc region" or "Fc portion" (which can be used interchangeably in this specification) refers to an antibody region containing at least one constant domain and which generally ( although not necessarily) it is glycosylated and which is capable of binding one or more Fc receptors and / or complement cascade components. The heavy chain constant region can be selected from any of five isotypes: α, δ, ε, γ, or μ. In addition, the heavy chains of different subclasses (as well as the heavy chains of the IgG subclass) are responsible for different effector functions, and thus, by selecting the desired heavy chain constant region, proteins having the desired effector function can be produced. Examples of heavy chain constant regions are gamma 1 (IgG1), gamma 2 (IgG2) and gamma 3 (IgG3), or hybrids thereof.

[0146] A "constant domain" is a domain in an antibody sequence that is very similar in antibodies / antibodies of the same type, e.g., IgG or IgM or IgE. The constant region of an antibody generally has multiple constant domains, e.g., the heavy, constant region γ, α or δ, has two constant domains.

[0147] As used herein, the term "specifically binds" should refer to an immunoglobulin or antibody, and means that the binding interactions between the immunoglobulin or antibody and the IL-3Rα chain depend on the presence of an antigenic determinant or IL-3 chain epitope 3Rα bound by an immunoglobulin or antibody. Accordingly, an immunoglobulin or antibody preferentially binds or recognizes an antigenic determinant or IL-3Rα chain epitope, even if it is present in a mixture of other molecules or organisms. In one example, an immunoglobulin or antibody reacts or binds more frequently, faster, for longer, and / or with greater affinity to IL-3Rα or to its expressed cells, than to alternative antigens or cells. After reading this definition, it is also understood that an immunoglobulin or antibody that specifically binds to IL-3Rα may or may not specifically bind to a second antigen. As such, "specific binding" does not necessarily require exclusive binding or undetectable binding to another antigen. The term "specific binding" is used interchangeably herein with the term "selective binding". Generally, in the specification reference to binding means specific binding, and each of the terms should be understood as supporting another term. In one example, "specific binding" to IL-3Ra or to its expressed cells means that the immunoglobulin or antibody binds to IL-3Ra or to its expressed cells with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, e.g. 20 nM or less, e.g., 1 nM or less, e.g. 0.8 nM or less.

[0148] The term "EU Kabat numbering system" should be understood to mean heavy chain numbering of an antibody in accordance with the EU index as given in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, ed. 5, United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on residue numbering of the human IgG1 EU antibody.

[0149] As used herein, the term "IL-3Ra mediated condition" should be understood to mean a condition associated with or caused by excessive expression of IL-3Ra and / or an excessive number of cells expressing IL-3Ra in a mammal , such as cancer cells (e.g. leukemia cells) and / or immune system cells (e.g. plasmacytoid dendritic cells).

[0150] As used herein, the term "myelodysplastic syndrome" or "MDS" should be understood to refer to a variety of hematological disease states that include inefficient production (or dysplasia) of hematopoietic stem cells. People with MDS often develop severe anemia and may require frequent blood transfusions. In many cases, as MDS progresses, patients develop cytopenia (low blood cell counts) due to progressive bone marrow damage. In about one third of patients with MDS, the disease develops into acute myeloid leukemia (AML). MDS can be diagnosed or classified according to various systems, including the French-American-British classification system (Bennett et al., Br. J. Haematol. 33: 451-458, 1976), The International Prognostic Sclubing System (Greenberg et al., Blood 89: 2079-88, 1997) or a system published by the World Health Organization.

[0151] As used herein, the term "treatment" refers to clinical intervention aimed at changing the natural history of a patient or cell being treated in the course of clinical pathology. Desirable effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and remission or improving prognosis. The patient is successfully "treated", for example, if one or more of the symptoms associated with the disease decreases or is eliminated.

[0152] As used herein, the term "prevention" includes providing prophylaxis regarding the occurrence or recurrence of a disease in a patient. You may have a predisposition or you may be at risk of developing a disease or a relapse, but you have not yet been diagnosed with the disease or a relapse.

[0153] As used herein, a mammal that is "at risk" of developing a disease or condition or recurrence may or may not have detectable disease or disease symptoms, and may or may not have detectable disease or disease symptoms before treatment according to this disclosure. "There is risk" means that the mammal has one or more risk factors that are measurable parameters that correlate with the development of the disease or condition according to the state of the art and / or as described herein.

[0154] "Effective amount" refers to at least an effective amount, in doses and over a period of time, to achieve the desired therapeutic or prophylactic result. An effective amount can be delivered using one or more dosages. In some examples of the invention, the term "effective amount" means the amount necessary for the effective treatment of a disease or condition, as previously described herein. The effective amount may vary depending on the disease or condition to be treated, and also on body weight, age, race, sex, health and / or physical condition, and other factors associated with the mammal to be treated. . Typically, the effective amount will remain in a relatively wide range (eg, the "dosage" range) that can be determined by the practicing physician through routine testing and experimentation. The effective amount can be administered in a single dose or in repeated doses once or several times throughout the treatment period.

[0155] A "therapeutically effective amount" is at least the minimum concentration necessary to obtain a measurable effect in the improvement of a particular disorder (e.g., SLE). The therapeutically effective amount determined herein may vary depending on factors such as the condition, age, sex and weight of the patient, and the ability of the immunoglobulin or antibody to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the immunoglobulin or antibody are offset by the therapeutically beneficial effects obtained.

[0156] "Prophylactically effective amount" refers to an effective amount, in doses and administered over a period of time, to achieve the desired prophylactic effect. Usually, but not necessarily, because the prophylactic dose is used in mammals before or at an early stage of the disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0157] Reference herein to "number of NK cells in a mammal" should be understood to include the number of NK cells in a mammalian sample and does not require determining the total number of NK cells in a mammal. This number can be expressed as, for example, the number of cells per ml or dl or as a percentage of the number of cells in a sample taken at various time points from the mammal.

[0158] For the purposes of nomenclature only, and not limitation, the amino acid sequence of the IL-3Ra chain is given in Gene ID under accession number 3563 and / or in SEQ ID NO: 1.

[0159] The "mammal" treated according to this disclosure may be a mammal, such as a non-human primate or human. In one example, the mammal is human.

[0160] The term "sequence identity" is used herein in its broadest sense and includes the number of exact matches of nucleotides or amino acids, including the appropriate match using a standard algorithm, taking into account the extent to which the sequences are identical in the comparison window. Sequence identity can be determined by assigning compared sequences using computer algorithm implementation (Geneworks Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by checking and best matching (i.e., which will result in the highest percentage of homology in the window comparisons), generated using any chosen method from among various methods. Reference may also 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 section 19.3 CURRENT PROTOCOLS IN MOLECULAR BIOLOGY edited by Ausubel et al. (John Wiley &amp; Sons Inc. NY, 1995-1999).

[0161] The disclosure also provides anti-IL-3Ra immunoglobulin derivatives, antibodies, or antigen binding fragments of the disclosure. As used herein, "derivative" proteins are altered, for example, by post-translational modifications (e.g., phosphorylation, acetylation, etc.), glycosylation modifications (e.g., addition, removal or change of glycosylation), and / or the inclusion of additional amino acid sequences, such as this is understood in the art.

[0162] The term "nucleic acid", as used herein, means single or double-stranded RNA and RNA capable of encoding an immunoglobulin, antibody or antigen binding fragment of the disclosure or the polyeptide component thereof. DNA includes genomic DNA and cDNA. RNA includes mRNA and cRNA. Nucleic acids can also be DNA-RNA hybrids. The nucleic acid contains a nucleotide sequence that typically includes nucleotides that contain bases A, G, C, T or U. However, the nucleotide sequences may include other bases such as inosine, methylcytosine, methylinosine, methyloadenosine and / or thiouridine, although not limited to those mentioned. [0163] The terms "hybridize and hybridize" are used in this specification to determine the pair formation of at least partially complementary nucleotide sequences to produce DNA-DNA, RNA-RNA or DNA-RNA hybrids. Hybrid sequences containing complementary nucleotide sequences are formed as a result of pairing between complementary purines and pyridimines according to the state of the art.

[0164] In this regard, it will be apparent that modified purines (e.g., inosine, methylinosine and methyloadenosine) and modified pyrimidines (thiouridine and methylcytosine) may also be involved in base pairing.

[0165] "Stringency of conditions", as used herein, refers to conditions of temperature and ionic strength, and the presence or absence of certain organic solvents and / or surfactants during hybridization. The higher the stringency, the higher the level of complementarity between hybridizing nucleotide sequences will be.

[0166] "Very stringent conditions" means those conditions under which only nucleic acids having a high frequency of complementary bases will hybridize.

[0167] Reference in the description to very harsh conditions includes the conditions of:

(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 hybridization at 42 ° C, and at least from about 0.01 M to at least about 0.15 M salt for rinsing 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, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at a temperature above 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.

[0168] Generally, washing is carried out at Tm = 69.3 + 0.41 (G + C)% -12 ° C. Generally, the Tm of the duplex DNA decreases by approximately 1 ° C with an increase of 1% in the number of bases changed.

[0169] Notwithstanding the above, stringent conditions are known in the art as described in Chapters 2.9 and 2.10 of Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY John Wiley &amp;

Sons, Inc. 1995-2009 [0170] An "expression vector" can be either a self-replicating extrachromosomal vector, such as a plasmid, or a vector that integrates into the host genome.

Immunoglobulins [0171] Accordingly, the immunoglobulin or antibody of the disclosure selectively binds to the IL-3Rα chain, whereby the interactions between the immunoglobulin or antibody and the IL-3Rα chain are understood to depend on the presence of binding of antigenic determinants or an IL-3Ra chain epitope. by immunoglobulin. Accordingly, an immunoglobulin or antibody preferentially binds or recognizes an antigenic determinant or IL-3Ra chain epitope, even when present in a mixture of other molecules or organisms.

Antibodies [0172] In one example, an immunoglobulin as described herein according to any example is an antibody, eg, comprising CDR regions and / or one or more of the variable regions described herein.

[0173] Suitably, the immunoglobulin is a humanized, chimeric or human antibody that comprises the light chain and heavy chain CDR amino acid sequences 1, 2 and 3, according to SEQ ID NO: 2-7, respectively. In one example, the antibody comprises the amino acid sequence of the heavy chain variable region in accordance with SEQ ID NO: 9, and the amino acid sequence of the light chain variable region in accordance with 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.

[0174] In one example, the antibody is a recombinant antibody. Methods for making antibodies comprising CDRs and / or variable regions described herein will be apparent to those skilled in the art based on the disclosure described herein and / or the documents to which this description relates.

[0175] In one example, the antibody of the disclosure comprises

VH and / or VL containing regions

CDRs as human as described herein and FR regions from the antibody. Optionally, the FR regions contain one or more amino acid substitutions, e.g. 2 or more or 3 or more or 4 or more or 5 or more or 10 or more or 15 or more. In one example, FRs contain no more than 30 amino acid substitutions, e.g., no more than 20 amino acid substitutions compared to human FRs. [0176] In one example, the antibody of the disclosure comprises VH and / or VL containing CDR regions as described herein, and FR regions from a non-human primate antibody, i.e., the antibody is synonymous with a humanized antibody, e.g. as described in

WO2007 / 019620.

[0177] In one example, the antibody of the disclosure is a composite antibody, e.g. as described in WO2006 / 082406. [0178] One exemplary antibody of the disclosure is a humanized antibody, e.g. as defined herein. In one example, the humanized antibody is an affinity matured antibody. In one example, a humanized antibody comprises:

(i) a VL containing:

a) CDR1 containing the sequence shown in SEQ ID NO: 2 (or encoded by the sequence shown in SEQ ID NO: 14);

b) CDR2 comprising the sequence shown in SEQ ID NO: 3v (or encoded by the sequence shown in SEQ IDNO: 15); and

c) CDR3 comprising the sequence shown in SEQ ID NO: 4 (or encoded by the sequence shown in SEQ ID NO: 16); and (ii) a VH containing:

d) CDR1 comprising the sequence shown in SEQ ID NO: 5 (or encoded by the sequence shown in SEQ ID NO: 17);

e) CDR2 comprising the sequence shown in SEQ ID NO: 6 (or encoded by the sequence shown in SEQ ID NO: 18); and

f) CDR3 comprising the sequence set forth in SEQ ID NO: 7 (or encoded by the sequence shown in SEQ ID NO: 19).

