HK1240948B - Cd123 binding agents and uses thereof - Google Patents

Description

CD123 binding agents and uses thereof

Technical Field

The disclosure provided herein relates to monoclonal antibodies that immunospecifically bind to cluster determinant 123 (CD123; also known as IL-3Rα), multispecific antibodies that immunospecifically bind to CD123 and cluster determinant 3 (CD3), and methods of making and using the same.

Background Art

Worldwide, a new blood cancer is diagnosed approximately every three minutes. The most common blood cancers are leukemia, lymphoma, and myeloma, with 156,420 Americans diagnosed with these three types of cancer in 2014. Approximately every 10 minutes, an American dies from a blood cancer. These diseases can affect the bone marrow, blood cells, lymph nodes, and other parts of the lymphatic system. These cancers are disproportionately affected by age, with a higher incidence among younger people. Leukemia is the most common cancer among children and adolescents under 20 years old.

One type of blood cancer cell expresses a cell marker called CD123 (IL-3Rα). Examples of blood cancer cells that express CD123 include blasts and leukemia stem cells. Diseases associated with CD123 expression include acute myeloid leukemia (AML), myelodysplastic syndrome (MDS; both low-risk and high-risk), acute lymphoblastic leukemia (ALL, all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), and blastic plasmacytoid dendritic cell neoplasm (DPDCN).

Currently, treatment options for these diseases include over 50 single drugs, as well as other drugs under investigation and in clinical trials. Radiation therapy (RT) is also commonly used to treat blood cancers, sometimes administered in conjunction with drug therapy. Immunotherapy, gene therapy, and personalized medicine are also available. However, these therapies can have significant side effects and cause adverse reactions. Therefore, new and improved therapies are needed to treat blood cancers that express CD123 (IL-3Rα).

Summary of the Invention

Provided herein are antibodies and antigen binding fragments thereof that immunospecifically bind to CD123. Also described herein are related polynucleotides capable of encoding provided CD123-specific antibodies and antigen binding fragments, cells expressing provided antibodies and antigen binding fragments, and related vectors and detectably labeled antibodies and antigen binding fragments. In addition, a method for using provided antibodies and antigen binding fragments is also described. For example, CD123-specific antibodies and antigen binding fragments can be used for diagnosing or monitoring the progress, regression, or stability of CD123-expressing cancers; for determining whether a patient should receive cancer treatment; or for determining whether a subject suffers from CD123-expressing cancers, and therefore can be suitable for treatment with CD123-specific anticancer therapeutic agents (such as multispecific antibodies for CD123 and CD3 as described herein).

Also provided herein are multispecific antibodies and multispecific antigen-binding fragments thereof that are immunospecifically bound to CD123 and CD3. Also described herein are related polynucleotides capable of encoding the CD123×CD3 multispecific antibodies provided, cells expressing the antibodies provided, and related vectors and detectably labeled multispecific antibodies. In addition, methods using the multispecific antibodies provided are also described. For example, CD123×CD3 multispecific antibodies can be used to diagnose or monitor the progression, regression, or stability of CD123-expressing cancers; for determining whether a patient should receive cancer treatment; or for determining whether a subject suffers from CD123-expressing cancers, and therefore may be suitable for treatment with CD123-specific anticancer therapeutics (such as CD123×CD3 multispecific antibodies described herein).

CD123-specific antibodies

Described herein are antibodies and antigen-binding fragments specific for separation of CD123. In some embodiments, CD123-specific antibodies and antigen-binding fragments bind to human CD123 SP1 (SEQ ID NO: 1). In some embodiments, CD123-specific antibodies and antigen-binding fragments bind to human CD123 SP2 (SEQ ID NO: 2). In some embodiments, CD123-specific antibodies and antigen-binding fragments bind to human CD123 SP1 and CD123 SP2. In some embodiments, CD123-specific antibodies and antigen-binding fragments bind to human CD123 SP1 and cynomolgus monkey CD123 (SEQ ID NO: 3). In some embodiments, the CD123-specific antibodies and antigen-binding fragments bind to an epitope that includes one or more residues from: (i) the CD123 SP2 extracellular domain (ECD) segment comprising residues 195-202 (RARERVYE (SEQ ID NO: 234)) and/or the CD123 SP2 ECD segment comprising residues 156-161 (RKFRYE (SEQ ID NO: 232)) and/or the CD123 SP2 ECD segment comprising residues 173-178 (TEQVRD (SEQ ID NO: 233)), or (ii) the CD123 SP2 ECD segment comprising residues 164-175 (IQKRMQPVITEQ (SEQ ID NO: 228)) and/or the CD123 SP2 ECD segment comprising residues 184-189 (LLNPGT (SEQ ID NO: 229)). The CD123-specific antibody or antigen-binding fragment may bind to CD123 with an affinity of 5×10 ⁻⁷ M or less, such as 1×10 ⁻⁷ M or less, 5× ^{10 −8 M} or less, 1×10 ⁻⁸ M or less, 5×10 −9 M or less, or 1×10 ⁻⁹ M or less.

