HK1181785B - Humanized anti-interleukin 3 receptor alpha chain antibodies - Google Patents

Abstract

The present disclosure provides antibodies that bind to interleukin-3 receptor alpha chain and uses thereof.

Description

Humanized anti-interleukin3receptor alpha chain antibodies

Information of related applications

This application is a national phase application according to 35USC371 of international application PCT/AU2011/001056 filed on 8/17/2011, which claims priority from U.S. patent application No. 61/374,489 entitled "Humanized Anti-Interleukin3Receptor Alpha Chain Antibodies (human Anti-Interleukin3Receptor Alpha Chain Antibodies)" filed on 8/17/2010 and international patent application No. PCT/AU2011/000155 entitled "Compositions and Methods for Targeting Type1Interferon Producing Cells". The entire contents of these applications are incorporated herein by reference.

Technical Field

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

Background

The functional interleukin-3 receptor is a receptor that comprises a specific alpha chain (IL-3 Ra; CD123) and shares a "common" IL-3 receptor beta chain (beta) with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-5 (IL-5)_c(ii) a CD 131).

IL-3R α is a type I transmembrane protein with a deduced molecular weight of about 41kDa containing an extracellular domain (involved in IL-3 binding), a transmembrane domain, and a short cytoplasmic tail of about 50 amino acids. The extracellular domain is composed of two regions: an N-terminal region of about 100 amino acids whose sequence is shown to be similar to the equivalent regions of the alpha chain of GM-CSF and IL-5 receptors; and a region proximal to the transmembrane domain that contains four conserved cysteine residues and a WSXWS motif common to other members of this cytokine receptor family.

The IL-3 binding domain comprises a Cytokine Receptor Motif (CRM) of about 200 amino acid residues consisting of two Ig-like folding domains. The extracellular domain of IL-3R α is highly glycosylated with N-glycosylation necessary for ligand binding and receptor signaling.

IL-3R α is widely expressed throughout the hematopoietic system, including hematopoietic progenitors, mast cells, erythrocytes, megakaryocytes, basophils, eosinophils, monocytes/macrophages, neutrophils, and CD5⁺B lymphocytes. Non-hematopoietic cells, such as plasmacytoid dendritic cells (pDC), Leydig cells, endothelial cells and stromal cells, also express IL-3R α.

IL-3 ra is also expressed by cells involved in certain disease conditions, including myelodysplastic syndromes, myeloid leukemias (e.g., Acute Myelogenous Leukemia (AML)), malignant lymphoproliferative disorders (e.g., lymphomas),Allergy and autoimmune diseases (e.g. lupus, Sjogren's syndrome)Or scleroderma). Thus, anti-IL-3 ra antibodies are needed for therapeutic applications.

Summary of The Invention

The present disclosure is based on the inventors' creation of humanized antibodies that specifically bind to IL-3R α. After humanization, the inventors found that the affinity of the antibody for IL-3R α was reduced. Thus, the inventors performed affinity maturation to increase the affinity of the antibody for IL-3R α. Unexpectedly, the affinity matured antibody was in the heavy chain variable region (V)_H) And light chain variable region (V)_L) And CDR1 of the light chain and the Framework Region (FR) of the light chain.

The inventors have also produced a form of such an antibody that is capable of inducing an enhanced level of effector function.

The inventors have also found that a particular modification that induces an increase in effector function results in another desirable property, namely that upon administration of the modified antibody to a mammal, the number of NK cells in the mammal is initially reduced, but then expanded to a level higher than before the administration. The inventors also demonstrated that there is a correlation between NK cell number and lysis of target cells (e.g. leukemia cells).

The present disclosure relates generally to murine antibody-based immunoglobulins capable of specifically binding to IL-3 ra chains.

In one embodiment, the present disclosure provides an isolated or recombinant immunoglobulin that specifically binds to an IL-3R α chain and comprises a V comprising the sequence set forth in SEQ ID NO 8_LAnd the Complementarity Determining Region (CDR) of SEQ ID NO. 9_HThe CDR of (1).

The present disclosure additionally or alternatively provides isolated or recombinant antibodies or antigen-binding fragments thereof capable of specifically binding to the IL-3 ra chainAnd comprises a V comprising the sequence shown in SEQ ID NO. 8_LCDR of (A) and V shown in SEQ ID NO. 9_HThe CDR of (1).

The present disclosure additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof capable of specifically binding to an IL-3 ra chain and comprising a V comprising the sequence set forth in SEQ ID No. 8_LCDR of (A) and V shown in SEQ ID NO. 9_HThe CDR of (1).

In one embodiment, the present disclosure provides an isolated or recombinant immunoglobulin that specifically binds to an IL-3R α chain and comprises the amino acid sequences of SEQ ID NOS: 2-7, respectively.

The present disclosure additionally or alternatively provides an isolated or recombinant antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment is capable of specifically binding to an IL-3 ra chain and comprises the amino acid sequence of SEQ id nos 2-7.

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

For example, an immunoglobulin or antibody comprises:

(i) light chain variable region (V) comprising CDR1, 2 and 3 as shown in SEQ ID NOs: 2, 3 and 4, respectively_L) (ii) a And

(ii) heavy chain variable region (V) comprising CDR1, 2 and 3 as shown in SEQ ID NOs: 5, 6 and 7, respectively_H)。

For example, an immunoglobulin or antibody comprises:

(i) v is as follows_L：

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

b) CDR2 comprising (or encoded by) the sequence shown in SEQ ID NO: 3; 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) v is as follows_H：

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 shown in SEQ ID NO:7 (or encoded by the sequence shown in SEQ ID NO: 19).

The present disclosure additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof capable of specifically binding to an IL-3 ra chain and comprising a V comprising an amino acid sequence according to SEQ ID NO:8 (or encoded by the sequence set forth in SEQ ID NO: 20)_LAnd/or V comprising the amino acid sequence according to SEQ ID NO:9 (or encoded by the sequence shown in SEQ ID NO: 21)_H。

The present disclosure additionally or alternatively provides an isolated or recombinant humanized antibody or antigen binding fragment thereof capable of specifically binding to an IL-3 ra chain and comprising a V comprising an amino acid sequence according to SEQ ID NO:8 (or encoded by the sequence set forth in SEQ ID NO: 20)_LAnd V comprising the amino acid sequence according to SEQ ID NO:9 (or encoded by the sequence shown in SEQ ID NO: 21)_H。

Exemplary antigen-binding fragments encompassed by the present disclosure include:

(i) domain antibodies (dabs);

(ii)Fv；

(iii) an scFv or a stable form thereof (e.g., a disulfide-stabilized scFv);

(iv) a dimeric scFv or a stable form thereof;

(v) bifunctional, trifunctional, tetrafunctional or higher multimers;

(vi) a Fab fragment;

(vii) a Fab' fragment;

(viii) a F (ab') fragment;

(ix)F(ab’)₂a fragment;

(x) Any one of (i) - (ix) fused to the Fc region of an antibody;

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

In one embodiment, the immunoglobulin or antibody removes or at least partially eliminates the cells to which it binds, such as leukemia cells and/or basophils and/or pdcs.

As will be apparent to those skilled in the art in light of the disclosure herein, exemplary immunoglobulins or antibodies are capable of removing or at least partially eliminating cells to which they bind without being conjugated to a toxic compound.

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

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

In one embodiment, the immunoglobulin or antibody comprises an antibody Fc region capable of inducing effector function. For example, the effector function is an Fc-mediated effector function. In one embodiment, the Fc region is an IgG1Fc region or an IgG3Fc region or a hybrid IgG1/IgG2Fc region.

In one embodiment, the immunoglobulin or antibody is capable of inducing effector function at levels similar (e.g., not significantly different or within about 10%) to or identical to wild-type human IgG1 and/or human IgG3Fc regions.

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

In one embodiment, the level of effector function induced by the immunoglobulin or antibody is enhanced relative to the level of the immunoglobulin or antibody comprising the wild-type IgG1Fc region.

In one embodiment, the immunoglobulin or antibody is afucosylated or comprises an afucosylated Fc region.

In another embodiment, the immunoglobulin or antibody has a lower level of fucosylation as compared to an immunoglobulin or antibody produced by a human or CHO cell that has not been engineered to reduce the level of fucosylation of the protein. According to this example, a lower level of fucosylation is understood to mean that in a composition comprising an immunoglobulin or antibody, the percentage of fucosylated immunoglobulin (e.g. the glycosyl attached to Asn297 of the antibody comprises fucose) is lower than the level produced by a human or CHO cell not engineered to reduce the level of fucosylation of the protein.

For example, the immunoglobulin or antibody is a V comprising a sequence shown in SEQ ID NO:8 (or encoded by a sequence shown in SEQ ID NO: 20)_LAnd V comprising (or encoded by) the sequence shown in SEQ ID NO:9_HThe afucosylated humanized antibody of (1). For example, the immunoglobulin or antibody is an afucosylated humanized antibody comprising a light chain comprising the sequence shown 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).

In one embodiment, 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 set forth in SEQ ID NO: 23) and a heavy chain comprising the sequence set forth in SEQ ID NO:10 (or encoded by the sequence set forth in SEQ ID NO: 22) expressed by a mammalian cell (e.g., a CHO cell) that does not express detectable levels (or that expresses lower levels) of alpha-1, 6-fucosyltransferase (FUT 8).

In one embodiment, the immunoglobulin or antibody comprises an Fc region comprising one or more amino acid sequence substitutions that enhance effector functions induced by the immunoglobulin. For example, one or more amino acid sequence substitutions increases the affinity of the Fc region for an Fc γ receptor (fcyr) as compared to an Fc region that does not comprise the substitution. For example, the one or more amino acid sequence substitutions increase the affinity of the Fc region for an Fc γ R selected from the group consisting of Fc γ RI, Fc γ RIIa, Fc γ RIIc, and Fc γ RIIIa as compared to an Fc region that does not comprise the substitutions. In one embodiment, the one or more amino acid sequence substitutions are:

(i) S239D, a330L and I332E according to EU numbering system of Kabat; or

(ii) S239D and I332E according to EU numbering system of Kabat.

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

(i) S239D, a330L and I332E according to EU numbering system of Kabat; and

(ii) S239D and I332E according to EU numbering system of Kabat.

In one embodiment, the Fc region comprises the sequence shown between residues 234 and 450 of SEQ ID NO:11 (comprising the S239D and I332E substitutions according to the EU numbering system of Kabat).

In one embodiment, the Fc region comprises the sequence shown between residues 234 and 450 of SEQ ID NO 12 (comprising the S239D, A330L and I332E substitutions according to the EU numbering system of Kabat).

In one embodiment, the immunoglobulin or antibody is selected from the group consisting of:

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

(ii) an antibody comprising a light chain comprising the sequence shown in SEQ ID NO. 13 and a heavy chain comprising the sequence shown in SEQ ID NO. 12;

as described above, the inventors have determined that following administration of an antibody described herein, the number of NK cells in circulation in the mammal is initially reduced and then increased as compared to the number of NK cells in circulation in the mammal prior to administration. The inventors have also demonstrated that increasing NK cells relative to target cells results in enhanced efficacy, i.e. killing a greater number of target cells. This effect was induced by an antibody comprising a constant region or Fc region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

Thus, in one embodiment, the present disclosure provides an isolated or recombinant antibody capable of specifically binding to an IL-3 ra chain and comprising:

(i) light chain variable region (V) comprising CDR1, 2 and 3 as shown in SEQ ID NOs: 2, 3 and 4, respectively_L)；

(ii) Heavy chain variable region (V) comprising CDR1, 2 and 3 as shown in SEQ ID NOs: 5, 6 and 7, respectively_H) (ii) a And

(iii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

In one embodiment, the present disclosure provides an isolated or recombinant humanized antibody capable of specifically binding to an IL-3 ra chain and comprising:

(i) v is as follows_L：

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

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

c) CDR3 comprising the sequence shown in SEQ ID NO. 4 (or encoded by the sequence shown in SEQ ID NO. 16);

(ii) v is as follows_H：

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 shown in SEQ ID NO:7 (or encoded by the sequence shown in SEQ ID NO: 19); and

(iii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

In one embodiment, the heavy chain constant region is a hybrid of human IgG1 and human IgG2 constant regions.

In one embodiment, the constant region comprises the sequence shown between residues 121-450 of SEQ ID NO. 11, including residues 121-450.

The present disclosure additionally or alternatively provides an isolated or recombinant humanized antibody capable of specifically binding to an IL-3 ra chain and comprising:

(i) v comprising the amino acid sequence according to SEQ ID NO:8 (or encoded by the sequence shown in SEQ ID NO: 20)_LAnd/or comprises a sequence according to SEQ ID NO:9 (or encoded by the sequence shown in SEQ ID NO: 21)_H(ii) a And

(ii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

In one embodiment, the heavy chain constant region is a hybrid of human IgG1 and human IgG2 constant regions.

In one embodiment, the constant region comprises the sequence shown between residues 121-450 of SEQ ID NO. 11, including residues 121-450.

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

In one embodiment, the heavy chain constant region is a hybrid of human IgG1 and human IgG2 constant regions.

In one embodiment, the constant region comprises the sequence shown between residues 121-450 of SEQ ID NO 11, including residues 121-450.

In one embodiment, the present disclosure provides an isolated or recombinant humanized antibody capable of specifically binding to an IL-3 Ra chain and comprising a light chain comprising the sequence set forth in SEQ ID NO 13 and a heavy chain comprising the sequence set forth in SEQ ID NO 11.