[0179] It will be apparent to those skilled in the art that the immunoglobulins or antibodies of the disclosure may include modifications that introduce non-naturally occurring sequences in humans, for example to increase affinity or effector functions.

[0180] The disclosure also provides the immunoglobulin or antibody variants of the disclosure. Accordingly, the variants exhibit at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the amino acid sequences shown in SEQ ID NOS: 213.

[0181] For example, it is known in the art that certain amino acids can be substituted or removed without adversely or significantly affecting the specificity and / or effector function of an immunoglobulin or antibody (e.g., conservative substitutions).

[0182] Antibody or immunoglobulin derivatives contemplated in the disclosure include, but are not limited to, modification of amino acid side chains, introduction of unnatural amino acids and / or derivatives thereof during peptide or polypeptide synthesis, and the use of cross-linking agents.

[0183] Exemplary antibodies and antigen-binding fragments thereof of the invention are described in Table 1.

Table 1: Exemplary antibodies and antigen binding fragments thereof

<td>Name</td><td>Heavy chain; SEQ ID NO:</td><td>Light chain; SEQ ID NO:</td>

<td>CSL362 Fv</td><td> 9¹</td><td> 8¹</td>

<td>CSL362</td><td> 10</td><td> 13</td>

<td>CSL362B</td><td> 10²</td><td> 13</td>

<td>CSL362X1</td><td> 11</td><td> 13</td>

<td>CSL362X2</td><td> 12</td><td> 13</td>

<td colspan="2">¹ variable region sequence only.</td><td></td>

<td colspan="2">² heavy chain constant region is</td><td>fucosylated.</td>

Antibody fragments

Antibodies Containing One Domain [0184] In some instances, the antigen binding antibody fragment of the disclosure is or includes an antibody containing one domain (the term is used interchangeably with the term "domain antibody" or "dAb"). An antibody containing one domain is a single polypeptide chain containing all or part of the antibody heavy chain variable domain.

Diabodies, trichodies, tetrabodies [0185] In some instances, the antigen binding fragment of the disclosure is or comprises a diabody, trichodyla, tetrabody or a higher protein complex such as those described in WO98 / 044001 and / or WO94 / 007921.

[0186] For example, the diabody is a protein containing two linked polypeptide chains, wherein each polypeptide chain has a VL-X-VH or VH-X-VL structure, wherein X is a linker having insufficient residues to provide a combination of VH and VL in a single polypeptide chain (or Fv formation) or is absent, and in which the VH of the polypeptide chain binds to the VL of another polypeptide chain, forming an antigen binding site, i.e. forming an Fv molecule capable of specifically binding one or more antigens. VL and VH may be the same in each polypeptide chain, or VL and VH may be different in each polypeptide chain so as to form a specific body (i.e. containing two Fvs having different specificity).

[0187] A diabody, trichodyla, 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. T cell (e.g. CD3).

Single chain Fv (scFv) fragments [0188] A person skilled in the art will know that scFvs contain VH and VL regions in a single polypeptide chain and a polypeptide linker between VH and VL that allows scFv to create the desired structure for antigen binding (i.e., binding) VH and VL of a single polypeptide chain with another to form Fv). For example, a linker with more than 12 amino acid residues from (Gly4Ser) 3 is one of the most preferred scFv linkers. [0189] This disclosure also contemplates Fv (or diFv or dsFv) disulfide stabilized bridges in which a single cysteine ​​residue has been introduced into FR VH and FR VL, and disulfide bonded cysteine ​​residues provide stable Fv.

[0190] Alternatively or additionally, this disclosure includes dimeric scFv, i.e. a protein containing two scFv molecules joined by a non-covalent or covalent bond, e.g. a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, the two scFvs are joined by a peptide linker of sufficient length to allow both scFvs formation and antigen binding, e.g. as described in US20060263367.

[0191] This disclosure also contemplates dimeric scFv capable of inducing effector activity. For example, one scFv binds to IL-3Ra and contains the CDRs and / or variable regions described herein, and the other scFv binds to a molecule on the cell surface of an immune system cell, e.g., T cells (e.g. CD3 or CD19). In one example, the dimeric protein is a combination of dAb and scFv. Examples of bispecific antibody fragments capable of inducing effector functions are described, for example, in US7235641.

Other Antibodies and Antibody Fragments [0192] This disclosure also contemplates other antibodies or antibody fragments, such as:

(i) bispecific key and lock proteins as described in

US5731168;

(ii) heteroconjugate proteins, e.g. as described in

US4676980;

(iii) heteroconjugate proteins prepared using a chemical crosslinker, e.g. as described in US4676980; and (iv) Fab3.

Constant Regions [0193] The disclosure includes immunoglobulins and antibodies, and antigen binding fragments comprising the antibody constant region and / or antibody Fc region.

[0194] The sequences of constant regions and / or Fc regions useful for producing immunoglobulins, antibodies or antigen binding fragments of the invention can be obtained from a variety of different sources. In some examples, the constant region, Fc or portion thereof, of an immunoglobulin, antibody or antigen-binding fragment is derived from a human antibody. The constant region, Fc or part thereof, may be derived from any class of antibody, including IgA, IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgG1, IgG2, IgG3 and IgG4. In one example, the constant or Fc region is a human IgG1 isotype or a human IgG2 isotype or a human IgG3 isotype or a hybrid of any of the above.

[0195] In one example, the constant region or Fc region is capable of inducing effector function. For example, the constant region or Fc region is a human IgG1 or IgG3 Fc region. In another example, the constant region or Fc region is a hybrid of the IgG1 and IgG2 constant region or Fc region or a hybrid of the IgG1 and IgG3 constant region or a hybrid of the IgG2 and IgG3 constant region or Fc region. Exemplary hybrids of the human IgG1 and IgG2 constant region or Fc regions are described in Chappel et al., Proc. Natl Acad. Sci. USA, 88: 90369040, 1991.

[0196] Methods for detecting whether or not the Fc region may induce effector functions will be apparent to those skilled in the art and / or described herein.

Effector function [0197] Accordingly, an anti-IL-3Ra immunoglobulin, antibody or antigen binding fragment of the disclosure has or exhibits an effector function that facilitates or allows at least partial reduction of the number, substantial reduction in the number or elimination of IL-3Ra + cells. Such effector function may be to enhance the affinity of binding to Fc receptors, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and / or complement dependent cytotoxicity (CDC).

[0198] As will be apparent to those skilled in the art, based on the description, some examples of the disclosure include an immunoglobulin, antibody or antigen binding fragment capable of inducing effector function.

[0199] In the case of IgG antibodies, these effector functions are regulated by the cooperation of the Fc region with a family of receptors called Fcy receptors (Fc? Rs), which are expressed on various cells of the immune system and / or complement, e.g. C1q. Formation of the Fc / FcyR complex attracts these cells to the sites of the bound antigen, resulting in signal transduction and subsequent immune responses. Methods to optimize the affinity of Fc? Rs binding to the Fc region of an antibody to enhance effector functions, in particular, altering ADCC and / or CDC activity relative to a "parent" Fc region, are known to those skilled in the art. These methods may include modifying the Fc region of the antibody to enhance interaction with the relevant Fc receptors and increase its effectiveness to facilitate ADCC and ADCP. Enhancement of ADCC activity after modification of oligosaccharide covalently bound to IgG1 antibodies by a conserved Asn297 residue in the Fc region has also been reported.

[0200] In this regard, it will be apparent that in some non-limiting examples, enhancement of effector function, such as ADCC, can be obtained by modification of an immunoglobulin or antibody with a normally glycosylated wild-type constant domain, including alteration or removal of glycosylation (see, for example, WO0061739 ) and / or mutations in the amino acid sequence (see for example WO2008036688). [0201] In one example, an immunoglobulin, antibody or antigen binding fragment binds to IL-3Rα in such a way that it is possible to induce effector function such as ADCC.

[0202] In one example, an immunoglobulin, antibody or antigen binding fragment binds to an epitope within IL-3R ", which allows induction of effector function such as ADCC.

[0203] In another example, an immunoglobulin, antibody or antigen binding fragment is capable of binding to IL-3Rα on a mammalian cell, thereby inducing an effector function such as ADCC.

[0204] For example, an immunoglobulin, antibody or antigen binding fragment remains bound to IL-3Rα on the cell surface for a time sufficient to induce effector function such as ADCC. For example, an immunoglobulin or antibody does not internalize too quickly to allow for ADCC induction.

[0205] Alternatively or additionally, the immunoglobulin, antibody or antigen-binding fragment is bound to IL3Ra on the cell surface in a manner that allows the immune effector cell to bind to the constant region or Fc region of the immunoglobulin, antibody or antigen binding fragment and inducing effector function such as ADCC. For example, when an immunoglobulin, antibody or antigen-binding fragment is bound to IL3Rα, the immunoglobulin Fc region, antibody or antigen-binding fragment is exposed in such a way that it is capable of interacting with an Fc receptor (e.g. Fc? R) on an immune effector cell . In the context of the invention, the term "immune effector cell" should be understood to mean any cell that expresses the Fc receptor and that is capable of killing the cell to which it is associated by ADCC or ADCP. In one example, the immune effector cell is an NK cell.

[0206] Each of the above paragraphs relating to the effector functions of an immunoglobulin, antibody or antigen-binding fragment should be regarded as regarding the use of mutatis mutandis to induce CDC. For example, an immunoglobulin, antibody or antigen binding fragment is bound to IL-3Rα on the cell surface in a manner that allows the complement component C1q to bind to the immunoglobulin constant or Fc region, antibody or antigen binding fragment, and induce CDC.

[0207] In one example, an immunoglobulin, antibody or antigen binding fragment is capable of inducing enhanced effector function.

[0208] In one example, there is an increase in effector function induced by the constant region or Fc region relative to the wild type IgG1 antibody constant or Fc region induced or the wild type IgG3 antibody constant region or Fc.

[0209] In another example, the constant region or Fc region is modified to induce an increased level of effector function as compared to the constant region or Fc region without modification. Such modifications may be at the amino acid level and / or at the secondary structure level and / or at the tertiary structure level and / or glycosylation of the constant region or Fc region.

[0210] A person skilled in the art will know that enhancement of effector function can be manifested in any of many ways, for example as a stronger effect, more lasting effect or faster effect.

[0211] For example, an anti-IL-3Ra immunoglobulin, antibody or antigen-binding fragment may possess or exhibit an effector function that includes antibody dependent cellular cytotoxicity (ADCC).

[0212] In one example, the constant region or Fc region contains 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 Fc? Rs. In one example, the constant region or Fc region has an affinity for Fc? R, which is more than 1-fold greater than the wild-type constant or Fc region, or more than 5-fold greater than the wild-type constant or Fc region, or between 5 -fold and 300-fold larger than the wild-type constant or Fc region. In one example, the constant region or Fc region contains 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 Kabat index. In one example, the constant region or Fc region contains at least one amino acid substitution selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D,

<td>L235S,</td><td>L235Y,</td><td>L235I,</td><td>S239D,</td><td>S239,</td><td>S239N,</td><td>S239Q,</td><td>S239T,</td>

<td>V240I,</td><td>V240M,</td><td>F243L,</td><td>V264I,</td><td>V264T,</td><td>V264Y,</td><td>V266I,</td><td>E272Y,</td>

<td>K274T,</td><td>K274,</td><td>K274R,</td><td>K274L,</td><td>K274Y,</td><td>F275W,</td><td>N276L,</td><td>Y278T,</td>

<td>V302I,</td><td>E318R,</td><td>S324D,</td><td>S324I,</td><td>S324V,</td><td>N325T,</td><td>K326I,</td><td>K326T,</td>

<td>L328M,</td><td>L328I,</td><td>L328Q,</td><td>L328D,</td><td>L328V,</td><td>L328T,</td><td>A330Y,</td><td>A330L,</td>

<td>A330I,</td><td>I332D,</td><td>I332E,</td><td>I332N,</td><td>I332Q,</td><td>T335D,</td><td>T335R, and</td><td>T335Y,</td>

numbered according to the index

EU Kabata.