In some embodiments, the CD123-specific antibody or antigen-binding fragment competes for binding to CD123 with a CD123-specific antibody or antigen-binding fragment that binds to an epitope that includes one or more residues from: (i) the CD123 SP2 ECD segment comprising residues 195-202 (RARERVYE (SEQ ID NO: 234)) or (ii) the CD123 SP2 ECD segment comprising residues 164-175 (IQKRMQPVITEQ (SEQ ID NO: 228)). Antibodies or fragments that bind to at least one residue in these epitopes may also bind to additional residues in the CD123 ECD, including (i) one or more residues from the CD123 SP2 ECD segment comprising residues 156-161 (RKFRYE (SEQ ID NO: 232)) and/or the CD123 SP2 ECD segment comprising residues 173-178 (TEQVRD (SEQ ID NO: 233)), or (ii) one or more residues from the CD123 SP2 ECD segment comprising residues 184-189 (LLNPGT (SEQ ID NO: 229)). The CD123-specific antibody or antigen-binding fragment can bind to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less.

In some embodiments, CD123-specific antibodies and antigen-binding fragments (such as those discussed in the first two paragraphs) are neutralizing antibodies. Neutralizing CD123-specific antibodies or antigen-binding fragments include those that can inhibit IL-3 from being bound to CD123 as determined by measuring the reduction in STAT5 phosphorylation when TF-1 cells are stimulated with rhIL-3.

In some embodiments, CD3123-specific antibodies and antigen-binding fragments can prevent IL-3 from binding to CD123 (IL3Ra) / CD131 (IL3Rb) receptors. In other embodiments, CD123-specific antibodies and antigen-binding fragments can prevent the association of the α chain and β chain of the IL3R receptor (CD123 (IL3Ra) / CD131 (IL3Rb)). Antibodies or antigen-binding fragments include those that can inhibit IL3 binding and/or inhibit CD123/CD133 heteromerization as determined by measuring the reduction in the degree of association between CD123 and CD131 and measuring the loss of heteromerization as the antibody concentration increases. Table 1 provides a summary of some CD123-specific antibody examples described herein:

Table 1. CDR sequences of mAbs generated by phage panning against human CD123

(SEQ ID NO:)

In some embodiments, CD123-specific antibodies or antigen-binding fragments thereof are provided, which comprise a heavy chain comprising CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1. In some embodiments, CD123-specific antibodies or antigen-binding fragments thereof are provided, which comprise a heavy chain comprising CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1 and a light chain comprising CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1. In some embodiments described herein, CD123-specific antibodies or antigen-binding fragments thereof compete for binding to CD123 with a heavy chain comprising CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1 and an antibody or antigen-binding fragment comprising a light chain comprising CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1.

Human IgG can be divided into four isotypes: IgG1, IgG2, IgG3 and IgG4. They have more than 95% homology in the amino acid sequence of the Fc region, but the main difference shown is the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of the antibody binds to Fc receptors (FcgR) on the surface of immune effector cells (such as natural killer cells and macrophages), resulting in phagocytosis or lysis of target cells. In CDC, the antibody kills the target cell by triggering the complement cascade on the cell surface. The antibodies described herein include antibodies having the described features of the variable domains combined with any IgG isotype, including modified versions in which the Fc sequence has been modified to achieve different effector functions.

For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions may be harmful and may pose a safety risk by causing off-mechanistic toxicity. Modification of effector functions can be achieved by modifying the Fc region to reduce the extent of its binding to FcgR or complement factors. The binding of IgG to activating FcgR (FcgRI, FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory FcgR (FcgRIIb) or the first component of complement (C1q) depends on residues located in the hinge region and CH2 domain. Mutations have been introduced into IgG1, IgG2 and IgG4 to reduce or silence Fc function. The antibodies described herein may include these modified forms.