In one embodiment, the immunoglobulin or antibody or antigen-binding fragment thereof of the present disclosure neutralizes IL-3 signaling.

In one embodiment, the immunoglobulin or antibody of the present disclosure is a naked immunoglobulin or the antibody or antigen binding fragment thereof of the present disclosure is a naked antibody or antigen binding fragment thereof.

In one embodiment, the immunoglobulin or antibody of the present disclosure is a full length antibody.

In one embodiment, the immunoglobulin or antibody of the disclosure binds to the equilibrium dissociation constant (K) of the IL-3 Ra chain_D) Is 1X 10^-8M or less, e.g. 5X 10^-9M or less, e.g. 3X 10^-9M or less, e.g. 2.5X 10^-9M or less.

In one embodiment, the immunoglobulins or antibodies of the present disclosure bind to K of the IL-3R α chain_DIs about 2.2X 10^-9M or less. In one embodiment, K_DAt about 1X 10^-9M and about 2.5X 10^-9Between M, e.g. about 2.2X 10^-9M。

In one embodiment, the immunoglobulins or antibodies of the present disclosure bind to K of the IL-3R α chain_DIs about 9X 10^-10M or less, e.g. about 8X 10^-10M or less. In one embodiment, K_DAt about 5X 10^-10M and about 9X 10^-10Between M, e.g. about 7.8X 10^-10M。

The disclosure also includes fragments, variants, and derivatives of the immunoglobulins or antibodies of the disclosure.

In one embodiment, the immunoglobulin or antibody of the disclosure is capable of reducing the number of NK cells when administered to a mammal (e.g., a non-human primate, such as cynomolgus monkey). For example, the immunoglobulin or antibody, when administered to a mammal, is capable of reducing the number of NK cells by at least about 20%, such as at least about 30% or 40%, within 12 hours or 10 hours or 8 hours of administration. For example, an immunoglobulin or antibody, when administered to a mammal, is capable of reducing the number of NK cells by at least about 50% within 6 hours of administration. In one embodiment, the immunoglobulin or antibody is capable of reducing the number of NK cells when administered at a dose of between 0.0001mg/kg and 50mg/kg, such as between about 0.0005mg/kg and about 40mg/kg, for example between about 0.001mg/kg and about 30mg/kg, such as about 0.005mg/kg and about 20mg/kg, such as about 0.01mg/kg and about 10 mg/kg. For example, the immunoglobulin or antibody, when administered to a mammal, is capable of reducing the number of NK cells by at least about 50% within 6 hours of administration when administered at a dose of about 0.01mg/kg or 0.1mg/kg or 1mg/kg or 10 mg/kg. In one embodiment, the dose is about 0.01mg/kg or 0.1 mg/kg.

In one embodiment, the number of NK cells in the mammal is increased at 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 the immunoglobulin or antibody as compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells increases at least about 8 or 11 or 17 or 22 or 29 days after administration. For example, the number of NK cells in the mammal is increased by about 10% or 20% or 30% or 50% or 60% or 70% or 80% compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells in the mammal is increased by about 20% at least 8 days after administration of the antibody or immunoglobulin at a dose of between about 0.001mg/kg and about 0.1mg/kg as compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. For example, the number of NK cells in the mammal is increased by about 50% at least 11, 17, 22, or 29 days after administration of the antibody or immunoglobulin at a dose of between about 0.001mg/kg and about 0.1mg/kg as compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody. In one embodiment, the antibody or immunoglobulin is administered at a dose of between 0.01mg/kg and 0.1 mg/kg. For example, the immunoglobulin or antibody is administered at a dose of 0.01mg/kg or 0.1 mg/kg.

Based on the disclosure herein, it will be apparent to those skilled in the art that the present disclosure provides immunoglobulins or antibodies that, when administered to a mammal, cause an increase in the number of NK cells in the mammal. For example, an immunoglobulin or antibody, when administered to a mammal, causes a decrease in the number of NK cells, followed by an increase in the number of NK cells, in the mammal. In one embodiment, the number of NK cells is increased or decreased compared to the number of NK cells in the mammal prior to administration of the immunoglobulin or antibody.

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

In one embodiment, the present disclosure provides a pharmaceutical composition comprising an immunoglobulin or antibody of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

The present disclosure also provides isolated nucleic acids encoding the immunoglobulins or antibodies of the present disclosure.

Exemplary sequences of nucleic acids are discussed in the context of encoding the antibodies or immunoglobulins of the present disclosure and should be understood to apply mutatis mutandis to the current embodiments of the present disclosure.

The disclosure also provides nucleic acids that are capable of hybridizing to the nucleic acids of the disclosure under high stringency hybridization conditions.

The disclosure also includes fragments, homologs, and derivatives of the isolated nucleic acids of the disclosure.

The disclosure also provides genetic constructs comprising an isolated nucleic acid of the disclosure and one or more additional nucleotide sequences (e.g., a promoter operably linked to the nucleic acid).

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

In one embodiment, a genetic construct of the disclosure comprises an encoded polypeptide (e.g., comprising V) operably linked to a promoter_H) And a promoter operably linked to the nucleic acidCode for another polypeptide (e.g., comprising V)_L) The nucleic acid of (1).

In another embodiment, the gene construct is a bicistronic gene construct, e.g., comprising in 5 'to 3' order the following operably linked components:

(i) a promoter;

(ii) a nucleic acid encoding a first polypeptide;

(iii) an internal ribosome entry site; and

(iv) a nucleic acid encoding a second polypeptide.

For example, the first polypeptide comprises V_HAnd the second polypeptide comprises V_LOr the first polypeptide comprises V_LAnd the second polypeptide comprises V_H。

The disclosure also encompasses separate genetic constructs, one of which encodes a first polypeptide (e.g., comprising V)_H) And wherein the other encodes a second polypeptide (e.g., comprising V)_L). For example, the present disclosure also provides a composition comprising:

(i) a first expression construct comprising an encoded polypeptide operably linked to a promoter (e.g., comprising V)_H) The nucleic acid of (1); and

(ii) a second expression construct comprising the encoded polypeptide operably linked to a promoter (e.g., comprising V)_L) The nucleic acid of (1).

The disclosure also provides host cells comprising the genetic constructs of the disclosure.

In one embodiment, the disclosure provides an isolated cell expressing an immunoglobulin or antibody or antigen-binding fragment of the disclosure or a recombinant cell genetically modified to express an immunoglobulin, antibody or antigen-binding fragment.

In one embodiment, the cell comprises a genetic construct of the disclosure or:

(i) A first gene construct comprising an encoded polypeptide operably linked to a promoter (e.g., comprising V)_H) The nucleic acid of (1); and

(ii) a second gene construct comprising an encoded polypeptide operably linked to a promoter (e.g., comprising V)_L) The nucleic acid according to (1),

wherein the first and second polypeptides form an immunoglobulin, antibody, or antigen-binding fragment of the disclosure.

The genetic construct may be integrated into the cell or remain in free form.

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

The present disclosure further provides methods for making an immunoglobulin, antibody, or antigen-binding fragment of the present disclosure, the methods comprising maintaining a genetic construct of the present disclosure under conditions sufficient to produce the immunoglobulin, antibody, or antigen-binding fragment.

In one embodiment, a method for making an immunoglobulin, antibody, or antigen-binding fragment of the present disclosure comprises culturing a cell of the present disclosure under conditions sufficient to produce and optionally secrete the immunoglobulin, antibody, or antigen-binding fragment.

In one embodiment, the method for making an immunoglobulin, antibody or antigen binding fragment of the present disclosure further comprises isolating the immunoglobulin, antibody or antigen binding fragment.

The present disclosure additionally provides methods for making a recombinant immunoglobulin or antibody of the present disclosure, the method comprising the steps of:

(i) culturing a host cell containing an expression vector of the present disclosure such that the recombinant immunoglobulin or antibody is expressed in the host cell; and

(ii) isolating the recombinant immunoglobulin.

In one embodiment, the method for making an immunoglobulin, antibody or antigen binding fragment of the present disclosure further comprises formulating the immunoglobulin, antibody or antigen binding fragment with a pharmaceutically acceptable carrier.

The present disclosure also provides a method for prophylactic or therapeutic treatment of a disease or disorder in a mammal, the method comprising the step of administering to the mammal an immunoglobulin or antibody of the present disclosure, thereby treating or preventing the disease or disorder.

In one embodiment, the mammal is a human.

In one embodiment, the mammal is in need of treatment or prevention.

In one embodiment, a mammal in need thereof is suffering from a disease or disorder.

In one embodiment, a mammal in need thereof is at risk of developing a disease or disorder or of relapse to said disease or disorder.

The present disclosure also provides for the use of an immunoglobulin, antibody, or antigen-binding fragment of the present disclosure or a composition of the present disclosure in a medicament.

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

The present disclosure also provides an immunoglobulin, antibody, or antigen-binding fragment of the present disclosure for use in treating a disease or disorder in a mammal.

In one embodiment, the disease or disorder is an IL-3 Ra mediated disease or disorder.

In one embodiment, the disease or disorder is myelodysplastic syndrome.

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

In one embodiment, the disease or disorder is an IL-3 ra-associated cancer, such as leukemia, i.e., the cancer (or leukemia) is characterized by cancer (or leukemia) cells expressing IL-3 ra.

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

In one embodiment, the disease or disorder is an autoimmune disorder or an inflammatory disorder. For example, the disorder is lupus (e.g., systemic lupus erythematosus), sjogren's syndrome, or scleroderma (e.g., systemic scleroderma).

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

In one embodiment, the method comprises administering to the mammal between about 0.0001mg/kg and 50mg/kg of an immunoglobulin, antibody or antigen-binding fragment. For example, the method comprises administering between about 0.0005mg/kg to about 40 mg/kg. For example, the method comprises administering between about 0.0005mg/kg to about 30 mg/kg. For example, the method comprises administering between about 0.001mg/kg to about 20 mg/kg. For example, the method comprises administering between about 0.001mg/kg to about 10 mg/kg. For example, the method comprises administering between about 0.01mg/kg and about 5 mg/kg. For example, the method comprises administering between about 0.001mg/kg to about 1 mg/kg.

In one embodiment, the method comprises administering between about 0.1mg/kg to about 10mg/kg of an immunoglobulin, antibody or antigen-binding fragment. For example, the method comprises administering between about 0.1mg/kg to about 5 mg/kg. For example, the method comprises administering between about 0.1mg/kg to about 1 mg/kg.

In one embodiment, the method comprises administering between about 10mg/kg to about 30mg/kg of an immunoglobulin, antibody or antigen binding fragment. For example, the method comprises administering between about 20mg/kg to about 30 mg/kg.

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

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

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

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

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

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

For example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of between 0.0001mg/kg and 5mg/kg, such as between 0.0005mg/kg and 5mg/kg, for example between 0.001mg/kg and 5mg/kg, and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 17 days or 21 days or 22 days or 28 days or 29 days or 30 days or 1 calendar month. For example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of between 0.01mg/kg and 5mg/kg, and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days. For example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of between 0.01mg/kg and 2mg/kg, and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days. For example, the immunoglobulin, antibody or antigen binding domain is administered at a dose of between 0.01mg/kg and 1mg/kg, and the period between administrations is at least about 7 days or 8 days or 9 days or 10 days or 11 days or 14 days. In some embodiments, the period between administrations is at least about 7 days and less than about 22 days, such as at least about 11 or 15 days and less than about 20 days, for example at least about 13 days and less than about 18 days.

In one embodiment, the immunoglobulin, antibody or antigen binding domain is administered at a dose of 0.01mg/kg and the period between administrations is 6 days or 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 15 days.

In one embodiment, the immunoglobulin, antibody or antigen binding domain is administered at a dose of 0.1mg/kg and the period between administrations is 6 days or 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 15 days.

In one embodiment, the immunoglobulin or antibody is administered at a dose of 1mg/kg and the period between administrations is 6 days or 7 days or 8 days or 9 days or 10 days or 11 days or 14 days or 15 days or 20 days or 21 days or 22 days.

For example, the immunoglobulin or antibody is administered at a dose of between 6mg/kg and 50mg/kg and the period between administrations is at least about 15 days. For example, the immunoglobulin or antibody is administered at a dose of between 10mg/kg and 30mg/kg and the period between administrations is at least about 14 or 15 days. For example, the immunoglobulin or antibody is administered at a dose of between 20mg/kg and 30mg/kg and the period between administrations is at least about 14 or 15 days. In some embodiments, the period between administrations is at least about 20 days and less than about 70 days, such as at least about 21 or 22 days and less than about 65 days, for example at least about 25 days and less than about 57 days.

In one embodiment, the immunoglobulin or antibody is administered at a dose of 10mg/kg and the period between administrations is 14 days or 15 days or 21 days or 22 days or 30 days or 48 days or 50 days or 56 days or 57 days or 60 days.