In one example, the constant region or Fc region contains at least one amino acid substitution selected from the group consisting of V264I, F243L / V264I, L328M, I332E, L328M / I332E,

V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E, L328Q / I332E, V264T, V240I, V266I, S239D3, S339 S239D / I332Q, S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D,

S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V2641 / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235I2, L235Y, L235Y, N325T,

L328D / I332E, L328V / I332E,

S239A / V264I / I332E,

S239A / V264I / A330Y / I332E,

L328T / I332E, L328I / I332E,

S239Q / V264I / I332E, S239D / A330Y / I332E,

S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E,

V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E,

S239D / V264I / I332E, S239D / V264I / S298A / I332E,

S239D / V264I / A330L / 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,

S239D / A330Y / I332E / L234I,

S239D / A330Y / I332E / V240I,

V240I / V266I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V264T,

S239D / A330Y / I332E / K326E, and S239D / A330Y / I332E / K326T, numbered according to the EU Kabat index.

[0213] In another example, the constant region or Fc region binds to FcYRIIIa more effectively than to FcYRIIb. For example, the constant region or Fc region contains at least one amino acid substitution at a position selected from the group consisting of: 234, 235, 239, 240, 264, 296, 330, and I332; numbered according to the EU Kabat index. In one example, the constant region or Fc region contains at least one amino acid substitution selected from the group consisting of: L234Y, L234I, L235I, S239D, S239E, S239N, S239Q, V240A, V240M, V264I, V264Y, Y296Q, A330L, A330Y, A330I, I332D, and I332E, numbered according to the EU Kabat index. For example, the constant region or Fc region contains at least one amino acid substitution selected from the group consisting of: I332E, V264I / I332E, S239E / I332E, S239Q / I332E, Y296Q, A330L, A330Y, I332D, S239D, S239D / I332E, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234Y,

L234I, L235I, V240A, V240M, V264Y, A330I, S239D / A330L / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264 / S239 / S239 / I332E, numbered according to the EU Kabat index.

[0214] In another example, the constant region or Fc region induces ADCC at a level greater than via a wild-type constant or Fc region. For example, a constant region or Fc region induces ADCC at a level that is more than 5-fold or between 5-fold and 1000-fold greater than that induced by the wild-type constant or Fc region. In one example, the constant region or Fc region contains 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 Kabat index. In one example, the constant region or Fc region contains at least one amino acid substitution selected from the group consisting of: P230A, E233D,

<td>L234A,</td><td>L234Y,</td><td>L234I,</td><td>L235D,</td><td>L235S,</td><td>L235Y,</td><td>L235I,</td><td>S239D,</td>

<td>S239,</td><td>S239N,</td><td>S239Q,</td><td>S239T,</td><td>V240I,</td><td>V240M,</td><td>F243L,</td><td>V264I,</td>

<td>V264T,</td><td>V264Y,</td><td>V266I,</td><td>E272Y,</td><td>K274T,</td><td>K274,</td><td>K274R,</td><td>K274L,</td>

<td>K274Y,</td><td>F275W,</td><td>N276L,</td><td>Y278T,</td><td>V302I,</td><td>E318R,</td><td>S324D,</td><td>S324I,</td>

<td>S324V,</td><td>N325T,</td><td>K326I,</td><td>K326T,</td><td>L328M,</td><td>L328I,</td><td>L328Q,</td><td>L328D,</td>

<td>L328V,</td><td>L328T,</td><td>A330Y,</td><td>A330L,</td><td>A330I,</td><td>I332D,</td><td>I332E,</td><td>I332N,</td>

<td>I332Q,</td><td>T335D,</td><td>T335R,</td><td>and</td><td>T335Y,</td><td colspan="3">numbered according to</td>

the EU Kabat index. In one example, the constant region or Fc region contains at least one amino acid substitution selected from the group consisting of: V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E, L328Q / I332E,

S239Q / I332D,

A330L / I332E,

V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332

A330Y / I332E, V264I / A330Y / I332E,

V264I / A330L / I332E, L234E, L234Y, L2341, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L32ET / L328 I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E2, S239E / S239 I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P230E2, K3 K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L2D3 / S3D3 A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E, and S239D / A330Y / I332E / K326T, numbered according to the EU Kabat index.

[0215] In one example, the constant region or Fc region comprises the following amino acid substitutions S239D / I332E, numbered according to the EU Kabat index. This constant region or Fc region shows an approximately 14-fold increase in affinity for Fc? RIIIs, compared to the wild type constant or Fc region, and about 3.3 increased ability to induce ADCC, compared to the wild type constant or Fc region. In one example, the constant region comprises sequences represented by residues 121-450 (inclusive) of SEQ ID

NO: 11. In one example, the Fc region comprises the sequence represented by residues 234-450 of SEQ ID NO: 11.

[0216] In one example, the constant region or Fc region comprises the following amino acid substitutions S239D / A330L / I332E, numbered according to the EU Kabat index. This constant region or Fc region shows about 138-fold increase in affinity for Fc? RIIIa, compared to the wild type constant or Fc region, and about 323 increased ability to induce ADCC, compared to the wild type constant or Fc region. In one example, the constant region comprises the sequence represented by residues 121-450 (inclusive) of SEQ ID NO: 12. In one example, the Fc region comprises the sequence represented by residues 234-450 of SEQ ID NO: 12.

[0217] Additional amino acid substitutions that increase the ability of the Fc region to induce effector functions are known in the art and / or described, for example, in

US6737056 or US7317091.

[0218] In one example, the glycosylation of the constant region or Fc region is changed to increase its ability to induce effector function. In this regard, native antibodies produced by mammalian cells typically contain a branched, biantennary oligosaccharide which is substantially N-linked to Asn297 of the CH2 domain of the constant region or Fc region. The oligosaccharide may include various carbohydrates, e.g. mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as GlcNAc associated fucose in the "core" of the biantennary structure of the oligosaccharide. In some examples, the constant regions or Fc regions of the disclosure contain a structure having a carbohydrate produced that lacks fucose attached (directly or indirectly) to the Fc region, ie, the Fc region is "non-fucosylated". Such variants may show better ability to induce ADCC. Methods for producing non-fucosylated antibodies include, expressing an immunoglobulin or antibody in a cell line unable to express α-1,6-fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et al., Biotechnol. Bioengineer., 87: 614-622 , 2004), expressing immunoglobulin or antibody in cells expressing small interference RNA against FUT8 (e.g. as described in Mori et al., Biotechnol. Bioengineer., 88: 901-908, 2004), expressing an immunoglobulin or antibody in cells unable to express guanosine diphosphate (GDP) -mannose-4,6-dehydrate (GMD) (e.g. as described in Kanda et al., J. Biotechnol., 130: 300-310, 2007). This disclosure also considers using immunoglobulins reduced levels of fucosylation, e.g., using a cell line modified to express β- (1,4) N-acetylglucosaminyltransferase III (GnT-III) (e.g. as described in Umana et al., Nat. Biotechnol., 17: 176-180, 1999). [0219] In one example, the immunoglobulin or antibody of the disclosure is non-fucosylated. For example, an immunoglobulin or antibody is produced in a cell (e.g., a mammalian cell, such as a CHO cell) that does not express FUT8.

[0220] Other methods include the use of cell lines that naturally produce antibodies capable of inducing Fc-mediated effector functions (e.g., duck embryonic stem cells for the production of antiviral vaccines, WO2008 / 129058; Production of recombinant proteins in EBX avian cells) ®, WO 2008/142124).

[0221] Immunoglobulins useful in the methods of the invention also include those having halved oligosaccharides, e.g., wherein the biantennary oligosaccharide attached to a constant region or Fc region is split in two by GlcNAc. Such immunoglobulins also have reduced fucosylation and / or have enhanced ADCC function. Examples of such immunoglobulins are described, e.g., in US6602684 and US20050123546.

[0222] Immunoglobulins having at least one galactose residue in the oligosaccharide attached to the constant region or Fc region are also contemplated. Such immunoglobulins have enhanced CDC functions. Such immunoglobulins are described, e.g., in WO1997 / 3008 and WO1999 / 22764.

[0223] Non-limiting examples of immunoglobulins that induce enhanced levels of ADCC activity include:

(i) an antibody comprising a heavy chain comprising the sequence depicted in SEQ ID NO: 11 and a light chain comprising the sequence depicted in SEQ ID NO: 13;

(ii) an antibody comprising a heavy chain comprising the sequence depicted in SEQ ID NO: 12 and a light chain comprising the sequence depicted in SEQ ID NO: 13; and (iii) an antibody comprising a heavy chain comprising the sequence depicted in SEQ ID NO: 10 and a light chain comprising the sequence depicted in SEQ ID NO: 13, wherein the heavy chain constant region is nonfucosylated.

[0224] A preferred antibody of the disclosure that induces enhanced levels of ADCC activity comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 13. As discussed in this specification, after administering this antibodies 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 to administration of the antibody;

(ii) the number of NK cells in the mammal that has been administered the heavy chain antibody comprising the sequence set forth in SEQ ID NO: 10 and the light chain comprising the sequence set forth in SEQ ID NO: 13, wherein the heavy chain constant region is nonfucosylated (e.g. as determined on the same day after administration); and (iii) the number of NK cells in a mammal that has been administered an antibody that binds specifically to the IL-3Ra chain and has a human IgG1 constant region (e.g. as determined on the same day after administration).

[0225] Methods for determining the ability of an immunoglobulin or antibody to induce effector functions are known in the art and / or described in more detail in this specification.

Additional Modifications [0226] The disclosure also contemplates additional immunoglobulin modifications.

[0227] For example, an immunoglobulin or antibody contains one or more amino acid substitutions that increase the half-life of the immunoglobulin. For example, an 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 example, the constant region or Fc region has increased affinity for FcRn at lower pH, e.g. around pH 6.0, to facilitate Fc / FcRn binding in the endosome. In one example, the constant region or Fc region has increased affinity for FcRn at a pH of about 6, compared to its affinity at pH 7.4, which facilitates releasing the constant region or Fc into the blood after cell recycling. These amino acid substitutions are useful for extending the half-life of immunoglobulin by reducing its clearance from the blood.

[0228] Exemplary amino acid substitutions include T250Q and / or M428L, according to the Kabat EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620.

Nucleic acids [0229] Another example of the disclosure provides an isolated nucleic acid that encodes an immunoglobulin or antibody of the disclosure, including fragments, variants, and derivatives of an immunoglobulin or antibody.

[0230] Some examples of nucleic acids include the nucleotide sequences set forth in one or more of SEQ ID NO: 14-19, which respectively encode the immunoglobulin CDR 1-6 regions or antibodies of the disclosure.

[0231] Other examples of nucleic acids include the nucleotide sequences set out in one or more of SEQ ID NO:

20-23.

[0232] The disclosure also contemplates nucleic acid homologues that encode the immunoglobulin variant or antibody of the disclosure as previously described.

[0233] For example, nucleic acid homologues have at least 80% or 85%, such as at least 90% or 95% or 99% identity of the nucleotide sequence with isolated nucleic acid that encodes any of SEQ ID NOS: 213. Accordingly, the nucleic acid homologue does not encode a protein containing a murine variable region capable of specifically binding to IL-3R ".

[0234] Nucleic acid homologues may hybridize with isolated nucleic acids of the disclosure under very stringent conditions.

Protein Production

Recombinant Expression [0235] In one example, the immunoglobulin or antibody described herein, according to any example, is obtained by recombinant methods.

[0236] By way of example only, the recombinant immunoglobulin or antibody of the disclosure can be produced using a method comprising the steps of:

(i) preparing an expression construct that contains the isolated nucleic acid of the disclosure operably linked to one or more regulatory nucleotide sequences (e.g.

promoter);

(ii) transfection or transformation of a suitable host cell with an expression construct;

(iii) expressing the recombinant immunoglobulin or antibody in a host cell; and (iv) isolating the recombinant immunoglobulin or antibody from the host cell.

[0237] In the case of recombinant immunoglobulin, the nucleic acid encoding it can be cloned into expression vectors, which are then transferred by transfection into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK), or bone marrow cells that do not themselves produce an immunoglobulin protein or antibody. [0238] Exemplary cells used to express an immunoglobulin or antibody are CHO cells, bone marrow cells or HEK cells. The cell may further contain one or more genetic mutations and / or deletions that facilitate expression of the modified immunoglobulin or antibody. One non-limiting example is the deletion of a gene encoding an enzyme necessary for fucosylation of an expressed immunoglobulin or antibody. For example, a delegated deletion gene encodes FUT8. The commercially available source of FUT8 deleted CHO cells is Biowa (Potelligent ™ cells). For example, the cells used to express a non-fucosylated immunoglobulin or antibody are CHO cells after FUT8 deletion, such as Potelligent ™ Biowa cells.