In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function compared to the parent Fc; (b) reduced affinity for FcgRI, FcgRIIa, FcgRIIb, FcgRIIIb and/or FcgRIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (e) reduced affinity for FcgRIIb; (f) reduced affinity for FcgRIIIb or (g) reduced affinity for FcgRIIIa.

In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, and IgG4 isotype. In some embodiments wherein the antibody has an IgG1 isotype, the antibody contains L234A, L235A, and/or K409R substitutions in its Fc region. In some embodiments wherein the antibody has an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may include these modified forms.

In some embodiments, the antibody can be bound to CD123 with a dissociation constant of 5nM or less as measured by surface plasmon resonance (SPR). In some embodiments, the antibody comprises the CDR of the antibody shown in Table 1 above. The determination of affinity measured by SPR includes the determination performed using a BIAcore 3000 or Biacore T200 machine, wherein the determination is performed at room temperature (e.g., at 25°C or close to 25°C), wherein the antibody capable of being bound to CD123 is captured to a level of about 75RU by anti-Fc antibodies (e.g., goat anti-human IgG Fc specific antibodies, Jackson ImmunoResearchlaboratories Prod#109-005-098) on a BIAcore sensor chip, and then association and dissociation data are collected at a flow rate of 40 μl/min.

In addition to the CD123 specific antibodies and Fab, a polynucleotide sequence capable of encoding the antibodies and Fab is also provided. A carrier comprising the polynucleotide is also provided, and a cell expressing the CD123 specific antibodies or Fab provided herein. The cell capable of expressing the disclosed carrier is also described. These cells can be mammalian cells (e.g., 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., Escherichia coli (E.coli)). The antibody can also be produced by hybridoma cells.

Methods using CD123-specific antibodies

Also disclosed is a method for using the CD123-specific antibody or antigen binding fragment. The specific antibodies used in the methods discussed in this section include those with the group of CDRs described for the antibodies in Table 1 above or competing with one of the antibodies in Table 1 for binding to CD123. For example, these antibodies or antigen binding fragments can be used to treat cancer, inhibiting the biological effects of IL-3 by preventing IL-3 from binding to IL-3R or wherein the antibody is conjugated to a toxin, thereby targeting the toxin to CD123 expression cancer. In addition, these antibodies or antigen binding fragments can also be used to detect the presence of CD123 in biological samples such as blood or serum; for quantitative analysis of the amount of CD123 in biological samples such as blood or serum; for diagnosing CD123 expression cancer; for determining a method for treating a subject with cancer; or for monitoring the progress of CD123 expression cancer in a subject. In some embodiments, CD123 expression type cancer can be hematological cancer, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastocyte plasmacytoid dendritic cell tumor (DPDCN). The method can be carried out before the subject receives the treatment of CD123 expression type cancer, for example, with the multispecific antibody treatment of anti-CD123 and CD3. In addition, the method can also be carried out after the subject receives the treatment of CD123 expression type cancer, for example, with the multispecific antibody treatment of anti-CD123 and CD3 described herein.

The method of detecting CD123 in a biological sample comprises exposing the biological sample to one or more CD123-specific antibodies or antigen-binding fragments described herein.

The method of diagnosing a CD123-expressing cancer in a subject also involves exposing a biological sample to one or more CD123-specific antibodies or antigen-binding fragments described herein; however, the method further comprises quantifying the amount of CD123 present in the sample; comparing the amount of CD123 present in the sample to a known standard or reference sample; and determining whether the subject's CD123 level falls within a range of CD123 levels associated with cancer.

Also described herein is a method for monitoring CD123-expressing cancer in a subject. The method includes exposing a biological sample to one or more CD123-specific antibodies or antigen-binding fragments described herein; quantitatively analyzing the amount of CD123 bound by the antibody or its antigen-binding fragment present in the sample; comparing the amount of CD123 present in the sample with the amount of CD123 in a known standard or reference sample or a similar sample previously obtained from the subject; and determining whether the CD123 level of the subject indicates cancer progression, regression, or stable disease based on the difference in the amount of CD123 in the compared samples.

A sample obtained from or derived from a subject is a biological sample, such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells, tissue, surgically resected tumor tissue, biopsy, fine needle aspiration, or histological preparation.

The CD123 specific antibody or Fab can be marked for use with the method or other methods known to those skilled in the art. For example, antibody as herein described or its Fab can be marked with radiolabels, fluorescent markers, epitope tags, biotin, chromophore labels, ECL labels, enzymes, ruthenium, 111In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine or similar such labels known in the art.