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

As described above, the inventors found that the number of NK cells increased in about 8 days after administration of the immunoglobulin or antibody of the present disclosure above the number present prior to administration. The inventors also demonstrated that an increase in NK cell number results in an increase in the efficacy of the antibodies or immunoglobulins of the present disclosure to induce target cell death. Thus, once the number of NK cells increases, another dose of antibody or immunoglobulin can be administered to induce a therapeutic/prophylactic effect. For example, the immunoglobulin, antibody or antigen binding fragment of the present disclosure is administered multiple times at a dose of between about 0.001mg/kg and about 1mg/kg, wherein the period between administrations is at least about 7 days, such as at least about 8 days, for example at least about 11 days, such as at least about 14 days, for example at least about 17 days, such as at least about 21 days, for example at least about 22 days, for example 28 days or 29 days or a calendar month or 56 or 57 or 60 days. In one embodiment, the period between doses is about 7 days. In one embodiment, the period between doses is about 8 days. In one embodiment, the period between doses is about 11 days. In one embodiment, the period between doses is about 14 days. In one embodiment, the period between doses is about 17 days. In one embodiment, the period between doses is about 21 days. In one embodiment, the period between doses is about 22 days. In one embodiment, the period between doses is about 28 days. In one embodiment, the period between doses is about 29 days. The dosage is between about 0.01mg/kg and about 0.1mg/kg, such as a dosage of about 0.01mg/kg or 0.1 mg/kg.

Brief description of the drawings

Figure 1A is a graph showing the number of NK cells in samples from non-human primate subjects at different time points (as shown) after administration of antibody CSL362X 2. Cell number is expressed as a percentage of the number of cells prior to administration of the antibody. The dosage of the antibody is indicated.

Figure 1B is a graph showing the number of NK cells in samples from non-human primate subjects at different time points (as shown) after administration of antibody CSL 362B. Cell number is expressed as a percentage of the number of cells prior to administration of the antibody. The dosage of the antibody is indicated.

Figure 1C is a graph showing the number of NK cells in samples from non-human primate subjects at different time points (as shown) after administration of a chimeric antibody comprising the constant domain of human IgG1 and the variable region of antibody 7G3 (designated CSL 360). Cell number is expressed as a percentage of the number of cells prior to administration of the antibody. The dosage of the antibody is indicated.

Figure 2A is a graph showing the percentage of TF-1 cells lysed in the presence of a specified cell population and different concentrations (as shown on the X-axis) of antibody CSL362X 1.

Figure 2B is a graph showing the percentage of AML cells in the presence of different numbers of NK cells and the antibody CSL362X 1. The ratio of effector cells (NK cells; E) to target cells (leukemia cells; T) is shown on the X-axis. Results generated using cells from two patients are depicted.

Detailed Description

Sequence Listing keywords

Amino acid sequence of SEQ ID NO 1-IL-3R alpha chain

Amino acid sequence of 2-humanized anti-IL-3R alpha chain antibody CSL362 and its modified form of LCDR1

Amino acid sequence of 3-humanized anti-IL-3R alpha chain antibody CSL362 and its modified form of LCDR2

Amino acid sequence of 4-humanized anti-IL-3R alpha chain antibody CSL362 and its modified form of LCDR3

Amino acid sequence of the 5-humanized anti-IL-3R alpha chain antibody CSL362 and its modified form HCDR1

Amino acid sequence of the 6-humanized anti-IL-3R alpha chain antibody CSL362 and its modified form HCDR2

Amino acid sequence of 7-humanized anti-IL-3R alpha chain antibody CSL362 and its modified form HCDR3

8-humanized anti-IL-3R alpha chain antibody CSL362 and modified forms thereof

9-humanized anti-IL-3R alpha chain antibody CSL362 and modified forms thereof

Amino acid sequence of heavy chains of 10-humanized anti-IL-3R alpha chain antibodies CSL362 and CSL362B

Amino acid sequence of heavy chain of the 11-humanized anti-IL-3R alpha chain antibody CSL362X1

Amino acid sequence of heavy chain of 12-humanized anti-IL-3R alpha chain antibody CSL362X2

Amino acid sequence of light chain of 13-humanized anti-IL-3R alpha chain antibody CSL362 and modified forms thereof

14-nucleotide sequence encoding humanized anti-IL-3R alpha chain antibody CSL362 and modified form thereof LCDR1

15-nucleotide sequence encoding humanized anti-IL-3R alpha chain antibody CSL362 and modified form thereof LCDR2

16-nucleotide sequence encoding humanized anti-IL-3R alpha chain antibody CSL362 and modified form thereof LCDR3

17-nucleotide sequence encoding humanized anti-IL-3R alpha chain antibody CSL362 and its modified form HCDR1

18-nucleotide sequence encoding humanized anti-IL-3R alpha chain antibody CSL362 and its modified form HCDR2

19-nucleotide sequence encoding humanized anti-IL-3R alpha chain antibody CSL362 and its modified form HCDR3

20-nucleotide sequence encoding the light chain variable region of humanized anti-IL-3R alpha chain antibody CSL362 and modified forms thereof

21-nucleotide sequence encoding the heavy chain variable region of humanized anti-IL-3R alpha chain antibody CSL362 and modified forms thereof

22-nucleotide sequence encoding the heavy chains of humanized anti-IL-3 Ra chain antibodies CSL362 and CSL362B

23-nucleotide sequence encoding the light chain of humanized anti-IL-3R alpha chain antibody CSL362 and modified forms thereof

General rule

In the present specification, a reference to a single step, composition of matter, group of steps, or group of compositions of matter shall be taken to encompass one or more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of compositions of matter, unless explicitly stated otherwise or the context requires otherwise.

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

The scope of the present disclosure is not limited by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any embodiment of the disclosure herein should be understood to apply mutatis mutandis to any other embodiment of the disclosure unless explicitly stated otherwise.

Unless otherwise specifically defined, all technical and scientific terms used herein are to be understood as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, recombinant proteins, cell culture and immunological techniques used in the present disclosure are standard procedures that are numerical to those of skill in the art. The techniques are described and illustrated in the following industry documents, such as: J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); molecular Cloning, A Laboratory Manual, Cold Spring harbor Laboratory Press (1989); A.Brown (eds.), Essential Molecular Biology: A Practical Approach, Vol.1 and Vol.2, IRL Press (1991); glover and B.D.Hames (eds.), DNA Cloning: A Practical Approach, volumes 1-4, IRL Press (1995 and 1996); and f.m. ausubel et al (eds.), Current Protocols in Molecular Biology, Greene pub. associates and Wiley-Interscience (1988, including all updates so far); the Harlow and David Lane (eds.) Antibodies, A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and j.e. coligan et al (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates so far).

The description and definition of the variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md.,1987 and 1991.

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

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

As used herein, the term "derived from" is understood to mean that the specified entity may be obtained from a particular source, but need not be obtained directly from that source.

Selected definition

As used herein, the term "immunoglobulin" includes any antigen binding protein product of an immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG, and IgE, and antigen binding fragments thereof. Exemplary immunoglobulins are antibodies. An exemplary immunoglobulin is a monoclonal immunoglobulin. Included within the term "immunoglobulin" are any immunoglobulins which have been suitably deimmunized thereby reducing or eliminating the immune response of a mammal to the immunoglobulin which has been administered to said mammal. In the case of treatment of humans, suitable immunoglobulins include chimeric, humanized or human immunoglobulins. Also included in the term "immunoglobulin" are modified, mutagenized, chimeric and/or humanized immunoglobulins comprising engineered or mutated amino acid residues, sequences or glycosylation, whether naturally occurring or produced by human intervention (e.g., by recombinant DNA techniques). Exemplary proteins include Fc receptor binding moieties. For example, proteins encompassed by the term "immunoglobulin" include domain antibodies, camelized antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin neoantigen receptor (IgNAR)). In general, camelid antibodies and IgNAR contain V_HHowever, lack of V_LAnd are commonly referred to as heavy chain immunoglobulins. Other "immunoglobulins" include T cell receptors and other immunoglobulin-like domain-containing proteins capable of binding antigen (e.g., via an antigen binding site comprising a variable region).

Those skilled in the art will appreciate that "antibody" is generally considered to comprise a plurality ofPolypeptide chains (e.g. comprising V)_LAnd a polypeptide comprising V_HPolypeptide) of (a) or (b) is used. Antibodies also typically comprise constant domains, some of which may be arranged as constant regions or constant fragments or crystallizable fragments (Fc). V_HAnd V_LThe interaction forms an Fv comprising an antigen-binding region capable of specifically binding to one or several closely related antigens. Typically, the light chain from the mammal is a kappa light chain or a lambda light chain, and the heavy chain from the mammal is alpha, delta, epsilon, gamma or mu. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG)₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂) Or a sub-category. The term "antibody" encompasses humanized antibodies.

The term "humanized antibody" is understood to mean an antibody comprising human-like variable regions, including the CDR-grafting of an antibody from a non-human species (e.g., mouse) onto or into the FR from a human antibody (this type of antibody is also referred to as "CDR-grafted antibody"). Humanized antibodies also include those in which one or more residues of a human antibody are modified by substitution of one or more amino acids and/or one or more FR residues of a human protein are substituted with the corresponding non-human residues. As exemplified herein, a humanized antibody can also comprise residues that are not found in either a human or non-human antibody. Any other region of the antibody (e.g., the Fc region) is typically human. Humanization may be performed using methods known in the art, for example US5225539, US6054297, US7566771 or US 5585089. The term "humanized antibody" also encompasses super-humanized proteins, e.g. as described in US 7732578.

The terms "full-length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, a full antibody includes an antibody having a heavy chain and a light chain (including an Fc region). The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody is capable of inducing one or more effector functions.

The term "naked antibody" refers to an antibody that is not conjugated to any compound (e.g., a toxic compound or a radiolabel).

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

In the context of the present disclosure, "effector functions" refer to those biological activities mediated by a cell or protein that binds to the Fc region (either the native sequence Fc region or the amino acid sequence variant Fc region) of an antibody that results in cell killing. Examples of effector functions induced by antibodies or immunoglobulins include: complement-dependent cytotoxicity; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); and B cell activation. In the context of the present disclosure, the term or "effector function induced by an antibody" or similar terms may be used interchangeably with "effector function of an immunoglobulin" or "effector function of an antibody" or similar terms and each provides literature support for each other.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors ("FcRs") present on certain cytotoxic cells (e.g., natural killer ("NK") cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed. Effector cells suitable for use in the assay include peripheral blood mononuclear cells ("PBMC") and NK cells.

As used herein, "variable region" refers to a portion of a light chain and/or heavy chain as defined herein that is capable of specifically binding an antigen, and for example includes the amino acid sequence of a CDR; namely CDR1, CDR2 and CDR3, and the Framework Region (FR). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. V_HRefers to the variable region of the heavy chain. V_LRefers to the variable region of the light chain.

As used herein, the term "complementarity determining regions" (synonymous CDRs; i.e., CDR1, CDR2, and CDR3) refer to amino acid residues in the variable region of an antibody whose presence is a major contributor to specific antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR 3. In one embodiment, the amino acid positions assigned to the CDRs and FRs are defined according to the following: kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md.,1987, and 1991 (also referred to herein as "Kabat numbering System". V._HThe FR and CDR positions were as follows: residues 1-30(FR1), 31-35(CDR1), 36-49(FR2), 50-65(CDR2), 66-94(FR3), 95-102(CDR3) and 103-113(FR 4). According to the Kabat numbering system, V_LThe FR and CDR positions are as follows: residues 1-23(FR1), 24-34(CDR1), 35-49(FR2), 50-56(CDR2), 57-88(FR3), 89-97(CDR3) and 98-107(FR 4).

"framework regions" (hereinafter FR) are those variable domain residues other than CDR residues.

The term "constant region" as used herein refers to the portions of the heavy and light chains of an antibody other than the variable regions. In a heavy chain, the constant region typically comprises multiple constant domains and hinge regions, for example an IgG constant region comprises the following linked components: constant heavy chain (C)_H)1, linker, C_H2 and C_H3. In the heavy chain, the constant region comprises Fc. In the light chain, the constant region typically comprises a constant domain (a C)_L1)。

The term "crystallizable fragment" or "Fc region" or "Fc portion" (which are used interchangeably herein) refers to a region of an antibody that comprises at least one constant domain and is generally (but not necessarily) glycosylated and capable of binding one or more Fc receptors and/or components of the complement cascade. The heavy chain constant region may be selected from any one of the following five isoforms: α, δ, ε, γ or μ. Furthermore, the heavy chains of various subclasses (e.g., the IgG subclasses of heavy chains) are responsible for different effector functions, and thus by selecting the desired heavy chain constant region, proteins with the desired effector functions can be made. Exemplary heavy chain constant regions are γ 1(IgG1), γ 2(IgG2), and γ 3(IgG3), or hybrids thereof.

A "constant domain" is a domain in an antibody that has a sequence that is highly similar to a sequence in an antibody/antibody of the same type (e.g., IgG or IgM or IgE). The constant region of an antibody typically comprises a plurality of constant domains, for example the constant region of a gamma, alpha or delta heavy chain comprises two constant domains.

As used herein, the term "specific binding" is understood to mean an immunoglobulin or antibody, meaning that the binding interaction between the immunoglobulin or antibody and the IL-3 Ra chain is dependent on the presence of an epitope or epitope of the IL-3 Ra chain bound by the immunoglobulin or antibody. Thus, the immunoglobulin or antibody preferentially binds or recognizes an IL-3 Ra chain epitope or epitope, even in mixtures present in other molecules or organisms. In one embodiment, the immunoglobulin or antibody reacts or associates with IL-3 Ra or cells expressing IL-3 Ra more often, more rapidly, with a longer duration, and/or with a higher affinity than it reacts or associates with a replacement antigen or cell. It will also be appreciated by reading this definition that an immunoglobulin or antibody that specifically binds IL-3R α may or may not specifically bind a second antigen. Thus, "specific binding" does not necessarily require exclusive binding or binding to another antigen that is not detectable. The terms "specific binding" and "selective binding" are used interchangeably herein. In general, reference herein to binding means specific binding, and each term should be understood to provide explicit support for the other term. ". in one embodiment," specific binding "to IL-3R α or IL-3R α expressing cells means the equilibrium constant (K) for an immunoglobulin or antibody to bind IL-3R α or IL-3R α expressing cells_D) Is 100nM or less, such as 50nM or less, e.g. 20nM or less, such as 1nM or less, e.g.0.8nM or less.