[0239] Molecular cloning techniques for achieving such purposes are known in the art and described, for example, in Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A variety of in vitro cloning and amplification methods are suitable for the construction of recombinant nucleic acids. Methods for producing recombinant antibodies are also known in science. See US4816567 or US5530101.

[0240] After isolation, the nucleic acid operably linked to the promoter is introduced into the genetic construct or expression vector for further cloning (DNA amplification) or for expression in a cell-free system or in cells. Thus, another example of the disclosure provides a genetic construct that contains the isolated nucleic acid of the disclosure and one or more additional nucleotide sequences. Accordingly, the genetic construct is in the form or contains the genetic components of a plasmid, bacteriophage, cosmid, artificial yeast or bacterial chromosome, according to the state of the art. Genetic constructs may be suitable for maintaining and propagating the isolated nucleic acid in bacteria or other host cells, for manipulation using recombinant DNA technology, and / or for expressing the nucleic acid or encoded immunoglobulin or antibody of the disclosure. To obtain expression in a host cell, the genetic construct is an expression construct. Accordingly, the expression construct comprises the nucleic acid of the disclosure operably linked to one or more additional sequences in the expression vector, such as a promoter sequence or regulatory sequence.

[0241] Typically, a regulatory nucleotide sequence may include, but is not limited to, promoter sequences, leader sequences or signal sequences, ribosomal binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activation sequences.

[0242] As used herein, the term "promoter" should be understood in its broadest context and includes transcriptional regulatory sequences of the genomic gene, including the TATA sequence or initiating element that is required for proper transcription initiation, with or without additional regulatory elements (eg. upstream activating sequences, transcription factor binding sites, enhancers and silencing sequences) that alter the expression of a nucleic acid, e.g. in response to a developmental and / or external stimulus, or in a tissue-specific manner. In this context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid or derivative that causes, activates or enhances the expression of the nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements for further enhancing transcription.

appropriate

Specialist sequences for enhancing expression and / or altering spatial expression and / or temporal expression of nucleic acid.

[0243] As used herein, the term "operably linked to" means that the promoter is positioned relative to the nucleic acid such that expression of that nucleic acid is controlled by the promoter.

[0244] A variety of cell expression vectors are available. Vector components generally include, but are not limited to, one or more of the following components: signal sequence, immunoglobulin coding sequence or antibody (e.g., derived from the information provided in this specification), a promoter element, and a termination sequence in the art will knew the expression of immunoglobulin.

Exemplary signal sequences include prokaryotic secretion signal sequences (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II), yeast secretion signal sequences (e.g., invertase leader, factor α leader or acid phosphatase leader), or mammalian secretion signal sequences ( e.g. herpes virus gD signal sequence).

[0245] Exemplary promoters active in mammalian cells include the cytomegalovirus direct early promoter (CMV-IE), human 1-α elongation factor promoter (EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chain promoter, promoter simian virus 40 (SV40), Rous sarcoma virus (RSV) promoter, major adenovirus late promoter, β-actin promoter; a hybrid regulatory element comprising the CMV enhancer sequence / β-actin promoter or immunoglobulin promoter or antibody, or an active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture; neonatal kidney cell line (BHK, ATCC CCL 10); or Chinese hamster ovary (CHO) cell line.

[0246] Typical promoters suitable for expression in yeast cells, such as, for example, yeast cells selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.

[0247] Methods for introducing an isolated nucleic acid or expression construct containing it into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on known effective techniques. Methods for introducing recombinant DNA into cells include, but are not limited to, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection, such as using lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), DNA collection via PEG, electroporation and microparticle bombardment, such as using tungsten or gold particles coated with DNA (Agracetus Inc., WI, USA).

[0248] Host cells used to produce an immunoglobulin or antibody can be cultured in a variety of media, depending on the type of cell used. Commercially available media such as F10 Hama (Sigma), minimal basic media ((MEM), (Sigma), RPMI-1640 (Sigma) and Eagle's modified Dulbecco's medium ((DMEM), Sigma) are suitable for growing mammalian cells. Media for growing other cell types discussed in this specification are known in the art.

Protein Isolation [0249] Methods for purifying immunoglobulin or antibodies are known in the art and / or described herein.

[0250] If the immunoglobulin or antibody is secreted into the medium, supernatants from such expression systems can be preconcentrated using commercially available protein concentration filters, for example, Amicon or Pellicon Millipore ultrafiltration units. Protease inhibitors such as PMSF can be introduced to inhibit proteolysis at any of the above stages, and antibiotics can be introduced to prevent the growth of unwanted organisms.

[0251] The ion isolated from the antibody can be purified by ion exchange chromatography, hydroxyapatite, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g. protein A affinity chromatography or protein G chromatography), or any combination of the above. These methods are known in the art and described, for example in WO99 / ​​57134 or Ed Harlow and David Lane (editors) of immunoglobulin cells or using, for example, chromatography on

Antibodies: A Laboratory Manual, Cold Spring Harbor

Laboratory, (1988).

[0252] One skilled in the art will also know that an immunoglobulin or antibody can be modified to include a tag to facilitate purification or detection, e.g. a polyhistidine tag, e.g. a hexahistidine tag, or an influenza virus (HA) hemagglutinin tag, or a simian virus tag 5 (V5) or FLAG tag, or glutathione S-transferase (GST) tag. The immunoglobulin or antibody thus obtained is then purified using methods known in the art, such as affinity-based purification. For example, an immunoglobulin or antibody containing a hexaHis tag is purified by contacting the sample containing the immunoglobulin or antibody with a nickel-nitrilotriacetic acid (Ni-NTA) complex that specifically binds a hex-His tag immobilized on a solid or semi-solid support, rinsing the sample for removal of unbound immunoglobulin, followed by elution of bound immunoglobulin. Alternatively or additionally, affinity-based purification methods use a ligand or antibody that binds to the label. [0253] In one example, the immunoglobulin or antibody also has a protease cleavage site, such as factor Xa or thrombin, which allows the appropriate protease to partially digest the immunoglobulin or antibody leading to the release of the immunoglobulin or antibody from the tag. The released antibody or immunoglobulin can then be separated from the fusion part by subsequent chromatographic separation.

Determination of Immunoglobulin Activity [0254] Immunoglobulins or antibodies according to can be readily tested for biological, e.g. as described below.

Activity disclosure binding assays [0255] One form of such assay is an antigen binding assay, e.g., as described in Scopes (W: Protein purification: principles and practice, 3rd edition, Springer Verlag, 1994). Such a method generally involves labeling an immunoglobulin or antibody and contacting it with the immobilized antigen. After washing to remove non-specific bound protein, the amount of label and consequently the amount of bound protein is detected. Of course, an immunoglobulin or antibody can be immobilized and the antigen labeled. Panning designations may also be used, e.g. as described or exemplified herein.

Determination of Signal Transition Suppression [0256] In some examples of the invention, an immunoglobulin or antibody is capable of suppressing signal transduction by IL-3.

[0257] Various assays are known in the art for assessing the ability of an immunoglobulin to withstand receptor signaling from a ligand.

[0258] In one example, an immunoglobulin or antibody reduces or prevents binding of IL-3 to the 3Rα chain and / or heterodimer of the IL-3Rα chain and IL-3Rβ chain. These assays can be performed as a competitive binding assay using labeled IL-3 and / or labeled immunoglobulin. For example, an IL-3Rα-labeled fusion protein or its detectable amount of bound extracellular region with the Fc region of an antibody or IL-3R-expressing, immobilized and labeled IL-3 cell, is then contacted with an immobilized receptor or cell in presence or absence of test immunoglobulin or antibody and associated label. A decrease in label in the presence of an antibody or immunoglobulin compared to a non-protein situation indicates that the immunoglobulin or antibody reduces or prevents binding of IL-3 to IL-3R. By testing multiple immunoglobulin or antibody concentrations, the IC50 is determined, i.e. protein concentration that reduces the amount of IL-3 that binds to IL-3R, or the EC50 can be determined, i.e., a protein concentration that achieves 50% of the maximum inhibition of IL-3 binding to IL-3R, achieved by an immunoglobulin or antibody.

[0259] In another example, an immunoglobulin or antibody reduces or prevents IL-3-mediated histamine release from basophils. For example, low density leukocytes containing basophils are incubated with IgE, IL-3 and various concentrations of immunoglobulin or antibody. Control cells do not contain immunoglobulin (positive control) or IL-3 (negative control). The level of histamine released is then determined using standard techniques, e.g. RIA. An immunoglobulin or antibody that reduces the level of histamine release to a level less than the positive control is considered to abolish IL-3 signaling. In one example, the level of reduction correlates with the concentration of immunoglobulin or antibody. An exemplary method for assessing IL-3 mediated histamine release is described, for example, in Lopez et al., J. Cell. Physiol., 145: 69, 1990.

[0260] In a further example, immunoglobulin or prevents overlapping antibody reduces or IL-3 mediated proliferation of the TF-1 leukemia cell line. For example, TF-1 cells are cultured without IL-3 or GMCSF for a time sufficient to stop cell proliferation (e.g. 24-48 hours). Cells are then cultured in the presence of IL-3 and various concentrations of immunoglobulin or antibody. Control cells are not contacted with immunoglobulin or antibody (positive control) or IL-3 (negative control). Then, using a standard technique, e.g. embedding³H-thymidine, cell proliferation is assessed. An immunoglobulin or antibody that reduces or prevents cell proliferation in the presence of IL-3 to a level lower than in the positive control is considered to suppress IL-3 signaling.

[0261] Another assay for assessing IL-3 signal transduction suppression involves determining whether or not immunoglobulin or antibody reduces or prevents IL-3 dependent effects on endothelial cells. For example, human umbilical vein endothelial cells (HUVEC) are cultured in the presence of IL-3 (optionally, with IFN-γ) and various concentrations of immunoglobulin or antibody. The amount of IL-6 secretion is then assessed, e.g. using an immunosorbent enzyme assay (ELISA). Control cultures do not contain immunoglobulin or antibody (positive control) or IL-3 (negative control). An immunoglobulin or antibody that reduces or prevents the production of IL-6 in the presence of IL-3 to a level lower than in the positive control is considered to abolish IL-3 signaling.

[0262] Other methods for assessing IL-3 signaling suppression are included in this disclosure.

Determination of effector functions [0263] Methods for assessing ADCC activity are known in the art.

[0264] In one example, the level of ADCC activity is assessed using a release assay ⁵¹Cr, Europium release assay or release assay ^{* 35}S. In each of these assays, IL-3Ra expressing cells are cultured with one or more of said compounds for a period of time and under conditions sufficient for cell uptake of the compound. In the case of a release mark³⁵S, cells expressing IL-3Ra can be cultured with labeled ³⁵S with methionine and / or cysteine ​​for a sufficient time for the labeled amino acid to be included in the newly synthesized proteins. The cells are then cultured in the presence or absence of immunoglobulin or antibody and in the presence of immune effector cells, e.g. peripheral blood mononuclear cells (PBMCs) and / or NK cells. Then the amount is detected⁵¹Cr, europium and / or ³⁵S in cell culture medium, and an increase in the presence of immunoglobulin or antibody compared to the absence of immunoglobulin indicates that the immunoglobulin performs effector function. Exemplary publications disclosing assays for determining the level of immunoglobulin induced ADCC include Hellstrom, et al. Natl Acad. Sci. USA 83: 7059-7063, 1986 and Bruggemann, et al., J. Exp. Med. 166: 1351-1361, 1987.

[0265] Other assays to assess the level of immunoglobulin or antibody induced ADCC include ACTI ™ non-radioactive cytotoxicity assay by flow cytometry (CellTechnology, Inc. CA, USA) or non-radioactive cytotoxicity assay CytoTox 96® (Promega, WI, USA).

[0266] Alternatively or additionally, the effector function of an immunoglobulin or antibody is assessed by determining its affinity for one or more Fc? Rs, e.g. as described in US7317091.

[0267] A C1q binding assay can also be performed to confirm that an immunoglobulin or antibody is capable of binding C1q and can induce CDC. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santlubo et al., J. Immunol. Methods 202: 163, 1996.

Determining the number of NK cells [0268] As discussed herein, the immunoglobulins and / or antibodies of the disclosure may affect the number of NK cells in mammals. Methods for determining NK cell numbers in mammals will be apparent to those skilled in the art.