CD123 specific antibody kit

The kit including the disclosed CD123-specific antibodies or their antigen-binding fragments is described herein. The kit can be used to implement the method using the CD123-specific antibodies or antigen-binding fragments provided herein or other methods known to those skilled in the art. In some embodiments, the kit may include antibodies or antigen-binding fragments as described herein and reagents for detecting whether CD123 exists in a biological sample. Therefore, the kit may include one or more antibodies or their antigen-binding fragments as described herein, and instructions for use of containers, antibodies or fragments for holding antibodies or fragments when not in use, antibodies or fragments attached to a solid support and/or antibodies or fragments as described herein or detectable labeling forms of the fragments.

CD123×CD3 multispecific antibody

Described herein are isolated multispecific antibodies that bind to CD123 and CD3 ("CD123×CD3 multispecific antibodies") and multispecific antigen-binding fragments thereof. In some embodiments, isolated antibodies or antigen-binding fragments thereof that immunospecifically bind to CD123 SP2 (IL3-Rα) and CD123 SP1 (IL3-Rα) are provided.

In some embodiments, the CD123-specific arm of the multispecific antibody binds to human CD123 and/or cynomolgus monkey CD123. In some embodiments, the CD123-specific arm of the CD123×CD3 multispecific antibody or antigen-binding fragment binds to the SP1 and/or SP2 fragments of human CD123. In a preferred embodiment, the CD123×CD3 multispecific antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment. In some embodiments, there is provided a) a first heavy chain (HC1), b) a second heavy chain (HC2), c) a first light chain (LC1) and d) a second light chain (LC2) separated CD123 (IL3-Rα) × CD3 bispecific antibody or its CD123 (IL3-Rα) × CD3 bispecific binding fragment, wherein HC1 and LC1 are paired to form a first antigen binding site that immunospecifically binds to CD123 (IL3-Rα), and HC2 and LC2 are paired to form a second antigen binding site that immunospecifically binds to CD3. In another embodiment, there is provided a cell expressing the separation of the antibody or bispecific binding fragment. In some embodiments, the CD123-binding arm (or "CD123-specific arm") of the CD123×CD3 multispecific antibody is derived from a CD123 antibody described herein (e.g., from an antibody having the CDR sequences listed in Table 1).

In some embodiments, the CD123-specific arm of the CD123×CD3 multispecific antibody or antigen-binding fragment is IgG or a derivative thereof. In some embodiments, the CD123×CD3 multispecific antibody can be bound to CD123 with a dissociation constant of 5 nM or less as measured by surface plasmon resonance or MSD-CAT.

In some embodiments, the CD3 binding arm (or "CD3-specific arm") of the CD123×CD3 multispecific antibody is derived from mouse monoclonal antibody SP34, a mouse IgG3/λ isotype (Pessano, S. et al., 1995. EMBO J. 4, 337-344). In some embodiments, the CD3 binding arm of the CD123×CD3 multispecific antibody comprises a VH domain and a VL domain selected from Table 2. Table 2 provides a summary of examples of some heavy and light chains of CD3-specific antibodies and antigen-binding fragments.

Table 2. Heavy and light chains of CD3-specific antibodies and antigen-binding fragments .

In some embodiments, the CD3-specific antibodies and antigen-binding fragments comprise a heavy chain in Table 3 and a light chain in Table 3. Table 3 provides a summary of the matrix of heavy and light chains of CD3-specific antibodies and antigen-binding fragments.

Table 3. Antibodies generated by combining heavy and light chains .

Human IgG can be divided into four isotypes: IgG1, IgG2, IgG3 and IgG4. They have more than 95% homology in the amino acid sequence of the Fc region, but the main difference they show is the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of the antibody binds to Fc receptors (FcgR) on the surface of immune effector cells (such as natural killer cells and macrophages), resulting in phagocytosis or lysis of target cells. In CDC, the antibody kills the target cell by triggering the complement cascade on the cell surface.

For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions may be detrimental and may pose a safety risk by causing off-mechanistic toxicity. Modifying effector function can be achieved by modifying the Fc region to reduce its binding to FcgR or complement factors. IgG binding to activating FcgR (FcgRI, FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory FcgR (FcgRIIb) or the first component of complement (C1q) depends on residues located in the hinge region and CH2 domain. Mutations have been introduced in IgG1, IgG2 and IgG4 to reduce or silence Fc function. Silent mutations may include, but are not limited to, IgG1 AA (F234A, L235A) or IgG4 PAA (S228P, F234A, L235A) or IgG2 AA (V234A, G237A) or IgG1 FEA (L234F, L235E, D265A) or IgG1 FES (L234F/L235E/P331S).