The term "EU numbering system of Kabat" is to be understood as meaning that the numbering of the heavy chains of the antibody is according to the EU index as taught in Kabat et al, 1991, Sequences of Proteins of Immunological Interest, 5 th edition, United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on residue numbering of human IgG1EU antibody.

As used herein, the term "IL-3 Ra-mediated disorder" is understood to mean a disorder associated with or caused by excessive IL-3 Ra expression and/or an excessive number of cells that express IL-3 Ra in a mammal, such as cancer cells (e.g., leukemia cells) and/or immune cells (e.g., plasmacytoid dendritic cells).

As used herein, the term "myelodysplastic syndrome" or "MDS" is understood to mean a diverse collection of hematological medical conditions involving inefficient production (or dysplasia) of myeloid blood cells. Subjects with MDS often develop severe anemia and may require frequent blood transfusions. In many cases, as MDS progresses, subjects develop cytopenia (low blood count) due to progressive bone marrow failure. In about one third of MDS patients, the disease is converted to Acute Myeloid Leukemia (AML). MDS can be diagnosed or classified as a variety of systems, including the French-American-British Classification System (Bennett et al, Br.J. Haematol.33: 451-458, 1976), the International Prognostic Scoring System (Greenberg et al, Blood89:2079-88,1997), or the systems promulgated by the world health organization.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated individual or cell during clinical pathology. Desirable effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease condition, and/or improving prognosis. For example, an individual is successfully "treated" when one or more symptoms associated with the disease are reduced or eliminated.

As used herein, the term "preventing" includes providing prevention with respect to the occurrence or recurrence of a disease in an individual. An individual may be susceptible to or at risk of developing a disease or disease recurrence, but the disease or recurrence has not yet been diagnosed.

As used herein, a mammal that is "at risk" of developing a disease or disorder or relapsing a disease or disorder or relapse may or may not have detectable disease or disease symptoms, and may or may not have shown detectable disease or disease symptoms, prior to treatment according to the present disclosure. By "at risk" is meant that the mammal has one or more risk factors, which are measurable parameters associated with the development of a disease or condition as known in the art and/or described herein.

An "effective amount" is an amount that is at least effective to achieve the desired therapeutic or prophylactic result at the requisite dosage and for the requisite period of time. An effective amount may be provided in one or more administrations. In some embodiments of the present disclosure, the term "effective amount" means an amount necessary to effect treatment of a disease or disorder as described previously. The effective amount may vary depending on the disease or condition to be treated and also depending on the weight, age, ethnic background, sex, health and/or physical condition and other factors associated with the mammal being treated. In general, an effective amount will be within a relatively wide range (e.g., a "dosage" range) that can be determined by routine experimentation and experimentation by a medical practitioner. An effective amount may be administered in a single dose or in doses that are repeated once or several times over a treatment period.

A "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a particular disorder (e.g., SLE). A therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex and weight of the patient and the ability of the immunoglobulin or antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount that outweighs any toxic or deleterious effects of the immunoglobulin or antibody.

A "prophylactically effective amount" is an amount effective, at the requisite dosage and for the requisite period of time, to achieve the desired prophylactic result. Typically, but not necessarily, because a prophylactic dose is administered to a mammal prior to or at an early stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

Reference herein to "the number of NK cells in the mammal" is to be understood as including the number of NK cells in a sample from the mammal and does not require determination of the total number of NK cells in the mammal. This amount can be expressed, for example, as a percentage of the number of cells per mL or dL or the number of cells in a sample taken from the mammal at various points over time.

For purposes of system nomenclature only and without limitation, the amino acid sequence of the IL-3R α chain is taught in Gene ID accession No. 3563 and/or SEQ ID NO: 1.

A "mammal" treated according to the present disclosure can be a mammal such as a non-human primate or human. In one embodiment, the mammal is a human.

The term "sequence identity" is used herein in its broadest sense, including the number of exact nucleotide or amino acid matches using standard algorithms in view of the appropriate alignment, in view of the degree of sequence identity over the window of comparison. Sequence identity can be determined by alignment of comparison sequences using Computer implementation of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package version 7.0, Genetics Computer Group,575Science Drive Madison, WI, USA, incorporated herein by reference) or by tests generated by any of a variety of selected methods and optimal alignment (i.e., the highest percentage of homology over the comparison window). Reference may also be made to the BLAST suite of programs, as disclosed, for example, in Altschul et al, 1997, Nucl. acids Res.253389. A detailed discussion of the sequence analysis can be found IN section 19.3 of Current PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons Inc NY, 1995) -1999) by Ausubel et al.

The disclosure also provides derivatives of the anti-IL-3R α immunoglobulins, antibodies or antigen-binding fragments of the disclosure. As used herein, a "derivative" protein has been altered, for example, by post-translational modifications (e.g., phosphorylation, acetylation, etc.), glycosylation modifications (e.g., addition, removal, or alteration of glycosylation), and/or inclusion of other amino acid sequences as understood in the art.

The term "nucleic acid" as used herein denotes single-or double-stranded DNA and RNA capable of encoding an immunoglobulin, antibody or antigen-binding fragment of the disclosure or a polypeptide component thereof. DNA includes genomic DNA and cDNA. RNA includes mRNA and cRNA. The nucleic acid may also be a DNA-RNA hybrid. A nucleic acid comprises a nucleotide sequence that typically includes nucleotides comprising A, G, C, T or U bases. However, the nucleotide sequence may include other bases such as inosine, methylcytosine, methylinosine, methyladenosine, and/or thiouridine, but is not limited thereto.

"hybridization" is used herein to mean the pairing of nucleotide sequences that are at least partially complementary to produce a DNA-DNA, RNA-RNA, or DNA-RNA hybrid. Hybridization sequences comprising complementary nucleotide sequences occur by base pairing between complementary purines and pyrimidines, as is well known in the art.

In this regard, it is understood that modified purines (e.g., inosine, methylinosine, and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine) can also be joined in base pairing.

"stringency" as used herein refers to the temperature and ionic strength during hybridization and the presence or absence of certain organic solvents and/or detergents. The higher the stringency, the higher the degree of complementarity desired between the hybridizing nucleotide sequences.

"high stringency conditions" refer to those conditions under which only nucleic acids with high complementary base frequencies can hybridize.

References herein to high stringency conditions include and encompass: -

(i) At least about 31% v/v to at least about 50% v/v formamide and at least about 0.01M to at least about 0.15M salt for hybridization at 42 ℃, and at least about 0.01M to at least about 0.15M salt for washing at 42 ℃;

(ii)1%BSA、1mM EDTA、0.5M NaHPO₄(ph7.2), 7% SDS for hybridization at 65 ℃, and (a)0.1 x SSC, 0.1% SDS; or (b)0.5% BSA, 1mM EDTA, 40mM NaHPO₄(ph7.2), 1% SDS for about one hour at a temperature in excess of 65 ℃; and

(iii)0.2 XSSC, 0.1% SDS was used for washing at 68 ℃ or above 68 ℃ for about 20 minutes.

Generally, the wash is at T_m=69.3+0.41(G + C)% -12 ℃. Typically, for every 1% increase in the number of mismatched bases, the Tm of the duplex DNA decreases by about 1 ℃.

Although described 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 & Sons, Inc.1995-2009.

An "expression vector" may be a self-replicating extra-chromosomal vector (e.g., a plasmid) or a vector that integrates into the host genome.

Immunoglobulins

Suitably, the immunoglobulin or antibody of the disclosure selectively binds to an IL-3 ra chain, by which is meant that the binding interaction between the immunoglobulin or antibody and an IL-3 ra chain is dependent on the presence of an epitope or epitope of the IL-3 ra chain to which the immunoglobulin binds. Thus, the immunoglobulin or antibody preferentially binds or recognizes an IL-3 Ra chain epitope or epitope, even in mixtures present in other molecules or organisms.

Antibodies

In one embodiment, the immunoglobulin described herein according to any embodiment is an antibody, e.g., an antibody comprising a CDR and/or one or more variable regions described herein.

Suitably, the immunoglobulin is a humanized, chimeric or human antibody comprising light and heavy chain CDR1, 2 and 3 amino acid sequences according to SEQ ID NOS: 2-7, respectively. In one embodiment, the antibody comprises the heavy chain variable region amino acid sequence of SEQ ID NO. 9 and the light chain variable region amino acid sequence of SEQ ID NO. 8. In one embodiment, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO 10, 11 or 12 and the light chain amino acid sequence of SEQ ID NO 13.

In one embodiment, the antibody is a recombinant antibody. Methods of making antibodies comprising CDRs and/or variable regions described herein will be apparent to those of skill in the art based on the disclosure herein and/or the references mentioned herein.

In one embodiment, an antibody of the disclosure comprises a V comprising a CDR shown herein and a FR from a human antibody_HAnd/or V_L. Optionally, the FR comprises one or more amino acid substitutions, for example 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 embodiment, the FR comprises no more than 30 amino acid substitutions, such as no more than 20 amino acid substitutions, as compared to a human FR.

In one embodiment, an antibody of the disclosure comprises a V comprising a CDR shown herein and a FR from a non-human primate antibody_HAnd/or V_LI.e. the antibody is a co-humanized antibody, e.g. as described in WO 2007/019620.

In one embodiment, the antibodies of the present disclosure are complex antibodies, e.g., as described in WO 2006/082406.

An exemplary antibody of the present disclosure is a humanized antibody, e.g., as defined herein. Exemplary humanized antibodies are described herein. In one embodiment, the humanized antibody has been affinity matured. In one embodiment, the humanized antibody comprises:

(i) v is as follows_L：

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

b) CDR2 comprising (or encoded by) the sequence shown in SEQ ID NO: 3; 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) v is as follows_H：

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 shown in SEQ ID NO:7 (or encoded by the sequence shown in SEQ ID NO: 19).

It will be appreciated by those skilled in the art that the immunoglobulins or antibodies of the present disclosure may include modifications introduced into sequences not naturally found in humans, for example, to enhance affinity or effector function.

The present disclosure also provides variants of the immunoglobulins or antibodies of the present disclosure. Suitably, the variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOS 2-13.

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

Derivatives of antibodies or immunoglobulins contemplated by the present disclosure include, but are not limited to, modification of amino acid side chains, incorporation of unnatural amino acids and/or derivatives thereof during peptide or polypeptide synthesis, and use of cross-linking agents.

Exemplary antibodies and antigen-binding fragments thereof of the present disclosure are described in table 1.

Table 1: exemplary antibodies and antigen-binding fragments thereof

Name (R)	Heavy chain SEQ ID NO:	light chain SEQ ID NO:
			CSL362Fv	91	81

CSL362	10	13
			CSL362B	102	13
CSL362X1	11	13
			CSL362X2	12	13

¹only variable region sequences.

²The heavy chain constant region is afucosylated.

Antibody fragments

Single domain antibodies

In some embodiments, an antigen-binding fragment of an antibody of the present disclosure is or comprises a single domain antibody (which is used interchangeably with the terms "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain comprising all or a portion of an antibody heavy chain variable domain.

Bifunctional, trifunctional, and tetrafunctional antibodies

In some embodiments, the antigen binding fragments of the present disclosure are or comprise bifunctional, trifunctional, tetrafunctional or higher protein complexes, such as those described in WO98/044001 and/or WO 94/007921.

For example, a bifunctional antibody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising structure V_L-X-V_HOr V_H-X-V_LWherein X is V which is not sufficiently contained to permit a single polypeptide chain_HAnd V_LA linker of residues associating (or forming Fv) or not, and V of one polypeptide chain_HIs combined with anotherV of a polypeptide chain_LTo form an antigen binding site, i.e., to form an Fv molecule capable of specifically binding to one or more antigens. V in each polypeptide chain_LAnd V_HV may be the same or in separate polypeptide chains_LAnd V_HMay be different in order to form a bispecific diabody (i.e., comprising two fvs of different specificity).

Bifunctional, trifunctional, tetrafunctional antibodies, and the like, capable of inducing effector activity can be made using an antigen binding domain capable of binding IL-3 ra and an antigen binding domain capable of binding a cell surface molecule on an immune cell (e.g., a T cell, such as CD 3).

Single chain fv (scFv) fragments

One skilled in the art will appreciate that scFv comprise V in a single polypeptide chain_HAnd V_LRegion and at V_HAnd V_LComprising a polypeptide linker therebetween that enables the scFv to form the desired structure for antigen binding (i.e., enables the V of a single polypeptide chain)_HAnd V_LCapable of associating with each other to form Fv). For example, the linker comprises more than 12 amino acid residues, of which (Gly)₄Ser)₃Is a more advantageous linker for scFv.

The disclosure also encompasses disulfide-bond stabilized Fv (or divv or dsFv) wherein at V_HFR and V of_LThe FR of (a) has a single cysteine residue introduced therein and the cysteine residues are linked by a disulfide bond to produce a stable Fv.