[0269] In one example, after administration of the immunoglobulin or antibody of the disclosure to a mammal (e.g., a non-human mammal such as a non-human primate, e.g. a macaque), a blood (or serum) sample is taken and the number of NK cells determined using sorting fluorescence activated cells (FACS). NK cells can be detected based on CD16 and / or CD56 expression and / or non-expression (or low expression levels) of CD20 and / or CD3. The percentage change in the number of NK cells can be determined by comparing with the number of NK cells in the sample obtained previously (e.g., before administration of the antibody or immunoglobulin.

Determination of affinity [0270] Optionally, the dissociation constant (Kd) or association constant (Ka) or equilibrium constant (KD) of the protein for IL3Ra or its epitope is determined. These immunoglobulin or antibody constants are measured, in one example, by an IL-3Ra binding assay using radioactive or fluorescent labeling. In this assay, the protein is equilibrated with a minimal concentration of labeled IL-3Ra (or its soluble form, e.g. containing the extracellular region of IL-3Ra fused to the Fc region) in the presence of a titrated series of unlabeled IL-3Ra. After washing to remove unbound IL-3Ra, the amount of label is determined.

[0271] Affinity measurements can be performed using standard methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Mouse Curr. Opin. Biotechnol 11:: 54, 2000; Englebienne Analyst. 123: 1599, 1998 ), isometric calorimetric titration (ITC) or other kinetic interaction assays known in the art.

[0272] In one example, constants are measured using surface plasmon 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. Determination of therapeutic efficacy

In vitro assays [0273] Various in vitro assays are available to assess the ability of an immunoglobulin or antibody to treat a disease or condition described herein.

[0274] For example, an immunoglobulin or antibody is evaluated for its ability to kill a cell, e.g. a cancer cell, such as a leukemia cell, using the method described herein.

[0275] In another example, immune cells, e.g. pDCs and / or basophils, or cell populations containing them (e.g. PBMC) are cultured in the presence or absence of an immunoglobulin or antibody and an inducer of those cells that are present in the disease or condition ( e.g. CpG oligonucleotides and / or immune complexes). The effectiveness of the immunoglobulin or antibody is then assessed in the treatment of the disease or condition, e.g. by determining the level of IFNa secreted into the cell culture medium using ELISA. Alternatively or additionally, the level of histamine or IL-4, IL-6 and / or IL-13 secretion is assessed. A decrease in the level of any of these cytokines compared to the absence of an immunoglobulin or antibody (or in the presence of a control immunoglobulin or antibody isotype) indicates that the immunoglobulin or antibody is suitable for treating a disease or condition. Alternatively or additionally, the level of dead cells is assessed. An increase in the frequency of cell death indicates that the immunoglobulin or antibody is suitable for treating a disease or condition.

In vivo assays [0276] In one example, the effectiveness of an immunoglobulin in the treatment of a disease or condition is assessed using an in vivo assay.

[0277] In one example, a tumor xenograft model is used to assess therapeutic efficacy. For example, NOD / SCID mice are irradiated and optionally anti-CD122 antibody is administered to remove NK cells. Mice are administered human leukemia cells (e.g. acute myeloid leukemia cells) and mouse or human bone marrow stem cells. After cell implantation, mice are administered the test immunoglobulin or antibody and the level of leukemia cells in the circulation and / or bone marrow and / or lymph nodes is assessed. Reduction in the number of leukemia cells in the circulation and / or bone marrow and / or lymph nodes in the presence of the antibody or immunoglobulin, compared to the situation of the absence of the antibody or immunoglobulin indicates therapeutic efficacy.

[0278] In another example, an 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 the circulation is assessed. An immunoglobulin or antibody that reduces the number / level of cells of the immune system, e.g. pDCs and / or basophils, compared to that prior to administration and / or to a control mammal to which no immunoglobulin or antibody was administered, are considered effective in the treatment of the disease or condition.

[0279] In another example, a level of a cytokine such as IFNa is detected in the circulation of a mammal, e.g., using the disclosure, a carrier, an example,

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 that has not received the immunoglobulin or antibody is considered effective in treating the disease or condition. Because cytokines such as IFNa are considered to play a role in certain diseases / conditions, e.g. in lupus, an immunoglobulin or antibody that reduces the production of IFNa is considered effective in treating such disease or condition.

Compositions [0280] Accordingly, in compositions or methods of administering an immunoglobulin or anti-IL-3Ra antibody to a mammal, the immunoglobulin or antibody is combined with a pharmaceutically acceptable carrier, diluent and / or excipient as known in the art. Accordingly, one example of this invention provides a pharmaceutical composition comprising an immunoglobulin or antibody of the combined with a pharmaceutically acceptable diluent and / or excipient. In another disclosure, there is provided a kit comprising a pharmaceutically acceptable carrier, diluent and / or excipient, suitable for combining or mixing with an immunoglobulin or antibody prior to administration to a mammal. In this example, the kit may further include instructions for use.

[0281] Generally, by "carrier, diluent or excipient" is meant a solid or liquid filler, binder, diluent, encapsulant, emulsifier, wetting agent, solvent, suspending agent, coating agent or lubricant that can be safely administered to any mammal, e.g. to man. Depending on the particular route of administration, a variety of acceptable carriers, diluents or excipients known in the art can be used, as for example described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991).

[0282] By way of example only, carriers, diluents or excipients may be selected from the group consisting of sugars (e.g. sucrose, maltose, trehalose, glucose), starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, oils, including vegetable oils, synthetic oils and synthetic mono- or diglycerides, lower alcohols, polyols, alginic acid, solutions phosphate buffered lubricants such as sodium or magnesium stearate, isotonic saline solutions, 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 via the gastrointestinal tract. Non-limiting examples of parenteral administration include injection, infusion and the like. For example, administration by injection includes intravenous, intraarterial, intramuscular and subcutaneous administration. Delivery in the form of a depot or slow release formulation which may be delivered, e.g., intradermally, intramuscularly and subcutaneously, is also contemplated.

Combination Therapies [0283] 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.

[0284] In one example, an immunoglobulin or antibody is administered, e.g., one month or two weeks or one week, prior to radiation therapy, e.g., in the treatment of cancers such as hematological cancer such as leukemia.

[0285] In one example, the other compound is a chemotherapeutic compound such as carboplatin, cisplatin, cyclophosphamide, docetaxal, doxorubicin, erlotinib, etoposide, fluorouracil, irinotecan, methotrexate, paclitaxel, topotecan, vincristine or vinblastine. In one example, the chemotherapeutic compound is selected from the group consisting of methotrexate, 1-asparaginase, vincristine, doxorubicin, danorubicin, cytarabine, idarubicin, mitoxantrone, cyclophosphamide, fludarabine, chlorambucil and combinations thereof.

[0286] In one example, another compound is a chemotherapeutic compound used in 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.

[0287] In one example, the other compound is a chemotherapeutic compound used in the treatment of acute lymphoblastic leukemia, such as a compound selected from the group consisting of methotrexate, 1-asparaginase, vincristine, doxorubicin, danorubicin, and combinations thereof.

[0288] In another example, another compound is a chemotherapeutic compound such as azacitidine.

[0289] In one example, the other compound is a biological compound useful in the treatment of cancer, e.g., rituximab, trastuzumab, bevacizumab, alemtuzumab, panitumumab, or cetuximab.

[0290] In one example, the other compound is an anti-inflammatory compound. Alternatively or additionally, another compound is an immunosuppressive compound. Alternatively or additionally, another compound is a corticosteroid such as prednisone and / or prednisolone. Alternatively or additionally, another compound is an anti-malarial compound, such as hydroxychloroquine or chloroquine. Alternatively or additionally, another compound is methotrexate. Alternatively or additionally, another compound is azathioprine. Alternatively or additionally, another compound is cyclophosphamide. Alternatively or additionally, the other compound is mycophenolate mofetil. Alternatively or additionally, another compound is an anti-CD20 antibody (e.g., rituximab or ofatumumab). Alternatively or additionally, another compound is an anti-CD22 antibody (e.g., epratuzumab). Alternatively or additionally, another compound is an anti-TNF antibody (e.g., infliximab or adalimumab or golimumab). Alternatively or additionally, another compound is a CTLA-4 antagonist (e.g., abatacept, CTLA4-Ig). Alternatively or additionally, another compound is an anti-IL-6 antibody. Alternatively or additionally, another compound is a BLys antagonist, such as for example an anti-BLys antibody (e.g. belimumab).

Dosage and Duration of Administration [0291] To prevent or treat a disease or condition or its recurrence, appropriate doses of the active agent (i.e., immunoglobulin or antibody of the disclosure) will depend on the type of disease being treated, the severity and course of the disease, and whether immunoglobulin or antibody is administered for preventive or curative purposes, prior therapy, patient's clinical history and response to immunoglobulin, and recognition of the attending physician. The specific dosage regimen, i.e., dose, time and repeat, will depend on the particular individual and the medical history of that individual in the judgment of the physician. Typically, your doctor will give you immunoglobulin until you get the dose that you want.

[0292] The methods of the invention are useful for treating, alleviating or preventing the symptoms of a disease or condition in a mammal, or improving prognosis in a mammal. The methods of the invention are also useful for retarding or preventing lupus in a patient who is at risk of developing lupus or its recurrence.

[0293] For administration of the immunoglobulin or antibodies described herein, normal dose amounts may range from about 10 ng / kg to about 100 mg / kg of the subject's body weight or more per day. Exemplary doses and ranges are provided in the description. For repeated dosages over several days or longer, depending on the severity of the disease or disorder being treated, treatment may be maintained until the desired symptom relief is obtained.

[0294] In some examples, the immunoglobulin or antibody is administered at an initial (or shock) dose of from about 1 mg / kg to about 30 mg / kg, such as from about 1 mg / kg to about 10 mg / kg, or about 2 mg / kg or about 3 mg / kg or 4 mg / kg or 5 mg / kg. The immunoglobulin or antibody may then be administered in a maintenance dose of from about 0.0001 mg / kg to about 1 mg / kg, such as from about 0.0005 mg / kg to about 1 mg / kg, for example from about 0.001 mg / kg to about 1 mg / kg, such as about 0.01 mg / kg to about 1 mg / kg, e.g. from about 0.01 mg / kg to about 0.1 mg / kg, e.g. 0.02 mg / kg or 0.03 mg / kg or 0.04 mg / kg or 0.05 mg / kg. Maintenance doses can be given every 7-30 days, such as every 10-15 days, e.g. every 10 or 11 or 12 or 13 or 14 or 15 days.

<td>[0295] W</td><td colspan="2">some</td><td>examples,</td><td colspan="2">immunoglobulin</td><td>or</td>

<td>antibody</td><td colspan="2">gives</td><td>in a dose</td><td>of</td><td>from</td><td>about</td>

<td>0.0001 mg / kg</td><td>down</td><td>about</td><td>50 mg / kg, as</td><td>e.g</td><td>from</td><td>about</td>

<td>0.0005 mg / kg</td><td>down</td><td colspan="2">about 50 mg / kg, on</td><td>example,</td><td>from</td><td>about</td>

<td>0.001 mg / kg</td><td>down</td><td>about</td><td>40 mg / kg, on</td><td>example,</td><td>from</td><td>about</td>

<td>0.005 mg / kg</td><td>down</td><td>about</td><td>30 mg / kg, as</td><td>e.g</td><td>from</td><td>about</td>

0.01 mg / kg to about 20 mg / kg. For example, immunoglobulin is administered at a dose of from about 0.01 mg / kg to about 10 mg / kg, such as from about 0.01 mg / kg to about 1 mg / kg, such as about 0.02 mg / kg or 0.03 mg / kg or 0.04 mg / kg or 0.05 mg / kg or 0.06 mg / kg or 0.07 mg / kg or 0.08 mg / kg or 0.09 mg / kg or 0.1 mg / kg or 0.2 mg / kg or 0.3 mg / kg or 0.4 mg / kg or 0.5 mg / kg or 0.6 mg / kg or 0.7 mg / kg or 0.8 mg / kg or 0.9 mg / kg (e.g. without a higher loading dose). In some examples, multiple doses are administered, e.g., every 7-30 days, such as, every 10-22 days, e.g., every 10-15 days, e.g., every 10 or 11 or 12, or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days. For example, an immunoglobulin or antibody is administered every 7 days or every 14 days or every 21 days.