In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function compared to the parent Fc; (b) reduced affinity for FcgRI, FcgRIIa, FcgRIIb, FcgRIIIb and/or FcgRIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (e) reduced affinity for FcgRIIb; (f) reduced affinity for FcgRIIIb or (g) reduced affinity for FcgRIIIa.

In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG or a derivative thereof. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG1 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG1 antibody from which the CD3-binding arm is derived comprises L234A, L235A, and F405L substitutions in its Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG4 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG4 antibody from which the CD3-binding arm is derived comprises S228P, L234A, L235A, F405L, and R409K substitutions in its Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG-AA Fc. In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody is IgG-AA Fc-L234A, L235A and F405L (wherein L234A, L235A and F405L are mutations). In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody binds to CD3ε on primary human T cells and/or primary cynomolgus monkey T cells. In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody activates primary human CD4+T cells and/or primary cynomolgus monkey CD4+T cells. In some embodiments, the CD123×CD3 multispecific antibody is capable of binding to CD3 on human or cynomolgus monkey T cells with a dissociation constant of less than 500nM or less than 100nM or less than 20nM as determined by competing with a labeled anti-CD3 antibody with a known affinity.

In addition to the CD123×CD3 multispecific antibody, a polynucleotide sequence capable of encoding the CD123×CD3 multispecific antibody is also provided. In some embodiments, there is provided a synthetic polynucleotide encoding the HCl, HC2, LC1 or LC2 of a bispecific binding fragment of CD123 (IL3-Rα)×CD3. A vector comprising the polynucleotide and a cell expressing the CD123×CD3 multispecific antibody provided herein are also provided. Cells capable of expressing the disclosed vectors are also described. These cells can be mammalian cells (e.g., 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The antibody can also be produced by hybridoma cells. In some embodiments, there is provided a method for producing CD123 (IL3-Rα)×CD3 bispecific antibodies or bispecific binding fragments by culturing cells.

Also provided herein are pharmaceutical compositions comprising a CD123 (IL3-Rα)×CD3 multispecific antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

Methods using CD123×CD3 multispecific antibodies

Also disclosed is a method for using the CD123×CD3 multispecific antibody and its multispecific antigen-binding fragment. For example, CD123×CD3 multispecific antibody and its multispecific antigen-binding fragment can be used to treat CD123 expression type cancer in a subject in need thereof. In some embodiments, CD123 expression type cancer is a hematological cancer, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

The method for treating a CD123-expressing cancer in a subject in need thereof comprises administering a therapeutically effective amount of the CD123×CD3 multispecific antibody or its multispecific antigen-binding fragment to the subject. In some embodiments, the subject is a mammal, preferably a human. In a preferred embodiment, a method for treating a subject with cancer is provided by administering a therapeutically effective amount of a CD123 (IL3-Rα)×CD3 bispecific antibody or bispecific antigen-binding fragment to a patient in need thereof for a period of time sufficient to treat the cancer.

Also provided herein are methods for inhibiting the growth or proliferation of cancer cells by administering a therapeutically effective amount of a CD123(IL3-Rα)×CD3 bispecific antibody or bispecific binding fragment.

Also provided herein are methods for redirecting T cells to CD123-expressing cancer cells by administering a therapeutically effective amount of a CD123(IL3-Rα)×CD3 bispecific antibody or bispecific binding fragment.

CD123×CD3 specific antibody kit

The kit comprising the disclosed CD123×CD3 multispecific antibodies is described herein. The kit can be used to implement the method using the CD123×CD3 multispecific antibodies provided herein or other methods known to those skilled in the art. In some embodiments, the kit may include antibodies as described herein and reagents for treating CD123-expressing cancers. Therefore, the kit may include one or more multispecific antibodies or multispecific antigen-binding fragments thereof as described herein, and instructions for use of containers and/or antibodies or fragments for holding antibodies or fragments when not in use, antibodies or fragments attached to a solid support and/or detectably labeled forms of antibodies or fragments as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Figure 1 shows the pDisplay vector used for cloning the CD123 extracellular domain.