Alternatively or additionally, the present disclosure encompasses dimeric scfvs, i.e., proteins comprising two scFv molecules linked by a non-covalent or covalent linkage (e.g., via a leucine zipper domain (e.g., derived from Fos or Jun)). Alternatively, the two scfvs are linked by a peptide linker of sufficient length to allow the two scfvs to form and bind antigen, e.g., as described in US 20060263367.

The present disclosure also encompasses dimeric scfvs capable of inducing effector activity. For example, one scFv binds IL-3 ra and comprises the CDRs and/or variable regions described herein and another scFv binds a cell surface molecule on an immune cell (e.g., a T cell, such as CD3 or CD 19). In one embodiment, the dimeric protein is a combination of a dAb and a scFv. Examples of bispecific antibody fragments capable of inducing effector functions are described in e.g. US 7235641.

Other antibodies and antibody fragments

Other antibodies and antibody fragments are also encompassed by the present disclosure, such as:

(i) a "keyhole and hole" bispecific protein as described in US5,731,168;

(ii) heteroconjugated proteins, such as described in US4,676,980;

(iii) heteroconjugated proteins made using chemical cross-linking agents, such as described in US4,676,980; and

(iv)Fab₃(e.g. as described in EP 19930302894).

Constant region

The present disclosure encompasses immunoglobulins and antibodies and antigen-binding fragments comprising the constant region of an antibody and/or the Fc region of an antibody.

Suitable sequences for making the constant regions and/or Fc regions of the immunoglobulins, antibodies or antigen-binding fragments of the present disclosure may be obtained from a variety of different sources. In some embodiments, the constant region, Fc, or portion thereof of the immunoglobulin, antibody, or antigen binding fragment is derived from a human antibody. The constant region, Fc or portion thereof may be derived from any antibody class, including IgA, IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG 4. In one embodiment, the constant region or Fc is human isotype IgG1 or human isotype IgG2 or human isotype IgG3 or a hybrid of any of the above isotypes.

In one embodiment, 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 IgG3Fc region. In another embodiment, the constant region or Fc region is a hybrid of IgG1 and IgG2 or IgG1 and IgG3 or Fc region or IgG2 and IgG 3. Exemplary hybrids of human IgG1 with the IgG2 constant region or Fc region are described in Chappel et al, Proc.Natl Acad.Sci.USA,88: 9036-.

Methods of determining whether an Fc region may or may not induce effector function will be apparent to those skilled in the art and/or described herein.

Effect function

Suitably, the anti-IL-3R α immunoglobulins, antibodies or antigen-binding fragments of the present disclosure have or are shown to contribute to IL-3R α⁺Cells or cells rendering IL-3R alpha⁺The cell is capable of at least partially removing, substantially removing, or eliminating an effector function. Such effector functions may be enhanced binding affinity to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).

As will be apparent to those of skill in the art based on the description herein, some embodiments of the present disclosure include an immunoglobulin, antibody, or antigen binding fragment capable of inducing an effector function.

For IgG class antibodies, these effector functions are limited by the binding of the Fc region to a family of receptors known as Fc γ receptors (Fc γ R) expressed on a variety of immune cells and/or to complement (e.g., C1 q). Formation of the Fc/Fc γ R complex recruits these cells to the site of the bound antigen, typically resulting in signaling and subsequent immune responses. Methods of optimizing the binding affinity of an Fc γ R to an antibody Fc region in order to enhance effector function, in particular to alter 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 an antibody to enhance its interaction with the relevant Fc receptor and increase its potential to promote ADCC and ADCP. It has also been described that ADCC activity is enhanced after modification of the oligosaccharide linked to the conserved Asn297 in the Fc region of IgG1 antibody.

In this regard, it is understood that, in some non-limiting embodiments, enhancing effector function (e.g., ADCC) may be achieved by modifying an immunoglobulin or antibody with a normally glycosylated wild-type constant domain, including altering or removing glycosylation (see, e.g., WO00/61739) and/or amino acid sequence mutations (see, e.g., WO 2008036688).

In one embodiment, the immunoglobulin, antibody or antigen binding fragment binds IL-3R α in a manner such that it is capable of inducing effector function (e.g., ADCC).

In one embodiment, the immunoglobulin, antibody or antigen binding fragment binds an epitope within IL-3R α that allows it to induce effector function (e.g., ADCC).

In another embodiment, the immunoglobulin, antibody or antigen binding fragment is capable of binding to IL-3R α on cells in a mammal, thereby inducing an effector function, such as ADCC.

For example, the immunoglobulin, antibody or antigen binding fragment remains bound to IL-3 Ra on the surface of a cell for a time sufficient to induce effector function (e.g., ADCC). For example, the immunoglobulin or antibody does not internalize too quickly, thereby allowing ADCC to be induced.

Alternatively or additionally, the immunoglobulin, antibody or antigen binding fragment binds to IL-3 ra on the surface of a cell in a manner that allows immune effector cells to bind to the constant region or Fc region in the immunoglobulin, antibody or antigen binding fragment and induce effector function (e.g., ADCC). For example, the Fc region of an immunoglobulin, antibody or antigen binding fragment is exposed in a manner that enables the immunoglobulin, antibody or antigen binding fragment to interact with an Fc receptor (e.g., fcyr) on an immune effector cell when bound to IL-3 ra. In the context of the present disclosure, the term "immune effector cell" is understood to mean any cell expressing an Fc receptor and capable of killing the cell to which it binds by ADCC or ADCP. In one embodiment, the immune effector cell is an NK cell.

The above paragraphs directed to immunoglobulins, antibodies or antigen-binding fragments are understood to apply mutatis mutandis to the induction of CDC. For example, an immunoglobulin, antibody or antigen-binding fragment binds to IL-3 ra on the surface of a cell in a manner that allows complement component C1q to bind to the constant region or Fc region in the immunoglobulin, antibody or antigen-binding fragment and induce CDC.

In one embodiment, the immunoglobulin, antibody or antigen binding fragment is capable of inducing an enhanced level of effector function.

In one embodiment, the level of effector function induced by the constant region or Fc region is enhanced relative to a wild-type IgG1 antibody constant region or Fc region or a wild-type IgG3 antibody constant region or Fc region.

In another embodiment, the constant region or Fc region is modified to enhance the level of effector function it is capable of inducing compared to a constant region or Fc region that is not modified. The modifications may be at the amino acid level and/or secondary structure level and/or tertiary structure level and/or glycosylation of the constant region or Fc region.

One skilled in the art will appreciate that higher effector function may be manifested in any of a variety of ways, such as by a higher level of action, more sustained action, or a faster rate of action.

For example, an anti-IL-3R α immunoglobulin, antibody or antigen-binding fragment has or exhibits effector functions including anti-junction-dependent cell-mediated cytotoxicity (ADCC).

In one embodiment, the constant region or Fc region comprises one or more amino acid modifications that enhance its ability to induce enhanced effector functions. In one embodiment, the constant region or Fc region binds one or more fcyr with higher affinity. In one embodiment, the constant region or Fc region has an affinity for fcyr that is greater than 1-fold or greater than 5-fold or between 5-fold and 300-fold the affinity of the wild-type constant region or Fc region. In one embodiment, the constant region or Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 230. 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332 and 335, numbered according to the EU index of Kabat. In one embodiment, the constant region or Fc region comprises at least one amino acid substitution selected from the group consisting of: p230, E233, L234, L235, S239, V240, F243, V264, V266, E272, K274, F275, N276, Y278, V302, E318, S324, N325, K326, L328, a330, I332, T335, and T335, numbered according to the EU index of Kabat. In one embodiment, the constant region or Fc region comprises an amino acid substitution selected from the group consisting of: v264, F243/V264, L328, I332, L328/I332, V264/I332, S298/I332, S239, A330, I332, L328/I332, V264, V240, V266, S239/I332, A330/I332, V264/A330/I332, L234, L235, S239, V240, V264, A330, N325, L328/I332, L264/I328, L328/I328, S328/I332, S239/, S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E, S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264I/I332E, S239D/V264I/S298A/I332E, S239E/V264E/A330E/I332E, S239E/I332E/A330E, P230E/E233E/I332E, E272E, K274E, K335, K E, K274F 275E, N E, Y36278E, V264E 302, K274E 72, K E/S239/S E/S239/S E/I332E/S239/S E/I E/S E/I E/S E/I E, S E/I E, S E/I E, S E/I E, S E/I E, S E/I E.

In another embodiment, the constant region or Fc region binds Fc γ RIIIa more efficiently than Fc γ RIIb. For example, the constant region or Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 234. 235, 239, 240, 264, 296, 330 and I332, numbering according to the EU index of Kabat. In one embodiment, the constant region or Fc region comprises at least one amino acid substitution selected from the group consisting of: L234Y, L234I, L235I, S239D, S239E, S239N, S239Q, V240A, V240M, V264I, V264Y, Y296Q, a330L, a330Y, a330I, I332D and I332E, numbered according to the EU index of Kabat. For example, the constant region or Fc region comprises amino acid substitutions selected from the group consisting of: i332, V264/I332, S239/I332, Y296, A330, I332, S239/I332, A330/I332, V264/A330/I332, L234, L235, V240, V264, A330, S239/A330/I332, S239/S298/I332, S239/V264/S298/I332 and S239/V264/A330/I332, numbering according to the EU index of Kabat.

In another embodiment, the constant region or Fc region induces ADCC at a level greater than the level of ADCC mediated by the wild type constant region or Fc region. For example, the level of ADCC induced by the constant region or Fc region is more than 5-fold or between 5-fold and 1000-fold greater than the level of ADCC induced by the wild-type constant region or Fc region. In one embodiment, the constant region or Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 230. 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332 and 335, numbered according to the EU index of Kabat. In one embodiment, the constant region or Fc region comprises at least one amino acid substitution selected from the group consisting of: p230, E233, L234, L235, S239, V240, F243, V264, V266, E272, K274, F275, N276, Y278, V302, E318, S324, N325, K326, L328, a330, I332, T335, and T335, numbered according to the EU index of Kabat. In one embodiment, the constant region or Fc region comprises an amino acid substitution selected from the group consisting of: v264, F243/V264, L328, I332, L328/I332, V264/I332, S298/I332, S239, A330, I332, L328/I332, V264, V240, V266, S239/I332, A330/I332, V264/A330/I332, L234, L235, S239, V240, V264, A330, N325, L328/I332, L264/I328, L328/I328, S328/I332, S239/, S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E, S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264I/I332E, S239D/V264I/S298A/I332E, S239E/V264E/A330E/I332E, S239E/I332E/A330E, P230E/E233E/I332E, E272E, K274E, K335, K E, K274F 275E, N E, Y36278E, V264E 302, K274E 72, K E/S239/S E/S239/S E/I332E/S239/S E/I E/S E/I E/S E/I E, S E/I E, S E/I E, S E/I E, S E/I E, S E/I E.

In one embodiment, the constant region or Fc region comprises the following amino acid substitutions S239D/I332E, numbered according to the EU index of Kabat. The affinity of this constant region or Fc region for fcyriiia is increased by about 14-fold compared to the wild-type constant region or Fc region and the ability to induce ADCC is increased by about 3.3-fold compared to the wild-type constant region or Fc region. In one embodiment, the constant region comprises the sequence shown between residues 121-450 of SEQ ID NO. 11, including residues 121-450. In one embodiment, the constant region comprises the sequence shown between residues 234 and 450 of SEQ ID NO 11.

In one embodiment, the constant region or Fc region comprises the following amino acid substitutions S239D/a330L/I332E, numbered according to the EU index of Kabat. The affinity of this constant region or Fc region for fcyriiia is increased by about 138-fold compared to the wild-type constant region or Fc region and the ability to induce ADCC is increased by about 323-fold compared to the wild-type constant region or Fc region. In one embodiment, the constant region comprises the sequence shown between residues 121-450 of SEQ ID NO 12, including residues 121-450. In one embodiment, the constant region comprises the sequence shown between residues 234 and 450 of SEQ ID NO 12.

Other amino acid substitutions that enhance the ability of the Fc region to induce effector functions are known in the art and/or described in, for example, US6737056 or US 7317091.

In one embodiment, the glycosylation of the constant region or Fc region is altered to enhance its ability to induce enhanced effector functions. In this regard, natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, typically linked by an N-type linkage to a C of a constant or Fc region_H2 domain Asn 297. Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the constant region or Fc region of the present disclosure comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region, i.e., the Fc region is "afucosylated". The variant may have an increased ability to induce ADCC. Methods for making afucosylated antibodies include expressing an immunoglobulin or antibody in a cell line incapable of expressing alpha-1, 6-fucosyltransferase (FUT8) (e.g., as described in Yunane-Ohnuki et al, Biotechnol. Bioengineer.,87:614-622, 2004), expressing an immunoglobulin or antibody in a cell expressing small interfering RNA against FUT8 (e.g., as described in Mori et al, Biotechnol. Bioen. Biongeneer, 88:901-908, 2004), expression of immunoglobulins or antibodies in cells incapable of expressing Guanosine Diphosphate (GDP) -mannose 4, 6-dehydratase (GMD) (e.g.as described by Kanda et al, J.Biotechnol.,130:300-310, 2007). The present disclosure also encompasses the use of immunoglobulins with reduced levels of fucosylation, such as immunoglobulins produced using cells modified to express β - (1,4) -N-acetylglucosaminyltransferase III (GnT-III) (e.g., as described in Um ā na et al, nat. Biotechnol.,17:176-180, 1999).