[0296] In some examples, the immunoglobulin or antibody is administered at a dose of from about 1 mg / kg to about 30 mg / kg, such as from about 1 mg / kg to about 10 mg / kg, or about 2 mg / kg or about 3 mg / kg or 4 mg / kg or 5 mg / kg, or such as from about 10 mg / kg to 30 mg / kg, such as for example about 10 mg / kg or 15 mg / kg or 20 mg / kg or 25 mg / kg (e.g. without a lower maintenance dose). In some examples, multiple doses are administered, e.g. every 10-70 days, such as every 14-70 days, such as every 14-60 days, e.g., every 14-50 days, such as every 14-40 days, or every 14-30 days. For example, dosages are given every 14 or 21 or 25

<td>or 28</td><td>or</td><td> 35</td><td>or</td><td> 40</td><td>or 42</td><td>or</td><td> 49</td><td>or</td><td>50 or</td><td> 55</td><td>or 57 or</td>

<td>63 or</td><td> 70</td><td>days</td><td>. On</td><td colspan="2">example,</td><td colspan="6">immunoglobulin or antibody</td>

<td>gives</td><td>himself</td><td>What</td><td> 21</td><td>days</td><td>or what</td><td> 28</td><td>days</td><td>or</td><td>every 35</td><td>days</td><td>or every 42</td>

<td>days or</td><td>What</td><td> 49</td><td>days</td><td>or</td><td>every 56</td><td>days.</td><td></td><td></td><td></td><td></td><td></td>

[0297] In some examples, the immunoglobulin or antibody causes or is associated with a decrease in the number of NK cells in a mammal after administration, e.g., within about 6 hours after administration. In some examples, a subsequent dose of immunoglobulin or antibody is administered when the number of NK cells in the mammal returns to 20% or 10% or 5% or 1% of the number of NK cells in the mammal prior to administration. In some examples, a subsequent dose of immunoglobulin or antibody is administered when the number of NK cells in the 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 in a mammal can be determined to determine when the next dose of immunoglobulin or antibody can be administered. Alternatively, the time of administration of the next dose of immunoglobulin or antibody is determined by prior analysis of the population or model organism (e.g. a non-human primate such as a macaque). For example, a subsequent dose of 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 after the previous dose.

[0298] In some examples, at the start of treatment, the mammal is administered immunoglobulin or antibody for no more than 7 consecutive days or 6 consecutive days, or 5 consecutive days, or 4 consecutive days.

[0299] In the case of a mammal that does not respond well to treatment, multiple doses per week may be administered. Alternatively, or in addition, increasing doses may be administered.

[0300] In another example, in mammals experiencing adverse reactions, the initial (or shock) dose may be divided into several days per week or multiple consecutive days.

[0301] Dosages of a particular immunoglobulin or antibody can be determined empirically in mammals that have received one or more doses of immunoglobulin. To assess the effectiveness of an immunoglobulin, clinical signs of a disease or condition may be monitored.

[0302] Administration of the immunoglobulin according to the methods of the invention may be continuous or discontinued depending, for example, on the physiological conditions of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art.

The administration of the immunoglobulin or antibody may be substantially continuous over a pre-selected period of time or it may be in a series of divided doses, e.g., either during or after the disease develops.

[0303] This disclosure includes the following non-limiting examples.

Non-limiting examples

Example 1: Humanized antagonist antibodies were prepared [0304] A humanized antibody was prepared that binds specifically to the human IL-3Rα chain that acts as IL-3 activities. Humanized antibody by grafting CDR sequences of antagonist mouse antibody (7G3) into human germline variable region sequences selected based on the canonical structure of the CDRs donor and recipient according to the procedure of Tan et al. (J Immunol. 169, 1119-1125,

2002); sometimes referred to as "superhumanization." This work was done using the scFv antibody. In this approach, the donor antibody CDR residues are then compared to those of the recipient germline variable sequences, and the sequence showing the highest correlation of CDR residues is selected as the recipient sequence. However, in this case the heavy chain sequence with the lower level of CDR correlation was chosen as the recipient sequence. The resulting humanized antibody contains, as a result of the humanization process, a completely human variable region sequence, however, the affinity for IL-3Rα is reduced compared to the parent murine antibody.

[0305] Affinity optimization was utilized using a ribosome-based mutagenesis-based presentation process (Kopsidas et al., BMC Biotechnol. 7, 18, 2007), carried out using the scFv antibody to increase the binding affinity of the humanized antibody. An affinity optimized scFv was generated which, when converted to ty [IgG1, showed a slightly increased binding affinity for IL-3Ra relative to the parent murine monoclonal antibody. Surprisingly, mutations in the VH and VL framework region as well as in the light chain CDR1 appeared as a result of the affinity optimization process. Thus, the CDR sequence set of the humanized affinity optimized antibody differs from that of the parent murine monoclonal antibody.

The affinity optimized antibody is referred to herein as CSL362.

[0306] Engineered Fc derivatives of humanized, affinity-optimized antibody, comprising antibody light chain and heavy chain variable regions and hybrid IgG1 / IgG2 constant domain hybrid with the three amino acid substitutions S239D / A330L / I332E (referred to herein as CSL362X2) or with two amino acid substitutions S239D / I332E (referred to in this CSL362X1) were produced by expressing the vector in CHO-S cells; positions of identified mutations were marked according to the EU numbering system.

description as appropriate [0307] A non-fucosylated version of a humanized, affinity-optimized antibody with a human IgG1 constant domain was produced by expressing the corresponding vector in FUT8-knocked CHO cells from Biowa (Potelligent® cells) (referred to herein as CSL362B).

Example 2: Determining binding affinity

Kinetics of full length monoclonal antibody:

[0308] To analyze full length antibodies, surface plasmon resonance (Biacore) assays were performed in a capture format in which a chemically immobilized anti-human or anti-Fc specific antibody (adsorbed goat anti-human antibody and mouse anti-human antibody IgG (gamma) (Invitrogen, cat. No. H10500) or specific anti-mouse Fc antibody (Jackson Immuno Research Labs inc. Cat. No. 515-005-071) was chemically immobilized on the surface of the CM-5 sensor using standard amine chemical coupling) and used for capture from the monoclonal antibody solution. Then, soluble human IL-3Ra at various concentrations was injected over the captured antibody. Responses were subtracted from those of the reference flow cell in which the antibody was not captured, but was also treated identically. These responses with the reference answer subtracted were then subtracted from the empty injection responses.

[0309] Finally corrected responses were fitted using non-linear regression to the model describing 1: 1 kinetics, taking into account mass transport constraints. The Rmax value was adjusted locally to account for slight variations in the level of captured antibody. Association rate (ka), dissociation rate (kd) and dissociation equilibrium constant (KD) were determined.

[0310] Antibodies were captured at 0.3 pg / ml for 180 seconds.

[0311] Soluble IL-3Ra was injected for 10 minutes and dissociation monitored over 30 minutes.

[0312] Soluble IL-3Ra was injected at 0, 0.62, 1.25, 2.5, 5, 10, 20 and 40 nM, with 2.5 and 5 nM in duplicate.

[0313] Regeneration was performed after each cycle using a 90 second injection of 100 mM H3PO4.

[0314] Significance was carried out at 25 ° C.

ScFv kinetics:

[0315] To analyze scFvs, soluble human IL-3Ra was chemically immobilized on the surface of the CM-5 sensor using standard amine chemical coupling, and scFvs were injected at various concentrations. Responses were subtracted from those derived from reference flow cells in which there was no immobilized IL-3Ra, but were also treated identically. These responses with the reference answer subtracted were then subtracted from the empty injection responses.

[0316] The finally corrected responses were fitted using non-linear regression to the model describing 1: 1 kinetics, taking into account mass transport restrictions. The Rmax value was adjusted globally and the association rate (ka), dissociation rate (kd) and dissociation equilibrium constant (KD) were determined.

[0317] scFvs were injected for 10 minutes and dissociation monitored over 20 minutes.

[0318] scFvs were injected at 0, 0.62, 1.25, 2.5, 5, 10, 20 and 40 nM, with 10 nM in duplicate [0319] Regeneration was performed after each cycle using 30 seconds 100 mM H3PO4 injection, 1 M NaCl and 15 second injection of 50 mM NaOH.

[0320] The assay was performed at 25 ° C.

[0321] Antibody and scFv affinities are summarized below in

Table 2:

Table 2: Dissociation affinity constants for antibodies and scFvs

Mouse mAb (7G3) parental antibody chimeric mouse mAb (human IgG1) superhumanized mAb (IgG1) scFv superhumanized scFv mAb CSL362

CSL362 (IgG1)

CSL362B

KD (M) ~ 9.2x10

1,0x10^-

1x10^- ~ 1,4x10^- ~ 2,2x10^- ~ 7.8x10 ~ 4.3x10

Example 3: NK cell levels after administration of humanized or chimeric antibodies [0322] Monkeys not previously immunized (non-human primate; NHPs) were administered a single dose of CSL362B or CSL362X1 by intravenous infusion. In a separate study, non-immunized monkeys were administered repeated doses (weekly x 4) of the chimeric antibody containing the variable regions of the mouse antibody used to produce the humanized antibody (7G3) and the human IgG1 constant region). Peripheral blood cells were harvested at various time points and analyzed for NHP NK cells by flow cytometry.

[0323] As shown in Fig. 1A, administration of CSL362X1 resulted in an initial reduction in NK cell number, e.g., approximately 6 hours after administration. At doses of 0.01 mg / kg and 0.1 mg / kg, this level exceeded the level observed prior to administration for approximately 8 days after administration and remained elevated to at least 22 or 29 days after administration. An increase in the number of NK cells was also observed at a dose of 1 mg / kg.

[0324] Contrary to the results described in the previous paragraph, administration of CSL362B reduced the number of NK cells, however the number of NK cells did not then exceed their number before administration (Fig. 1B).

[0325] Repeated administration of the chimeric antibody did not significantly change the number of NK cells detected in the circulation (Fig. 1C).

Example 4: Enhanced ADCC of humanized antibodies in the presence of NK cells [0326] Human PBMC or NK cells were isolated and incubated with TF-1 cells in the presence of different concentrations of CSL362X1. Effector cells (E; PBMC) and target cells (T; TF-1 cells) were combined to obtain a 50: 1 ratio (E: T ratio) or when the purified NK cells were used as effector cells, the ratio was 20: 1. Cell lysis was measured using a non-radioactive LDH cytotoxicity kit CytoTox 96 (Promega).

Specific lysis was determined according to the following formula: Specific lysis = [sample lysis - spontaneous lysis / [maximum lysis - spontaneous lysis] x 100% [0327] Maximum lysis was assessed by adding Extran ™ to a final concentration of 0.75% (v / v) .). Spontaneous lysis was that which occurred in wells with cells alone (without antibody).

[0328] As shown in Fig. 2A, lysis of TF-1 cells occurred in the presence of PBMCs and in the presence of NK cells, however it was significantly reduced in PBMCs from which NK cells were removed.

[0329] In a separate experiment, leukemia cells from two different AML patients were used as target cells. A single antibody concentration (10 μg / ml) was used in this assay and purified NK cells were added to form different E: T ratios. FIG. 2B shows that the higher the number of NK cells relative to the target cells (peripheral blood blasts from two different AML patients) (i.e., the E: T ratio), the higher the specific lysis of the target cells by the humanized antibody.