Figure 2. Figure 2 (Figure 2A, Figure 2B and Figure 2C) shows a cell binding assay demonstrating the binding potential of the phage panel positive binders to CD123 expressing cells.

Figure 3. Figure 3 shows a competition ELISA between the antibody panel and anti-CD123 antibody 7G3.

Figure 4. Figure 4 (Figure 4A and Figure 4B) shows a STAT5 functional assay based on CD123 cells. Figure 4C shows a dose-dependent STAT5 functional assay based on CD123 cells for I3RB18 and 7G3 antibodies.

Figure 5. Figure 5 (Figures 5A, 5B and 5C) shows that Mabs I3RB2, I3RB18 and 7G3 bind to endogenous CD123 expressed on the AML cell line OCI-AML5.

Figure 6. Figure 6 (Figure 6A and Figure 6B) shows competitive binding assays between labeled I3RB2 and I3RB18 mAbs and other anti-CD123 Abs identified in the screen.

Figure 7. Figure 7 (Figure 7A (SEQ ID NO: 232) and Figure 7B (SEQ ID NO: 232)) shows the results of epitope mapping studies showing differences in deuterium levels of CD123 SP2 in the presence or absence of Fab by hydrogen/deuterium exchange mass spectrometry (HDX-MS).

Figure 8. Figure 8 shows the antibody residues involved in CD123 sp2 binding observed in the co-crystal structure of I3RB18-derived scFv and CD123 sp2 ECD. Numbering: CD123 sp2 is oval; I3RB18 CDRs are square.

Figure 9. Figure 9A shows the co-crystal structure of CD123 sp2:I3RB18 (labeled B18) and Figure 9B shows the co-crystal structure of CD123sp1:CSL362 Fab (humanized version of mAb 7G3 from PDB entry number 4JZJ).

Figure 10. Figure 10 shows the amino acid sequence of SP34 with consecutive numbering. The CDRs defined by AbM (K.R. Abhinandan and A.C. Martin, 2008. Mol. Immunol. 45, 3832-3839) are underlined. Ser230 is the last HC residue present in papain-cleaved Fab. Residues 231-455 are from IGHG3_MOUSE (mouse IgG3, isotype 2).

Figure 11. Figure 11 shows the variable domain of SP34, showing the key residues at the VL/VH interface. Residues 38, 48 and 51 in VL (labeled) make contact with CDR-H3.

Figure 12. Figure 12 shows human framework adapted ("HFA") variants of the VH (SEQ ID NOs 5 and 184-187, respectively, in order of appearance) and VL (SEQ ID NOs 4 and 188-190, respectively, in order of appearance). Numbering is sequential; CDRs under the AbM definition are underlined; residues that differ from SP34 are highlighted in bold; back mutations in the HFA variants are bold and underlined.

Figure 13. Figure 13 shows the binding of SP34 HFA variants to primary human T cells.

Figure 14. Figure 14 shows the binding of SP34 HFA variants to cynomolgus monkey primary T cells.

Figure 15. Figure 15 shows that SP34 HFA variants activate primary human T cells in vitro. Negative controls are shown in white and positive controls are shown in black.

Figure 16. Figure 16 shows that SP34 HFA variants activate primary cynomolgus monkey T cells in vitro. Negative controls are shown in white and positive controls are shown in black.

Figure 17. Figure 17 shows the correlation of binding and activation of SP34 HFA variants. Mean binding and CD69 mean fluorescence intensity ("MFI") values for humans (Figure 17A) and cynomolgus monkeys (Figure 17B) are plotted relative to each other.

Figure 18. Figure 18 shows T cell mediated cytotoxicity assays using the MV4-11 cell line for donor M6587 (Figure 18A) and donor M7020 (Figure 18B).

Figure 19. Figure 19 shows T cell mediated cytotoxicity assays using the OCI-M2 cell line for donor M6587 (Figure 19A) and donor M7020 (Figure 19B).

Figure 20. Figure 20 shows T cell mediated cytotoxicity assays using the OCI-AML cell line for donor M6587 (Figure 20A) and donor M7020 (Figure 20B).

Figure 21. Figure 21 shows the efficacy of I3RB186 in the KG-1 tumor xenograft model.

Figure 22. Figure 22 shows the efficacy of I3RB186 in the KG-1 tumor xenograft model as determined by fluorescence activated cell sorting (FACS) analysis of CD45+ (Figure 22A) and CD8+/CD4+ (Figure 22B) of peripheral blood on day 30.