In one embodiment, the immunoglobulin or antibody of the present disclosure is afucosylated. For example, the immunoglobulin or antibody is produced in a cell (e.g., a mammalian cell, such as a CHO cell) that does not express FUT 8.

Other methods include the use of cell lines that inherently produce antibodies capable of inducing enhanced Fc-mediated effector function (e.g., duck embryo-derived stem cells for the manufacture of viral vaccines, WO 2008/129058; in aviansRecombinant protein production in cells, WO 2008/142124).

Immunoglobulins suitable for use in the methods of the present disclosure also include those having bisected oligosaccharides, for example where the biantennary oligosaccharide attached to the constant region or Fc region is bisected by GlcNAc. The immunoglobulin may have reduced fucosylation and/or improved ADCC function. Examples of such immunoglobulin proteins are described in e.g. US6602684 and US 20050123546.

Immunoglobulins having at least one galactose residue in an oligosaccharide linked to a constant region or Fc region are also contemplated. The immunoglobulin may have increased CDC function. Such immunoglobulins are described, for example, in WO1997/30087 and WO 1999/22764.

Non-limiting examples of immunoglobulins that induce enhanced levels of ADCC activity include

(i) An antibody comprising a heavy chain comprising the sequence shown in SEQ ID NO. 11 and a light chain comprising the sequence shown in SEQ ID NO. 13;

(ii) an antibody comprising a heavy chain comprising the sequence shown in SEQ ID NO. 12 and a light chain comprising the sequence shown in SEQ ID NO. 13; and

(iii) an antibody comprising a heavy chain comprising the sequence shown in SEQ ID NO. 10 and a light chain comprising the sequence shown in SEQ ID NO. 13, wherein the heavy chain constant region is afucosylated.

Advantageous antibodies of the present disclosure that induce enhanced levels of ADCC activity comprise a heavy chain comprising the sequence shown in SEQ ID NO. 11 and a light chain comprising the sequence shown in SEQ ID NO. 13. As discussed herein, following administration of such an antibody to a mammal (e.g., at least 7 or 8 or 11 or 17 or 22 or 29 days following administration), the number of NK cells in the mammal is increased as compared to one or more of:

(i) the number of NK cells in the mammal prior to administration of the antibody;

(ii) number of NK cells in a mammal administered an antibody comprising a heavy chain comprising the sequence shown in SEQ ID NO. 10 and a light chain comprising the sequence shown in SEQ ID NO. 13, wherein the heavy chain constant region is afucosylated (e.g., as assessed on the same day after administration); and

(iii) the number of NK cells in a mammal administered an antibody that specifically binds to IL-3 ra chain and has a human IgG1 constant region (e.g., as assessed on the same day following administration).

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

Other modifications

Other modifications to the immunoglobulin are also contemplated by the present disclosure.

For example, an immunoglobulin or antibody comprises one or more amino acid modifications 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 enhance the affinity of the constant region or Fc region for a neonatal Fc region (FcRn). For example, the constant region or Fc region has an increased affinity for FcRn at lower pH (e.g., about pH6.0), thereby facilitating Fc/FcRn binding in endosomes. In one embodiment, the affinity of the constant region or Fc region for FcRn at about pH6 is enhanced compared to its affinity at about pH7.4, which facilitates the re-release of the constant region or Fc into the blood following cell recirculation. These amino acid substitutions are useful for extending the half-life of immunoglobulins by reducing blood clearance.

Exemplary amino acid substitutions include T250Q and/or M428L, according to the EU numbering system of Kabat. Other or alternative amino acid substitutions are described, for example, in US 20070135620.

Nucleic acids

Another embodiment of the disclosure provides isolated nucleic acids encoding the immunoglobulins or antibodies of the disclosure (including fragments, variants, and derivatives of immunoglobulins or antibodies).

Certain examples of nucleic acids comprise the nucleotide sequences set forth in one or more of SEQ ID NOS 14-19, SEQ ID NOS 14-19 encode the CDRs 1-6, respectively, of the immunoglobulins or antibodies disclosed.

Other examples of nucleic acids include the nucleotide sequences shown in one or more of SEQ ID NOS 20-23.

The present disclosure also encompasses nucleic acid homologs encoding variants of the immunoglobulins or antibodies of the present disclosure as described above.

Exemplary nucleic acid homologs share at least 80% or 85%, such as at least 90% or 95% or 99% nucleotide sequence identity with an isolated nucleic acid encoding any of SEQ ID NOs 2-13. Suitably, the nucleic acid homologue does not encode a protein comprising a murine variable region capable of specifically binding IL-3 ra.

Nucleic acid homologs can hybridize to an isolated nucleic acid of the disclosure under high stringency conditions.

Protein production

Recombinant expression

In one embodiment, the immunoglobulin or antibody described herein according to any embodiment is recombinant.

By way of example only, the recombinant immunoglobulins or antibodies of the present disclosure may be made by a method comprising the steps of:

(i) preparing an expression construct comprising an isolated nucleic acid of the present disclosure operably linked to one or more regulatory nucleotide sequences (e.g., a promoter);

(ii) transfecting or transforming a suitable host cell with the expression construct;

(iii) expressing a recombinant immunoglobulin or antibody in the host cell; and

(iv) isolating the recombinant immunoglobulin or antibody from the host cell.

In the case of recombinant immunoglobulins, the nucleic acid encoding it may be cloned into an expression vector, which is then transfected into a host cell, such as an escherichia coli (e.coli) cell, a yeast cell, an insect cell, or a mammalian cell, such as a simian COS cell, a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a myeloma cell that does not otherwise produce immunoglobulin or antibody proteins.

Exemplary cells for expressing immunoglobulins or antibodies are CHO cells, myeloma cells, or HEK cells. The cell may further comprise one or more mutations and/or deletions that facilitate expression of the modified immunoglobulin or antibody. One non-limiting example is a deletion of a gene encoding an enzyme required for fucosylation of the expressed immunoglobulin or antibody. For example, the deleted gene encodes FUT 8. One commercially available source of FUT 8-deficient CHO cells isBiowa(Potelligent^TMA cell). For example, cells for expression of afucosylated immunoglobulins or antibodies are FUT 8-deleted CHO cells, such as the Potelligent of Biowa^TMA cell.

Molecular Cloning techniques for achieving these objectives are known in the art and are described in, for example, Ausubel et al (eds.), 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 cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods of making recombinant antibodies are also known in the art. See US4816567 or US 5530101.

After isolation, the nucleic acid is inserted into a promoter operably linked to a gene construct or expression vector for further cloning (amplification of the DNA) or expression in a cell-free system or cell. Thus, another embodiment of the disclosure provides a genetic construct comprising an isolated nucleic acid of the disclosure and one or more additional nucleotide sequences. Suitably, the genetic construct is in the form of or comprises a genetic component in the form of a plasmid, phage, cosmid, yeast or bacterial artificial chromosome, as is well known in the art. The genetic constructs may be suitable for maintaining and propagating the isolated nucleic acid in a bacterial or other host cell for manipulation by recombinant DNA techniques and/or expression of the nucleic acids of the disclosure or the encoded immunoglobulins or antibodies. For the purpose of host cell expression, the genetic construct is an expression construct. Suitably, an expression construct comprises a nucleic acid of the disclosure operably linked to one or more other sequences (e.g., a promoter or regulatory sequence) in an expression vector.

In general, regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences.

As used herein, the term "promoter" is to be understood in its broadest sense and includes transcriptional regulatory sequences of genomic genes, including the TATA box or initiator element required for precise initiation of transcription, with or without other regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter nucleic acid expression (e.g., in response to developmental and/or external stimuli, or in a tissue-specific manner). In the context of the present disclosure, the term "promoter" is also used to describe a recombinant, synthetic or fused nucleic acid, or derivative that confers, activates or enhances expression of the nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more particular regulatory elements to further enhance expression and/or alter spatial expression and/or temporal expression of the nucleic acid.

As used herein, the term "operably linked" means that a promoter is positioned relative to a nucleic acid such that expression of the nucleic acid is under the control of the promoter.

A variety of vectors are available for expression in cells. Carrier components typically include, but are not limited to, one or more of the following: signal sequences, immunoglobulin or antibody encoding sequences (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. One skilled in the art will appreciate sequences suitable for expression of immunoglobulins. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II), yeast secretion signals (e.g., invertase leader, alpha factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammals include cytomegalovirus very early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), micronucleus RNA promoter (U1a and U1b), alpha myosin heavy chain promoter, simian virus 40 promoter (SV40), rous sarcoma virus promoter (RSV), adenovirus major late promoter, beta actin promoter; a hybridization regulatory element comprising a CMV enhancer/beta actin promoter or an immunoglobulin or antibody promoter or active fragment thereof. Examples of suitable host cell lines are monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or chinese hamster ovary Cells (CHO).

Typical promoters suitable for expression in a yeast cell, such as a yeast selected from the group consisting of Pichia pastoris (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Schizosaccharomyces pombe (S.pombe), include, but are not limited to, the ADHl promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.

The manner for introducing an isolated nucleic acid or an expression construct comprising the nucleic acid into a cell for expression is known to those of skill in the art. The technique used to specify the cells depends on known successful techniques. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran-mediated transfection, transfection mediated by liposomes (e.g., using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA)), PEG-mediated DNA uptake, electroporation, and microprojectile bombardment (e.g., using DNA-coated tungsten or gold particles (Agracetus inc., WI, USA)), among others.

Host cells for the production of immunoglobulins or antibodies may be cultured in a variety of media, depending on the cell type used. Commercially available media (e.g., Ham's F10(Sigma), minimal essential Medium ((MEM), (Sigma)), RPMl-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing mammalian cells.

Isolation of proteins

Methods for purifying immunoglobulins or antibodies are known in the art and/or described herein.

When the immunoglobulin or antibody is secreted into the culture medium, the supernatant from the expression system can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (e.g., PMSF) may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

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

Those skilled in the art will also appreciate that the immunoglobulin or antibody may be modified to include a tag to facilitate purification or detection, such as a polyhistidine tag, for example, a hexa-polyhistidine tag, or an influenza Hemagglutinin (HA) tag, or a simian virus 5(V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting immunoglobulin or antibody is then purified using methods known in the art, such as affinity purification. For example, an immunoglobulin or antibody comprising a hexahistidine tag can be purified by contacting a sample comprising the immunoglobulin or antibody with nickel-nitrilotriacetic acid (Ni-NTA) specifically binding to the hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound immunoglobulin, and then eluting bound immunoglobulin. Alternatively or additionally, a ligand or antibody that binds to a tag is used in the affinity purification method.

In one embodiment, the immunoglobulin or antibody further has a protease cleavage site, such as factor X_aOr a protease cleavage site of thrombin, which allows partial digestion of immunoglobulins by the relevant proteaseOr the antibody and thereby release the immunoglobulin or antibody from the tag. The released antibody or immunoglobulin can then be separated from the fusion partner by subsequent chromatographic separation.

Analysis of immunoglobulin Activity

The immunoglobulins or antibodies of the present disclosure can be readily screened for biological activity, e.g., as described below.

Binding assays

One form of such assay is an antigen binding assay, such as described in Scopes (Protein purification: principles and practice, third edition, Springer Verlag, 1994). Such methods typically involve labeling an immunoglobulin or antibody and contacting it with an immobilized antigen. After washing to remove non-specifically bound proteins, the amount of label and thus the amount of bound protein is detected. Of course, it is possible to immobilize either immunoglobulins or antibodies and label the antigen. Panning-type analysis, such as described or exemplified herein, may also be used.

Determination of neutralization

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

Various assays for assessing the ability of an immunoglobulin neutralizing ligand to signal through a receptor are known in the art.

In one embodiment, the immunoglobulin or antibody reduces or prevents binding of IL-3 to a 3R α chain and/or a heterodimer of an IL-3R α chain and an IL-3R β chain. These assays can be performed using labeled IL-3 and/or labeled immunoglobulin as competitive binding assays. For example, the labeled IL-3R α or extracellular region thereof fused to the Fc region of the antibody or immobilized IL-3R-expressing cells and labeled IL-3 are then contacted with the immobilized receptor or cells in the presence or absence of the test immunoglobulin or antibody and the amount of bound label is detected. In antibodies or immunizationsA decrease in the amount of label bound in the presence of immunoglobulin compared to that in the absence of protein indicates that the immunoglobulin or antibody reduces or prevents IL-3 from binding to IL-3R. Determination of IC by testing various concentrations of immunoglobulin or antibody₅₀I.e., the concentration of protein that reduces the amount of IL-3 that binds IL-3R, or EC can be determined₅₀I.e., a protein concentration that achieves 50% of the maximal inhibition of IL-3 binding to IL-3R achieved by the immunoglobulin or antibody.

In another embodiment, the immunoglobulin or antibody reduces or prevents IL-3 mediated release of histamine from basophils. For example, low density leukocytes comprising basophils are incubated with IgE, IL-3, and various concentrations of immunoglobulin or antibody. Control cells did not contain immunoglobulin (positive control) or IL-3 (negative control). The level of histamine released is then assessed using standard techniques (e.g., RIA). Immunoglobulins or antibodies that reduce histamine release levels to levels below the positive control are considered to neutralize IL-3 signaling. In one embodiment, the reduced level is correlated with immunoglobulin or antibody concentration. An exemplary method for assessing IL-3 mediated histamine release is described, for example, in Lopez et al, j.cell.physiol.,145:69,1990.