SEQUENCE LIST [0330] &lt;110&gt; CSL Limited Panousis, Con &lt;120&gt; Humanized antibodies directed against the interleukin 3 receptor alpha chain &lt;130&gt; 510921 &lt;150&gt; USSN 61/374489 &lt;151&gt; 2010-08-17 &lt;150&gt; PCT / AU2011 / 000155 &lt;151&gt; 2011-02-17 &lt;160&gt; 23

<td> &lt;170&gt;</td><td>PatentIn version</td>

<td> &lt;210&gt;</td><td> 1</td>

<td> &lt;211&gt;</td><td> 359</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Homo sapiens</td>

<td> &lt;400&gt;</td><td> 1</td>

Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met Lys Ala Lys 15 10 15

Ala Gin Gin Leu Thr Trp Asp Leu Asn Arg Asn Val Thr Asp Ile Glu 20 25 30

Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn Asn Ser Tyr 35 40 45

Cys Gin Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn Tyr Thr Val 50 55 60

Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe Pro Glu Asn 65 70 75 80

Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys Trp Ile His 85 90 95

Asp Val Asp Phe Leu Cheese Cys Cheese Trp Ala Val Gly Pro Gly Ala Pro 100 105 110

Ala Asp Val Gin Tyr Asp Leu Tyr Leu Asn Val Ala Asn Arg Arg Gin 115 120 125

Gin Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gin Gly Thr Arg Ile 130 135 140

Gly Cys Arg Phe Asp Asp Ile Cheese Arg Leu Cheese Cheese Gly Cheese Gin Cheese

100

145

150

155

160

Cheese His Ile Leu Val Arg Gly Arg Cheese Ala Ala Phe Gly Ile Pro Cys 165 170 175

Thr Asp Lys Phe Val Val Phe Ser Gin Ile Glu Ile Leu Thr Pro Pro 180 185 190

Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met His Trp Lys 195 200 205

Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu Gin Ile Gin 210 215 220

Lys Arg Met Gin Pro Val Ile Thr Glu Gin Val Arg Asp Arg Thr Ser 225 230 235 240

Phe Gin Leu Leu Asn Pro Gly Thr Tyr Thr Val Gin Ile Arg Ala Arg 245 250 255

Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro Gin Arg Phe 260 265 270

Glu Cys Asp Gin Glu Glu Gly Ala Asn Thr Arg Ala Trp Arg Thr Ser 275 280 285

Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Val Cys Val Phe Val 290 295 300

Ile Cys Arg Arg Tyr Leu Val Met Gin Arg Leu Phe Pro Arg Ile Pro 305 310 315 320

His Met Lys Asp Pro Ile Gly Asp Ser Phe Gin Asn Asp Lys Leu Val 325 330 335

Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu Val Thr Glu 340, 345 350

Val Gin Val Val Gin Lys Thr 355 &lt;210&gt; 2 &lt;211&gt; 17

101

<td> &lt;212&gt;</td><td colspan="3">PRT</td>

<td> &lt;213&gt;</td><td>artificial</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>sequence</td><td>The amino acid</td><td>Humanized LCDR1</td>

<td colspan="2">CSL362 antibodies</td><td>directed</td><td>against the IL- chain</td>

<td>3Ralfa</td><td colspan="2">and its modified forms</td><td></td>

<td> &lt;400&gt;</td><td> 2</td><td></td><td></td>

Glu Cheese Cheese Gin Cheese Leu Leu Asn Cheese Gly Asn Gin Lys Asn Tyr Leu 15 10 15

Thr

<td> &lt;210&gt;</td><td colspan="3"> 3</td>

<td> &lt;211&gt;</td><td> 7</td><td></td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td><td></td>

<td> &lt;213&gt;</td><td>artificial</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>sequence</td><td>The amino acid</td><td>LCDR2</td>

<td colspan="2">CSL362 antibodies</td><td>directed</td><td>against</td>

humanized IL3Ralf chain and its modified forms &lt;400&gt; 3

Trp Ala Ser Thr Arg Glu Ser 1 5 &lt;210&gt; 4 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt;

amino acid sequence

Humanized LCDR3

102 CSL362 antibody directed against the IL3Ralpha chain and its modified forms &lt;400&gt; 4

Gin Asn Asp Tyr Ser Tyr Pro Tyr Thr 1 5

<td> &lt;210&gt;</td><td colspan="3"> 5</td>

<td> &lt;211&gt;</td><td> 5</td><td></td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td><td></td>

<td> &lt;213&gt;</td><td>artificial</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>sequence</td><td>The amino acid</td><td>HCDR1</td>

<td colspan="2">CSL362 antibodies</td><td>directed</td><td>against</td>

<td>3Ralfa</td><td colspan="2">and its modified forms</td><td></td>

<td> &lt;400&gt;</td><td> 5</td><td></td><td></td>

Asp Tyr Tyr Met Lys 1 5 humanized IL-chain

<td> &lt;210&gt;</td><td colspan="3"> 6</td>

<td> &lt;211&gt;</td><td> 17</td><td></td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td><td></td>

<td> &lt;213&gt;</td><td>artificial</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>sequence</td><td>The amino acid</td><td>HCDR2</td>

<td colspan="2">CSL362 antibodies</td><td>directed</td><td>against</td>

3Ralpha and its modified forms of the humanized IL &lt;400&gt; chain 6

Asp Ile Ile Pro Cheese Asn Gly Ala Thr Phe Tyr Asn Gin Lys Phe Lys 15 10 15

Gly

103 &lt;210&gt; 7 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; amino acid sequence of HCDR3 of a humanized antibody directed against the IL-3Ralpha chain &lt;400&gt; 7

Cheese His Leu Leu Arg Ala Cheese Trp Phe Ala Tyr 15 10 &lt;210&gt; 8 &lt;211&gt; 114 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; amino acid sequence of the variable region in the light chain of the humanized CSL362 antibody directed against the IL-3Ralpha chain and its modified forms &lt;400&gt; 8

<td>Asp 1</td><td>How much</td><td>val</td><td colspan="3">Met Thr Gin 5</td><td>Cheese</td><td colspan="5">Pro Asp Cheese Leu Ala 10</td><td>val</td><td colspan="3">Leu Gly cheese 15</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>How much</td><td>own</td><td>Cys</td><td>Glu</td><td>Cheese</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>own</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>own</td><td>Gin</td><td>lys</td><td>own</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Pro</td><td>Pro</td><td>lys</td><td>Pro</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Trp</td><td>ala</td><td>Cheese</td><td>Thr</td><td>Arg</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>val</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

104

Ile Ser Cheese Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile 100 105 110

Lys Arg &lt;210&gt; 9 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; amino acid sequence of the variable region in the heavy chain of the humanized CSL362 antibody directed against the IL-3Ralpha chain and its modified forms &lt;400&gt; 9

<td>Glu</td><td>val</td><td>Gin</td><td>Leu</td><td>val</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>ala</td><td>Glu</td><td>val</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly</td><td>Glu</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>How much</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Tyr</td><td>Cheese</td><td>phe</td><td>Thr</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Underworld</td><td>lys</td><td>Trp</td><td>ala</td><td>Arg</td><td>Gin</td><td>Underworld</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Trp</td><td>Underworld</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gly</td><td>Asp</td><td>How much</td><td>How much</td><td>Pro</td><td>Cheese</td><td>own</td><td>Gly</td><td>ala</td><td>Thr</td><td>phe</td><td>Tyr</td><td>own</td><td>Gin</td><td>lys</td><td>phe</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>lys</td><td>Gly</td><td>Gin</td><td>val</td><td>Thr</td><td>How much</td><td>Cheese</td><td>ala</td><td>Asp</td><td>lys</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Thr</td><td>Thr</td><td>Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>Gin</td><td>Trp</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>lys</td><td>ala</td><td>Cheese</td><td>Asp</td><td>Thr</td><td>ala</td><td>Underworld</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>ala</td><td>Arg</td><td>Cheese</td><td>His</td><td>Leu</td><td>Leu</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Trp</td><td>phe</td><td>ala</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Underworld</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

105 &lt;210&gt; 10 &lt;211&gt; 450 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; amino acid sequence of the humanized CSL362 antibody heavy chain directed against IL-3Ralpha and CSL362B &lt;400&gt; 10

Glu Val Gin Leu Val Gin Cheese Gly Ala Glu Val Lys Lys Pro Gly Glu

5 10 15

Cheese Leu Lys Ile Cheese Cys Lys Gly Cheese Gly Tyr Cheese Phe Thr Asp Tyr

25 30

Tyr Met Lys Trp Ala Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45

Gly Asp Ile Ile Pro Cheese Asn Gly Ala Thr Phe Tyr Asn Gin Lys Phe 50 55 60

Lys Gly Gin Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Thr Tyr 65 70 75 80

Leu Gin Trp Cheese Cheese Leu Lys Ala Cheese Asp Thr Ala Met Tyr Tyr Cys 85 90 95

Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gin 100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125

Phe Pro Leu Ala Pro Cheese Cheese Lys Cheese Thr Cheese Gly Gly Thr Ala Ala 130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

106

<img file="PL2606069T3_D0001.tif" />

107

<img file="PL2606069T3_D0002.tif" />

&lt;210&gt; 11 &lt;211&gt; 450 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; amino acid sequence of the humanized CSL362 antibody heavy chain directed against the IL-3Ralph X1 chain &lt;400&gt; 11

Glu Val Gin Leu Val Gin Cheese Gly Ala Glu Val Lys Lys Pro Gly Glu 15 10 15

Cheese Leu Lys Ile Cheese Cys Lys Gly Cheese Gly Tyr Cheese Phe Thr Asp Tyr 20 25 30

Tyr Met Lys Trp Ala Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45

Gly Asp Ile Ile Pro Cheese Asn Gly Ala Thr Phe Tyr Asn Gin Lys Phe 50 55 60

Lys Gly Gin Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Thr Tyr 65 70 75 80

Leu Gin Trp Cheese Cheese Leu Lys Ala Cheese Asp Thr Ala Met Tyr Tyr Cys 85 90 95

Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gin 100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125

108

Phe Pro Leu Ala Pro Cheese Cheese Lys Cheese Thr Cheese Gly Gly Thr Ala Ala 130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro val Thr Val Ser 145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175

Leu Gin Cheese Cheese Gly Leu Tyr Cheese Leu Cheese Cheese Val Val Thr Val Pro 180 185 190

Cheese Cheese Cheese Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270

Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg 290 295 300

Val Val Ser Val Leu Thr Val Val His Gin Asp Trp Leu Asn Gly Lys 305 310 315 320

Glu Tyr Lys Cys Lys Val Cheese Asn Lys Ala Leu Pro Ala Pro Glu Glu 325 330 335

Lys Thr Ile Ser Lys Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr 340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu 355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Cheese Asp Ile Ala Val Glu Trp 370 375 380

109

<img file="PL2606069T3_D0003.tif" />

&lt;210&gt; 12 &lt;211&gt; 450 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; amino acid sequence of the humanized CSL362 antibody heavy chain directed against the IL-3Ralph X2 chain &lt;400&gt; 12

110

<td>Glu 1</td><td>val</td><td>Gin</td><td>Leu</td><td>val 5</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>ala</td><td>Glu 10</td><td>val</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly 15</td><td>Glu</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>how much twenty</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Gly</td><td>Cheese 25</td><td>Gly</td><td>Tyr</td><td>Cheese</td><td>phe</td><td>Thr thirty</td><td>Asp</td><td>Tyr</td>

<td>Tyr</td><td>Underworld</td><td>lys 35</td><td>Trp</td><td>ala</td><td>Arg</td><td>Gin</td><td>Underworld 40</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu 45</td><td>Glu</td><td>Trp</td><td>Underworld</td>

<td>Gly</td><td>Asp 50</td><td>How much</td><td>How much</td><td>Pro</td><td>Cheese</td><td>own 55</td><td>Gly</td><td>ala</td><td>Thr</td><td>phe</td><td>Tyr 60</td><td>own</td><td>Gin</td><td>lys</td><td>phe</td>

<td>lys 65</td><td>Gly</td><td>Gin</td><td>val</td><td>Thr</td><td>How much 70</td><td>Cheese</td><td>ala</td><td>Asp</td><td>lys</td><td>Cheese 75</td><td>How much</td><td>Cheese</td><td>Thr</td><td>Thr</td><td>Tyr 80</td>

<td>Leu</td><td>Gin</td><td>Trp</td><td>Cheese</td><td>Cheese 85</td><td>Leu</td><td>lys</td><td>ala</td><td>Cheese</td><td>Asp 90</td><td>Thr</td><td>ala</td><td>Underworld</td><td>Tyr</td><td>Tyr 95</td><td>Cys</td>

<td>ala</td><td>Arg</td><td>Cheese</td><td>His 100</td><td>Leu</td><td>Leu</td><td>Arg</td><td>ala</td><td>Cheese 105</td><td>Trp</td><td>phe</td><td>ala</td><td>Tyr</td><td>Trp 110</td><td>Gly</td><td>Gin</td>

<td>Gly</td><td>Thr</td><td>Underworld 115</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese 120</td><td>ala</td><td>Cheese</td><td>Thr</td><td>lys</td><td>Gly 125</td><td>Pro</td><td>Cheese</td><td>val</td>

<td>phe</td><td>Pro 130</td><td>Leu</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Cheese 135</td><td>lys</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Gly 140</td><td>Gly</td><td>Thr</td><td>ala</td><td>ala</td>

<td>Leu 145</td><td>Gly</td><td>Cys</td><td>Leu</td><td>val</td><td>lys 150</td><td>Asp</td><td>Tyr</td><td>phe</td><td>Pro</td><td>Glu 155</td><td>Pro</td><td>val</td><td>Thr</td><td>val</td><td>Cheese 160</td>