Figure 23. Figure 23 shows the efficacy of I3RB186 in the KG-1 tumor xenograft model by FACS analysis of CD45+ (Figure 23A) and CD8+/CD4+ (Figure 23B) in peripheral blood on day 53 after tumor implantation.

Figure 24. Figure 24 shows the efficacy of I3RB186 in the KG-1 tumor xenograft model by showing changes in body weight after treatment.

Figure 25. Figure 25 shows the efficacy of CD123 x CD3 bispecific Ab I3RB186 versus control empty-arm bispecific Abs I3RB191 and I3RB192 in the KG-1 tumor xenograft model.

Figure 26. Figure 26 shows the efficacy of CD123×CD3 bispecific Ab I3RB186 and control empty-arm bispecific Abs I3RB191 and I3RB192 in the KG-1 tumor xenograft model by FACS analysis of CD45+ (Figure 26A) and CD8+/CD4+ (Figure 26B) results at day 36 after tumor implantation.

Figure 27. Figure 27 shows the efficacy of CD123×CD3 bispecific Ab I3RB186 and control empty-arm bispecific Abs I3RB191 and I3RB192 in the KG-1 tumor xenograft model by FACS analysis of CD45+ (Figure 27A) and CD8+/CD4+ (Figure 27B) results at day 63 after tumor implantation.

Figure 28. Figure 28 shows the efficacy of CD123xCD3 bispecific Ab I3RB186 compared to control empty-arm bispecific Abs I3RB191 and I3RB192 in the KG-1 tumor xenograft model by showing changes in body weight after treatment.

Figure 29. Figure 29 shows saturation binding curves used to determine the cell binding affinity (Kd) of SP34-2 on primary human T cells (Figure 29A) and cynomolgus monkey T cells (Figure 29B).

Figure 30. Figure 30 shows competition binding experiments using labeled antibody Alexa FluorR 488B146 and increasing concentrations of unlabeled CD123xCD3 antibody on primary human T cells (Figure 30A) and cynomolgus monkey T cells (Figure 30B).

Figure 31. Figure 31 shows T cell mediated cytotoxicity assays using the OCI-AML cell line for donor M6948 (Figure 31A) and donor M6521 (Figure 31B).

Figure 32. Figure 32 shows T cell mediated cytotoxicity assays using the KG-1 cell line for donor M6948 (Figure 32A) and donor M6521 (Figure 32B).

Figure 33. Figure 33 shows T cell mediated cytotoxicity assays using the JIM3 cell line for donor M6948 (Figure 33A) and donor M6521 (Figure 33B).

Figure 34. Figures 34A, 34B, 34C and 34D show the effects of CD123xCD3 antibodies to I3RB218 (Figure 34A), 8747 (Figure 34B), I3RB217 (Figure 34C) and 7959 (Figure 34D) on IL-3-induced heteromerization of CD123 and CD131.

Figure 35. Figure 35 shows the efficacy of CD123 x CD3 Ab 7959 and Ab 9958 in the KG-1 tumor xenograft model by comparing mean tumor volumes.

Figure 36. Figure 36 shows the efficacy of CD123xCD3Ab 3978 in the KG-1 tumor xenograft model by comparing mean tumor volumes.

Figure 37. Figure 37 shows the efficacy of CD123xCD3 Ab 8747 in the KG-1 tumor xenograft model by comparing mean tumor volumes.

Figure 38. Figure 38 shows the efficacy of CD123xCD3 Ab 8876 in the KG-1 tumor xenograft model by comparing mean tumor volumes.

Figure 39. Figure 39 shows the efficacy of CD123xCD3 Ab 7959 and Ab 9958 in the KG-1 tumor xenograft model by comparing body weight changes after treatment.

Figure 40. Figure 40 shows the efficacy of CD123xCD3 Ab 3978 in the KG-1 tumor xenograft model by comparing body weight changes after treatment.

Figure 41. Figure 41 shows the efficacy of CD123xCD3 Ab 8747 in the KG-1 tumor xenograft model by comparing body weight changes after treatment.

Figure 42. Figure 42 shows the efficacy of CD123xCD3 Ab 8876 in the KG-1 tumor xenograft model by comparing body weight changes after treatment.

Figure 43 shows the in vivo PK of CD123×CD3 bispecific antibodies 3978, 7955, 7959, and 9958 in mice

DETAILED DESCRIPTION

definition

Various terms related to the various aspects of the description are used throughout the specification and claims. Unless otherwise indicated, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes reference to two or more cells, and so forth.