In another embodiment, the immunoglobulin or antibody reduces or prevents IL-3 mediated proliferation of leukemia cell line TF-1. For example, TF-1 cells are cultured in the absence of IL-3 or GM-CSF for a time sufficient to stop their proliferation (e.g., 24-48 hours). The cells were then cultured in the presence of IL-3 and various concentrations of immunoglobulin or antibody. Control cells were not contacted with immunoglobulin or antibody (positive control) or IL-3 (negative control). Followed by standard techniques (e.g. using³H-thymidine incorporation) to assess cell proliferation. An immunoglobulin or antibody that reduces or prevents cell proliferation in the presence of IL-3 to a level below that of the positive control is considered to neutralize IL-3 signaling.

Another assay for assessing neutralization of IL-3 signaling involves determining whether an immunoglobulin or antibody reduces or prevents IL-3 mediated effects on endothelial cells. For example, Human Umbilical Vein Endothelial Cells (HUVECs) are cultured in the presence of IL-3 (optionally containing IFN-. gamma.) and various concentrations of immunoglobulin or antibody. The amount of secreted IL-6 is then assessed using, for example, an enzyme-linked immunosorbent assay (ELISA). Control cultures did not contain immunoglobulin or antibody (positive control) or IL-3 (negative control). An immunoglobulin or antibody that reduces or prevents IL-6 production in the presence of IL-3 to a level below that of the positive control is considered to neutralize IL-3 signaling.

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

Determination of Effect function

Methods for assessing ADCC activity are known in the art.

In one embodiment, use is made of⁵¹Cr release analysis, europium release analysis or³⁵S release assay to assess the level of ADCC activity. In each of these assays, cells expressing IL-3 Ra are cultured with one or more of the compounds under conditions and for a time sufficient for the compounds to be taken up by the cells. In that³⁵In the case of S release assays, IL-3R α -expressing cells can be combined with³⁵The S-labeled methionine and/or cysteine are incubated together for a time sufficient to incorporate the labeled amino acid into the newly synthesized protein. The cells are then cultured in the presence or absence of immunoglobulin or antibodies and in the presence of immune effector cells, such as Peripheral Blood Mononuclear Cells (PBMCs) and/or NK cells. Followed by detection in cell culture medium⁵¹Cr, europium and/or³⁵S, and an increase in the presence of the immunoglobulin or antibody compared to in the absence of the immunoglobulin indicates that the immunoglobulin has effector function. Exemplary publications disclosing methods for assessing the level of ADCC induced by immunoglobulins include Hellstrom et al, Proc. Natl Acad. Sci. USA83: 7059. 7063,1986 and Bruggemann et al, J.exp. Med.166: 1351. 1361, 1987.

Water for evaluating ADCC induced by immunoglobulin or antibodyOther analyses of the flat include ACTI for flow cytometry^TMNonradioactive cytotoxicity assay (Celltechnology, Inc. CA, USA) or CytoToxNon-radioactive cytotoxicity assay (Promega, WI, USA).

Alternatively or additionally, the effector function of an immunoglobulin or antibody is assessed by determining the affinity of the immunoglobulin or antibody for one or more fcyr, for example as described in US 7317091.

C1q binding assays may also be performed to confirm that the immunoglobulin or antibody is capable of binding C1q and can induce CDC. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, j.

Determination of NK cell number

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

In one embodiment, after administering an immunoglobulin or antibody of the disclosure to a mammal (e.g., a non-human mammal, such as a non-human primate, e.g., cynomolgus monkey), a sample of blood (or serum) is obtained and the number of NK cells is assessed using Fluorescence Activated Cell Sorting (FACS). NK cells may be detected based on expression of CD16 and/or CD56 and/or lack of expression (or low level of expression) of CD20 and/or CD 3. The percentage change in NK cell number can be determined by comparison with the number of NK cells in a sample obtained earlier (e.g. before administration of antibody or immunoglobulin).

Determination of affinity

Optionally, determining the dissociation constant (Kd) or association constant (Ka) or equilibrium constant (K) of the protein for IL-3R alpha or an epitope thereof_D)。In one embodiment, these constants of the immunoglobulin or antibody are measured by a radiolabelled or fluorescently labeled IL-3 Ra binding assay. This assay balances the protein with the lowest concentration of labeled IL-3 Ra (or a soluble form thereof, e.g., an extracellular region comprising IL-3 Ra fused to an Fc region) in the presence of a series of titrations of unlabeled IL-3 Ra. After washing to remove unbound IL-3R α, the amount of label is determined.

Affinity measurements can be determined by standard methods for antibody reactions, such as immunoassays, Surface Plasmon Resonance (SPR) (Rich and Myszka curr. Opin. Biotechnol11::54,2000; Englebenne analysis. 123:1599,1998), Isothermal Titration Calorimetry (ITC), or other kinetic interaction assays known in the art.

In one embodiment, the constants are measured by using surface plasmon resonance analysis, for example using BIAcore surface plasmon resonance (BIAcore, inc., Piscataway, NJ) with immobilized IL-3 ra or regions thereof. An exemplary SPR method is described in US 7229619.

Evaluating treatment efficacy

In vitro assay

A variety of in vitro assays can be used to assess the ability of an immunoglobulin or antibody to treat a disease or disorder described herein.

For example, the ability of an immunoglobulin or antibody to kill a cell (e.g., a cancer cell, such as a leukemia cell) is assessed using the methods described herein.

In another embodiment, immune cells (e.g., pdcs and/or basophils) or cell populations comprising the same (e.g., PBMCs) are cultured in the presence or absence of immunoglobulins or antibodies and inducers of those cells present in the disease or disorder (e.g., CpG oligonucleotides and/or immune complexes). The efficacy of the immunoglobulin or antibody in treating a disease or disorder is then assessed by measuring the level of IFN α secreted into the cell culture medium, e.g., using ELISA. Alternatively or additionally, the level of histamine secretion or IL-4, IL-6 and/or IL-13 secretion is assessed. A decrease in the level of any of these cytokines as compared to the level in the absence of an immunoglobulin or antibody (or in the presence of an isotype control immunoglobulin or antibody) indicates that the immunoglobulin or antibody is suitable for treating a disease or disorder. Alternatively or additionally, the level of cell death is assessed. An increase in cell death indicates that the immunoglobulin or antibody is suitable for treating a disease or disorder.

In vivo analysis

In one embodiment, an in vivo assay is used to assess the efficacy of an immunoglobulin for treating a disease or disorder.

In one embodiment, a xenograft model of cancer is used to assess treatment efficacy. For example, NOD/SCID mice are irradiated with radiation and optionally treated with anti-CD 122 antibody to remove NK cells. Human leukemia cells (e.g., acute myelocytic leukemia cells) and mouse or human bone marrow stem cells are administered to the mouse. Following cell transplantation, mice are administered a test immunoglobulin or antibody and levels of leukemia cells in circulation and/or bone marrow and/or lymph nodes are assessed. A decrease in the number of leukemic cells in circulation and/or bone marrow and/or lymph nodes in the presence of the antibody or immunoglobulin as compared to in the absence of the antibody or immunoglobulin indicates efficacy of the treatment.

In another embodiment, 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., pDC and/or basophils) in circulation is assessed. Immunoglobulins or antibodies that reduce the number/level of immune cells (e.g., pDC and/or basophils) compared to that in a control mammal prior to and/or not to which the immunoglobulins or antibodies have been administered are considered suitable for treating a disease or disorder.

In another embodiment, the level of a cytokine (e.g., IFN α) in the circulation of the mammal is detected, for example, using ELISA. An immunoglobulin or antibody that reduces the level of a cytokine compared to the level in a control mammal prior to administration and/or to which the immunoglobulin or antibody has not been administered is considered suitable for treating a disease or condition. Because cytokines such as IFN α are considered to play a role in some diseases/disorders (e.g., lupus), immunoglobulins or antibodies that reduce IFN α production are considered suitable for treating the disorder.

Composition comprising a metal oxide and a metal oxide

Suitably, in the compositions or methods for administering an anti-IL-3 ra immunoglobulin or antibody to a mammal, the immunoglobulin is in combination with a pharmaceutically acceptable carrier, diluent and/or excipient, as understood in the art. Accordingly, one embodiment of the present disclosure provides a pharmaceutical composition comprising an immunoglobulin or antibody of the present disclosure in combination with a pharmaceutically acceptable carrier, diluent and/or excipient. In another embodiment, the present disclosure provides a kit comprising a pharmaceutically acceptable carrier, diluent and/or excipient suitable for combination or mixing with an immunoglobulin or antibody prior to administration to a mammal. In this embodiment, the kit can further comprise instructions for use.

By "carrier, diluent or excipient" is meant, in general, a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating or lubricant that can be safely administered to any mammal (e.g., a human). Depending on the particular route of administration, a variety of acceptable carriers, diluents or excipients known in the art may be used, for example as described in Remington's Pharmaceutical Sciences (Mack Publishing co.n.j.usa, 1991).

By way of example only, the carrier, diluent or excipient may be selected from the group comprising: sugars (e.g., sucrose, maltose, trehalose, glucose), starch, 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, phosphate buffer solutions, lubricants (such as sodium stearate or magnesium stearate), isotonic saline, and pyrogen-free water. For example, the carrier, diluent or excipient is compatible with or suitable for parenteral administration. Parenteral administration includes any route of administration that does not pass through the digestive tract. Non-limiting examples of parenteral administration include injection, infusion, and the like. For example, administration by injection includes intravenous, intraarterial, intramuscular, and subcutaneous injections. Delivery via depot or slow release formulations which can be delivered, for example, intradermally, intramuscularly and subcutaneously is also contemplated.

Combination therapy

In one embodiment, the immunoglobulin or antibody of the present disclosure is administered with another compound or therapeutic treatment suitable for treating a disease or disorder.

In one embodiment, the immunoglobulin or antibody is administered one month or two weeks or one week prior, e.g., prior to radiation therapy (e.g., for treating cancer, such as hematological cancer, such as leukemia).

In one embodiment, the other compound is a chemotherapeutic compound, such as carboplatin (carboplatin), cisplatin (cispain), cyclophosphamide (cyclophosphamide), docetaxel (docetaxel), doxorubicin (doxorubicin), erlotinib (erlotinib), etoposide (etoposide), fluorouracil (fluorouracil), irinotecan (irinotecan), methotrexate (methotrexate), paclitaxel (paclitaxel), topotecan (topotecan), vincristine (vincristine), or vinblastine (vinblastine). In one embodiment, the chemotherapeutic compound is selected from the group consisting of: methotrexate, l-asparaginase, vincristine, doxorubicin, daunomycin, cytarabine, idarubicin, mitoxantrone, cyclophosphamide, fludarabine, chlorambucil, and combinations thereof.

In one embodiment, the other compound is a chemotherapeutic compound for treating acute leukemia, such as a compound selected from the group consisting of: methotrexate, l-asparaginase, vincristine, doxorubicin, daunomycin, cytarabine, idarubicin, mitoxantrone, and combinations thereof.

In one embodiment, the other compound is a chemotherapeutic compound for treating acute lymphoblastic leukemia, such as a compound selected from the group consisting of: methotrexate, l-asparaginase, vincristine, doxorubicin, daunomycin, and combinations thereof.

In another embodiment, the other compound is a chemotherapeutic compound, such as azacitidine.

In one embodiment, the other compound is a biologic agent suitable for treating cancer, such as rituximab (rituximab), trastuzumab (trastuzumab), bevacizumab (bevacizumab), alemtuzumab (alemtuzumab), panitumumab (panitumumab), or cetuximab (cetuximab).

In one embodiment, the other compound is an anti-inflammatory compound. Alternatively or additionally, the other compound is an immunosuppressant. Alternatively or additionally, the other compound is a corticosteroid, such as prednisone and/or prednisolone. Alternatively or additionally, the other compound is an antimalarial compound, such as hydroxychloroquine or chloroquine. Alternatively or additionally, the other compound is methotrexate. Alternatively or additionally, the other compound is azathioprine. Alternatively or additionally, the other compound is cyclophosphamide. Alternatively or additionally, the other compound is mycophenolate mofetil. Alternatively or additionally, the other compound is an anti-CD 20 antibody (e.g., rituximab or ofatumumab). Alternatively or additionally, the other compound is an anti-CD 22 antibody (e.g., epratuzumab). Alternatively or additionally, the other compound is an anti-TNF antibody (e.g., infliximab or adalimumab or golimumab). Alternatively or additionally, the other compound is a CTLA-4 antagonist (e.g., abatacept, CTLA 4-Ig). Alternatively or additionally, the other compound is an anti-IL-6 antibody. Alternatively or additionally, the other compound is a BLys antagonist, such as an anti-BLys antibody (e.g., belimumab).

Dosage and time course of administration

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

The methods of the present disclosure are useful for treating, ameliorating, or preventing symptoms of a disease or disorder in a mammal, or for improving the prognosis of a mammal. The methods of the present disclosure are also useful for delaying onset of lupus or preventing lupus in an individual at risk of developing lupus or a recurrence thereof.

For administration of the immunoglobulins or antibodies described herein, the normal dosage may vary from about 10ng to about 100mg or more per kg of body weight of the individual per day. Exemplary dosages and ranges thereof are described herein. For repeated administration over several days or longer, depending on the severity of the disease or condition to be treated, treatment may be continued until the desired suppression of symptoms is achieved.