<td>Trp</td><td>own</td><td>Cheese</td><td>Gly</td><td>ala 165</td><td>Leu</td><td>Thr</td><td>Cheese</td><td>Gly</td><td>val 170</td><td>His</td><td>Thr</td><td>phe</td><td>Pro</td><td>ala 175</td><td>val</td>

<td>Leu</td><td>Gin</td><td>Cheese</td><td>Cheese 180</td><td>Gly</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Leu 185</td><td>Cheese</td><td>Cheese</td><td>val</td><td>val</td><td>Thr 190</td><td>val</td><td>Pro</td>

<td>Cheese</td><td>Cheese</td><td>Cheese 195</td><td>Leu</td><td>Gly</td><td>Thr</td><td>Gin</td><td>Thr 200</td><td>Tyr</td><td>How much</td><td>Cys</td><td>own</td><td>val 205</td><td>own</td><td>His</td><td>lys</td>

<td>Pro</td><td>Cheese 210</td><td>own</td><td>Thr</td><td>lys</td><td>val</td><td>Asp 215</td><td>lys</td><td>lys</td><td>val</td><td>Glu</td><td>Pro 220</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Asp</td>

<td>lys 225</td><td>Thr</td><td>His</td><td>Thr</td><td>Cys</td><td>Pro 230</td><td>Pro</td><td>Cys</td><td>Pro</td><td>ala</td><td>Pro 235</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Gly 240</td>

<td>Pro</td><td>Asp</td><td>val</td><td>phe</td><td>Leu</td><td>phe</td><td>Pro</td><td>Pro</td><td>lys</td><td>Pro</td><td>lys</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Underworld</td><td>How much</td>

245 250 255

111

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270

Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg 290 295 300

Val Val Ser Val Leu Thr Val Val His Gin Asp Trp Leu Asn Gly Lys 305 310 315 320

Glu Tyr Lys Cys Lys Val Cheese Asn Lys Ala Leu Pro Leu Pro Glu Glu 325 330 335

Lys Thr Ile Ser Lys Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr 340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu 355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Cheese Asp Ile Ala Val Glu Trp 370 375 380

Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met 385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415

Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430

Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445

Gly Lys 450 &lt;210&gt; 13 &lt;211&gt; 220 &lt;212&gt; PRT &lt;213&gt; Artificial

112 &lt;220&gt;

&lt;223&gt; amino acid sequence of the humanized CSL362 antibody light chain directed against the IL3Ralpha chain and its modified forms &lt;400&gt; 13

Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 15 10 15

Glu Arg Ala Thr Ile Asn Cys Glu Cheese Cheese Gin Cheese Leu Leu Asn Cheese 20 25 30

Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

Ile Ser Cheese Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Asn 85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile 100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125

Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140

113

<td>phe</td><td>Tyr</td><td>Pro</td><td>Arg</td><td>Glu</td><td>ala</td><td>lys</td><td>val</td><td>Gin</td><td>Trp</td><td>lys</td><td>val</td><td>Asp</td><td>own</td><td>ala</td><td>Leu</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Gin</td><td>Cheese</td><td>Gly</td><td>own</td><td>Cheese</td><td>Gin</td><td>Glu</td><td>Cheese</td><td>val</td><td>Thr</td><td>Glu</td><td>Gin</td><td>Asp</td><td>Cheese</td><td>lys</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Cheese</td><td>Thr</td><td>Tyr</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>lys</td><td>ala</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Glu</td><td>lys</td><td>His</td><td>lys</td><td>val</td><td>Tyr</td><td>ala</td><td>Cys</td><td>Glu</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Cheese</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Cheese</td><td>Pro</td><td>val</td><td>Thr</td><td>lys</td><td>Cheese</td><td>phe</td><td>own</td><td>Arg</td><td>Gly</td><td>Glu</td><td>Cys</td><td></td><td></td><td></td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 14 &lt;211&gt; 51 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for LCDR1 of the humanized CSL362 antibody directed against the IL3Ralpha chain and its modified forms &lt;400&gt; 14 gagagcagcc agagcctgct gaacagcggc aaccagaaga actacctgac c &lt;210&gt; 15 &lt;211&gt; 21 &lt;212&gt; DNA &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the LCDR2 of the humanized CSL362 antibody directed against the IL3Ralpha chain and its modified forms &lt;400&gt; tgggccagca cccgggagag c

114 &lt;210&gt; 16 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence encoding the LCDR3 of the humanized CSL362 antibody directed against the IL3Ralfa chain and its modified forms &lt;400&gt; 16 cagaacgact acagctaccc ctacacc &lt;210&gt; 17 &lt;211&gt; 15 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for HCDR1 of the humanized CSL362 antibody directed against the IL3Ralfa chain and its modified forms &lt;400&gt; 17 gactactaca tgaag &lt;210&gt; 18 &lt;211&gt; 51 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the HCDR2 humanized CSL362 antibody directed against the IL3Ralpha chain and its modified forms &lt;400&gt; 18 gacatcatcc ccagcaacgg cgccaccttc tacaaccaga agttcaaggg c

115 &lt;210&gt; 19 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the HCDR3 humanized CSL362 antibody directed against the IL3Ralpha chain and its modified forms &lt;400&gt; 19 tcccacctgc tgagggccag ctggttcgcc tac &lt;210&gt; 20 &lt;211&gt; 342 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the variable region in the light chain of the humanized CSL362 antibody directed against the IL-3Ralpha chain and its modified forms &lt;400&gt;

<td>gacatcgtga</td><td>tgacccagag</td><td>ccccgacagc</td><td>ctggccgtga</td><td>gcctgggcga</td><td>gagggccacc</td><td> 60</td>

<td>atcaactgcg</td><td>agagcagcca</td><td>gagcctgctg</td><td>aacagcggca</td><td>accagaagaa</td><td>ctacctgacc</td><td> 120</td>

<td>tggtatcagc</td><td>agaagcccgg</td><td>ccagcccccc</td><td>aagccactga</td><td>tctactgggc</td><td>cagcacccgg</td><td> 180</td>

<td>gagagcggcg</td><td>tgcccgacag</td><td>gttcagcggc</td><td>agcggctccg</td><td>gcaccgactt</td><td>caccctgacc</td><td> 240</td>

<td>atcagcagcc</td><td>tgcaggccga</td><td>ggacgtggcc</td><td>gtgtactact</td><td>gccagaacga</td><td>ctacagctac</td><td> 300</td>

<td>ccctacacct</td><td>tcggccaggg</td><td>caccaagctg</td><td>gaaatcaaga</td><td>gg</td><td></td><td> 342</td>

&lt;210&gt; 21 &lt;211&gt; 360 &lt;212&gt; DNA &lt;213&gt; Artificial

116 &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the variable region in the heavy chain of the humanized CSL362 antibody directed against the IL-3Ralpha chain and its modified forms &lt;400&gt; 21

<td>gaggtgcagć</td><td>tggtgcagag</td><td>cggagccgag</td><td>gtgaagaagc</td><td>ccggcgagag</td><td>cctgaagatc</td><td> 60</td>

<td>agctgcaagg</td><td>gcagcggcta</td><td>cagcttcacc</td><td>gactactaca</td><td>tgaagtgggc</td><td>ccggcagatg</td><td> 120</td>

<td>cccggcaagg</td><td>gcctggaatg</td><td>gatgggcgac</td><td>atcatcccca</td><td>gcaacggcgc</td><td>caccttctac</td><td> 180</td>

<td>aaccagaagt</td><td>tcaagggcca</td><td>ggtcaccatc</td><td>agcgccgaca</td><td>agagcatcag</td><td>caccacctac</td><td> 240</td>

<td>ctgcagtgga</td><td>gcagcctgaa</td><td>ggccagcgac</td><td>accgccatgt</td><td>actactgcgc</td><td>caggtcccac</td><td> 300</td>

<td>ctgctgaggg</td><td>ccagctggtt</td><td>cgcctactgg</td><td>ggccagggca</td><td>caatggtgac</td><td>cgtgagcagc</td><td> 360</td>

&lt;210&gt; 22 &lt;211&gt; 1353 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the heavy chain of the humanized CSL362 antibody directed against the IL-3Ralpha chain and CSL362B &lt;400&gt; 22

117

<td>gaggtgcagc tggtgcagag cggagccgag gtgaagaagc ccggcgagag cctgaagatc</td><td> 60</td>

<td>agctgcaagg gcagcggcta cagcttcacc gactactaca tgaagtgggc ccggcagatg</td><td> 120</td>

<td>cccggcaagg gcctggaatg gatgggcgac atcatcccca gcaacggcgc caccttctac</td><td> 180</td>

<td>aaccagaagt tcaagggcca ggtcaccatc agcgccgaca agagcatcag caccacctac</td><td> 240</td>

<td>ctgcagtgga gcagcctgaa ggccagcgac accgccatgt actactgcgc caggtcccac</td><td> 300</td>

<td>ctgctgaggg ccagctggtt cgcctactgg ggccagggca caatggtgac cgtgagcagc</td><td> 360</td>

<td>gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc</td><td> 420</td>

<td>ggcacagcag ccctgggatg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc</td><td> 480</td>

<td>tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagcagc</td><td> 540</td>

<td>ggcctgtatt ctctgagcag cgtcgtgaca gtgcccagca gcagcctggg cacccagacc</td><td> 600</td>

<td>tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc</td><td> 660</td>

<td>aagagctgcg acaagaccca cacctgtcct ccatgcccag ccccagagct gctgggcgga</td><td> 720</td>

<td>ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag caggaccccc</td><td> 780</td>

<td>gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc cagaggtgaa gttcaactgg</td><td> 840</td>

<td>tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagagagga acagtacaac</td><td> 900</td>

<td>agcacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag</td><td> 960</td>

<td>gagtacaagt gcaaagtctc caacaaggcc ctgccagccc ccatcgagaa aaccatcagc</td><td> 1020</td>

<td>aaggccaagg gccagccacg ggagccccag gtgtacaccc tgcccccctc ccgggacgag</td><td> 1080</td>

<td>ctgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc cagcgacatt</td><td> 1140</td>

<td>gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac ccccccagtg</td><td> 1200</td>

<td>ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gagcaggtgg</td><td> 1260</td>

<td>cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc</td><td> 1320</td>

<td>cagaagagcc tgagcctgtc ccccggcaag tga</td><td> 1353</td>

&lt;210&gt; 23 &lt;211&gt; 663 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; nucleotide sequence coding for the light chain of the humanized CSL362 antibody directed against the IL-3Ralpha chain and its modified forms &lt;400&gt; 23

118 gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc

<td>atcaactgcg</td><td>agagcagcca</td><td>gagcctgctg</td><td>aacagcggca</td><td>accagaagaa</td><td>ctacctgacc</td><td> 120</td>

<td>tggtatcagc</td><td>agaagcccgg</td><td>ccagcccccc</td><td>aagccactga</td><td>tctactgggc</td><td>cagcacccgg</td><td> 180</td>

<td>gagagcggcg</td><td>tgcccgacag</td><td>gttcagcggc</td><td>agcggctccg</td><td>gcaccgactt</td><td>caccctgacc</td><td> 240</td>

<td>atcagcagcc</td><td>tgcaggccga</td><td>ggacgtggcc</td><td>gtgtactact</td><td>gccagaacga</td><td>ctacagctac</td><td> 300</td>

<td>ccctacacct</td><td>tcggccaggg</td><td>caccaagctg</td><td>gaaatcaaga</td><td>ggaccgtggc</td><td>tgccccatct</td><td> 360</td>

<td>gtcttcatct</td><td>tcccccccag</td><td>cgacgagcag</td><td>ctgaagagcg</td><td>gcaccgccag</td><td>cgtggtgtgc</td><td> 420</td>

<td>ctgctgaata</td><td>acttctaccc</td><td>ccgggaggcc</td><td>aaggtgcagt</td><td>ggaaggtgga</td><td>caacgccctg</td><td> 480</td>

<td>cagagcggca</td><td>acagccagga</td><td>aagcgtcacc</td><td>gagcaggaca</td><td>gcaaggactc</td><td>cacctacagc</td><td> 540</td>

<td>ctgagcagca</td><td>ccctgaccct</td><td>gagcaaggcc</td><td>gactacgaga</td><td>agcacaaggt</td><td>gtacgcctgc</td><td> 600</td>

<td>gaggtgaccc</td><td>accagggcct</td><td>gtccagcccc</td><td>gtgaccaaga</td><td>gcttcaacag</td><td>gggcgagtgc</td><td> 660</td>

tga 663

119