In some embodiments, the immunoglobulin or antibody is administered at an initial (or loading) dose of between about 1mg/kg to about 30mg/kg, such as about 1m/gkg to about 10mg/kg, or about 2mg/kg or about 3mg/kg or 4mg/kg or 5 mg/kg. The immunoglobulin or antibody may then be administered at a maintenance dose of between about 0.0001mg/kg and about 1mg/kg, such as from about 0.0005mg/kg to about 1mg/kg, for example from about 0.001mg/kg to about 1mg/kg, such as from about 0.01mg/kg to about 1mg/kg, for example from about 0.01mg/kg to about 0.1mg/kg, such as from about 0.02mg/kg or 0.03mg/kg or 0.04mg/kg or 0.05 mg/kg. Maintenance doses may be administered once every 7-30 days, such as once every 10-15 days, for example once every 10 or 11 or 12 or 13 or 14 or 15 days.

In some embodiments, the immunoglobulin or antibody is administered at a dose of between about 0.0001mg/kg and about 50mg/kg, such as between about 0.0005mg/kg and about 50mg/kg, for example between about 0.001mg/kg and about 40mg/kg, for example between about 0.005mg/kg and about 30mg/kg, such as between about 0.01mg/kg and about 20 mg/kg. For example, the immunoglobulin is administered at a dose (e.g., without a higher loading) of between about 0.01mg/kg and about 10mg/kg, such as from about 0.01mg/kg to about 1mg/kg, such as from about 0.02mg/kg or 0.03mg/kg or 0.04mg/kg or 0.05mg/kg or 0.06mg/kg or 0.07mg/kg or 0.08mg/kg or 0.09mg/kg or 0.1mg/kg or 0.2mg/kg or 0.3mg/kg or 0.4mg/kg or 0.5mg/kg or 0.6mg/kg or 0.7mg/kg or 0.8mg/kg or 0.9 mg/kg. In some embodiments, multiple doses are administered, e.g., once every 7-30 days, such as once every 10-22 days, e.g., once every 10-15 days, e.g., once every 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days. For example, the immunoglobulin or antibody is administered once every 7 days or once every 14 days or once every 21 days.

In some embodiments, the immunoglobulin or antibody is administered at a dose (e.g., without a lower maintenance dose) of between about 1mg/kg to about 30mg/kg, such as about 1mg/kg to about 10mg/kg, or about 2mg/kg or about 3mg/kg or 4mg/kg or 5mg/kg, or such as about 10mg/kg to 30mg/kg, such as about 10mg/kg or 15mg/kg or 20mg/kg or 25 mg/kg. In some embodiments, multiple doses are administered, e.g., once every 10-70 days, such as once every 14-60 days, such as once every 14-50 days, such as once every 14-40 days, or once every 14-30 days. For example, the dose is administered once every 14 or 21 or 25 or 28 or 35 or 40 or 42 or 49 or 50 or 55 or 57 or 63 or 70 days. For example, the immunoglobulin or antibody is administered once every 21 days or every 28 days or every 35 days or every 42 days or every 49 days or every 56 days.

In some embodiments, the immunoglobulin or antibody causes or is associated with a reduction in NK cells in the mammal within, for example, about 6 hours after administration. In some embodiments, another dose of immunoglobulin or antibody is administered when the number of NK cells in the mammal returns to within 20% or 10% or 5% or 1% of the number of NK cells in the mammal prior to administration. In some embodiments, another 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 the mammal can be evaluated to determine when to administer another dose of immunoglobulin or antibody. Alternatively, the timing of administration of another dose of immunoglobulin or antibody is determined by prior analysis of the population or model organism (e.g., a non-human primate, such as cynomolgus monkey). For example, another 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.

In some embodiments, the immunoglobulin or antibody is administered to the mammal no more than 7 consecutive days or 6 consecutive days or 5 consecutive days or 4 consecutive days at the start of therapy.

In the case of mammals that do not respond adequately to treatment, multiple doses may be administered over a week. Alternatively or additionally, increased doses may be administered.

In another embodiment, for mammals experiencing an adverse reaction, the initial (or loading) dose may be divided into multiple days within a week or consecutive days.

The dosage for a particular immunoglobulin or antibody may be determined empirically in a mammal to which one or more immunoglobulin administrations have been given. To assess the efficacy of the immunoglobulin, the clinical symptoms of the disease or disorder may be monitored.

Administration of the immunoglobulin according to the methods of the present disclosure may be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to skilled practitioners. Administration of the immunoglobulin or antibody may be substantially continuous over a preselected period of time, or may be a series of spaced doses, for example during or after the development of the condition.

The present disclosure includes the following non-limiting examples.

Non-limiting examples

Example 1: humanized antibodies

Humanized antibodies that specifically bind to human IL-3R α chain were made that act as antagonists of IL-3 activity. Humanized antibodies were made according to the procedure of Tan et al (J immunol.169,1119-1125,2002) by grafting CDR sequences of antagonistic murine antibodies (7G3) onto human variable framework germline sequences selected based on the classical structure of the donor and acceptor CDRs; sometimes referred to as "super-humanization". This work was performed using antibodies in scFv format. This method then compares the CDR residues of the donor antibody to those of the variable framework germline acceptor sequence and selects the sequence with the highest correlation of CDR residues as the acceptor sequence. However, in the context of the present disclosure, heavy chain acceptor sequences with lower levels of CDR correlation are selected. The resulting humanized antibody has a fully human variable framework sequence due to the humanization process, however the affinity for IL-3R α is reduced compared to the parent murine antibody.

Affinity optimization was performed using a ribosome display based mutagenesis procedure (Kopsidas et al, bmcbiotechnol.7,18,2007) with antibodies in scFv format in an attempt to increase the binding affinity of the humanized antibody. Produced when converted to IgG₁Affinity optimized scFv that when formatted show slightly improved IL-3 Ra binding affinity compared to that of the parent murine monoclonal antibody. Unexpectedly, the affinity optimization procedure was at V_HAnd V_LAnd in CDR1 of the light chain. Thus, the set of CDR sequences of the humanized affinity optimized antibody is different from the set of CDR sequences of the parent murine monoclonal antibody. Herein, the affinity optimized antibody is referred to herein as CSL 362.

Fc engineered derivatives of humanized affinity-optimized antibodies were made by expression of appropriate vectors in CHO-S cells, comprising the light and heavy chain variable regions of the antibody and having three amino acid substitutions S239D/A330L/I332E (referred to herein as CSL362X2) or two amino acid substitutions S239D/I332E (referred to herein as CSL362X2)CSL362X1) hybrid IgG₁An IgG2 constant domain; the positions of the identified mutations are based on the EU numbering system.

By using FUT8 knockout CHO cells from Biowa: (Cells) expressing appropriate vectors to generate humanized affinity optimized antibodies with human IgG₁An afucosylated version of the constant domain (referred to herein as CSL 362B).

Example 2: binding affinity assay

Full length mAb kinetics:

to analyze the full-length antibodies, surface plasmon resonance (Biacore) analysis was performed in a capture format in which chemically immobilized anti-human or anti-mouse Fc specific antibodies (adsorbed anti-human goat anti-human IgG (γ) mouse (Invitrogen, cat # H10500) or anti-mouse Fc specific antibodies (Jackson Immuno Research Labs inc. cat # 515 @ -005) 071) were chemically immobilized on the CM-5 sensor surface using standard amine coupling chemistry and used to capture the mabs from solution. Soluble human IL-3R α was then injected at various concentrations onto the captured antibody. Reactions were subtracted from those from a reference flow cell that did not capture antibody, but otherwise handled the same. The reference subtracted reaction was then subtracted from the reaction from the blank injection.

The final corrected response was fitted to a model describing 1:1 kinetics (including mass transport limitation aspects) using non-linear regression. Rmax values were fitted locally to account for slight deviations in captured antibody levels. Determination of association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K)_D)。

The antibody was captured at 0.3. mu.g/ml for 180 seconds.

Soluble IL-3 ra was injected for 10 minutes and dissociation was monitored for 30 minutes.

Soluble IL-3 Ra was injected at 0, 0.62, 1.25, 2.5, 5, 10,20 and 40nM, with two replicates at 2.5 and 5 nM.

After each cycle by injection of 100mM H₃PO₄The regeneration is carried out for 90 seconds.

The analysis was performed at 25 ℃.

scFv kinetics:

to analyze the scFv, soluble human IL-3 ra was chemically immobilized on the CM-5 sensor surface using standard amine coupling chemistry, and various concentrations of scFv were injected. The reactions were subtracted from those from a reference flow cell that was not immobilized with IL-3R α, but otherwise treated identically. The reference subtracted reaction was then subtracted from the reaction from the blank injection.

The final corrected response was fitted to a model describing 1:1 kinetics (including mass transport limitation aspects) using non-linear regression. Rmax values were fit to a full scale and the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) were determined_D)。

scFv injection was performed for 10 min and dissociation was monitored for 20 min.

scFv were injected at 0, 0.62, 1.25, 2.5, 5, 10,20 and 40nM, with duplicate replicates at 10 nM.

By injection of 100mM H after each cycle₃PO₄Regeneration was performed with 1M NaCl for 30 seconds and injection of 50mM NaOH for 15 seconds.

The analysis was performed at 25 ℃.

The antibody and scFv affinities are listed in table 2 below:

table 2: affinity dissociation constants of antibodies and scFv

Antibodies	KD(M)
		Parent murine mAb (7G3)	About 9.2X 10-10
Chimeric versions of parent murine mabs) (human IgG1)	About 1.0X 10-9
		Super-humanized mAbs (IgG)1)	About 1X 10-8
Hyperhumanized mAb scFv	About 1.4X 10-8
		CSL362scFv	About 2.2X 10-9
CSL362(IgG1)	About 7.8X 10-10

Antibodies	KD(M)
		CSL362B	About 4.3X 10-10

Example 3: NK cell levels following administration of humanized or chimeric antibodies

Untreated monkeys (non-human primates; NHPs) were administered a single dose of CSL362B or CSL362X1 by intravenous infusion. In another study, repeated doses (weekly × 4) of variable regions comprising murine antibody (7G3) used to make humanized antibodies and human IgG were administered to untreated monkeys₁Chimeric antibodies to constant regions. Peripheral blood was collected at different time points and analyzed by flow cytometry for NHP NK cells.

As shown in figure 1A, administration of CSL362X1 resulted in initial depletion of NK cells, e.g., about 6 hours after administration. At doses of 0.01mg/kg and 0.1mg/kg, this level exceeded that observed prior to administration at about day 8 post-administration and remained high until at least 22 or 29 days post-administration. An increase in the number of NK cells was also observed at the 1mg/kg dose.

In contrast to the results described in the preceding paragraph, administration of CSL362B resulted in NK cell depletion, however NK cell numbers did not later exceed the numbers before administration (fig. 1B).

Repeated administration of the chimeric antibody did not produce a substantial change in the number of NK cells detected in the circulation (fig. 1C).

Example 4: ADCC enhancement of humanized antibodies in the presence of NK cells

Human PBMC or NK cells were isolated and incubated with TF-1 cells in the presence of various concentrations of CSL362X 1. Effector cells (E; PBMC) and target cells (T; TF-1 cells) are combined to achieve a ratio of 50:1 (E: T ratio), or 20: 1in the case of using purified NK cells as effector cells. Cell lysis was measured using the ldhcytotox 96 non-radioactive cytotoxicity kit (Promega).

Specific lysis was determined by the following calculation:

specific lysis = [ sample lysis-spontaneous lysis ]/[ maximum lysis-spontaneous lysis ] × 100%.

By adding Extran^TMTo a final concentration of 0.75% (v/v) to assess maximum dissolution. Spontaneous lysis is lysis that occurs in wells with cells only (no abs).

As shown in figure 2A, lysis of TF-1 cells occurred in the presence of PBMCs and in the presence of NK cells, however, substantially decreased in PBMCs from which NK cells were removed.

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

Claims (4)

1. An isolated or recombinant antibody capable of specifically binding Interleukin (IL) -3 ra chain, wherein the antibody comprises:

(i) light chain variable region (V) comprising CDR1, 2 and 3 as shown in SEQ ID NOs: 2, 3 and 4, respectively_L)；

(ii) Heavy chain variable region (V) comprising CDR1, 2 and 3 as shown in SEQ ID NOs 5, 6 and 7, respectively_H) (ii) a And

(iii) a heavy chain constant region comprising amino acid substitutions S239D and I332E according to the EU numbering system of Kabat.

2. The isolated or recombinant antibody of claim 1, which is a humanized antibody comprising:

(i) light chain variable region (V) consisting of the amino acid sequence according to SEQ ID NO 8_L) And a heavy chain variable region (V) consisting of the amino acid sequence according to SEQ ID NO 9_H) (ii) a And

(ii) a heavy chain constant region consisting of amino acid substitutions S239D and I332E according to EU numbering system of Kabat.

3. The isolated or recombinant antibody of claim 1, wherein the heavy chain constant region consists of the sequence shown between residues 121 and 450 (including residues 121 and 450) of SEQ ID NO: 11.

4. The isolated or recombinant antibody of claim 1, wherein the antibody is a humanized antibody comprising: a light chain consisting of the sequence shown in SEQ ID NO. 13 and a heavy chain consisting of the sequence shown in SEQ ID NO. 11.