As used herein, the term "about" when referring to a measurable value such as an amount, a time interval, etc., is meant to encompass variations of up to ±10% from the specified value, as such variations are suitable for performing the methods disclosed herein. Unless otherwise indicated, all numbers used in the specification and claims expressing the amounts of ingredients, properties such as molecular weight, reaction conditions, etc. are to be understood as being modified by the term "about" in all instances. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values that may vary depending on the desired properties sought to be obtained by the present invention. At a minimum, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of significant digits reported and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

"Isolated" means that a biological component (such as nucleic acid, peptide or protein) has been substantially separated from other biological components (i.e. other chromosomes and extrachromosomal DNA and RNA and protein) of a naturally occurring organism of a component, is obtained separately or purified therefrom. Therefore, nucleic acids, peptides and proteins "isolated" include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides and proteins can be a part of a composition, and if such a composition is not a part of the nucleic acid, peptide or protein's own environment, it is still isolated. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids. As used herein, "isolated" antibodies or antigen-binding fragments are intended to mean antibodies or antigen-binding fragments that are substantially free of other antibodies or antigen-binding fragments with different antigenic specificities (for example, antibodies that specifically bind to the separation of CD123 are substantially free of antibodies that specifically bind to antigens other than CD123). However, antibodies that specifically bind to the separation of an epitope, isotype or variant of CD123 can have cross-reactivity with other related antigens, for example, antigens from other species (such as CD123 species homologues).

"Polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA having a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA having a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA having a backbone that has been modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and rare bases such as inosine. DNA and RNA can be modified in a variety of ways; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides that typically occur in nature, as well as chemical forms of DNA and RNA that are unique to viruses and cells. "Polynucleotide" also includes relatively short nucleic acid chains, often referred to as oligonucleotides.

The meaning of "substantially identical" can vary depending on the context in which the term is used. Due to the natural sequence variations that may exist between heavy and light chains and the genes encoding them, it is expected that a certain degree of variation will be found in the amino acid sequences or genes encoding the antibodies or antigen-binding fragments described herein, with little or no effect on their unique binding properties (e.g., specificity and affinity). This expectation is due in part to the degeneracy of the genetic code and the successful evolution of conservative amino acid sequence variations, but this does not significantly change the properties of the encoded protein. Therefore, in the context of nucleic acid sequences, "substantially identical" means that there is at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or higher identity. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, which parameters need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The degree of variation that may occur in the amino acid sequence of a protein and has no substantial effect on the function of the protein is much lower than that of the nucleic acid sequence, because the same principle of degeneracy does not apply to amino acid sequences. Therefore, in the context of an antibody or antigen-binding fragment, "substantially the same" means an antibody or antigen-binding fragment with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity. Other embodiments include CD123-specific antibodies or antigen-binding fragments with a framework, a scaffold or other non-binding regions, which do not have significant homogeneity with the antibodies and antigen-binding fragments described herein, but do incorporate one or more CDRs or confer other sequences required for binding with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity with such sequences described herein. A "vector" is a replicon, such as a plasmid, phage, cosmid, or virus, into which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of that segment.

A "clone" is a group of cells derived from a single cell or a common progenitor cell by mitosis. A "cell line" is a clone of primary cells that is capable of stable growth in vitro for many generations. In some examples provided herein, cells are transformed by transfecting them with DNA.

The terms "expression" and "production" are used synonymously herein to refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass the translation of RNA into one or more polypeptides, and also encompass all naturally occurring post-transcriptional and post-translational modifications. Expression or production of an antibody or antigen-binding fragment thereof can occur within the cytoplasm of a cell, or in an extracellular environment, such as a growth medium in a cell culture.

The terms "treat" or "treatment" refer to any success or indication of success in alleviating or ameliorating an injury, condition, or disorder, including any objective or subjective parameter, such as symptom relief, remission, weakening, or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, reducing the degree of failure at a degenerative endpoint, improving the physical or mental well-being of the subject, or prolonging survival. Treatment can be assessed by objective or subjective parameters, including the results of a physical examination, neurological examination, or psychiatric evaluation.

An "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the dosage and for the period of time required. A therapeutically effective amount of a CD123×CD3 antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

Unless otherwise indicated, "antibody" refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY), including the various monomeric, polymeric, and chimeric forms. The term "antibody" specifically encompasses polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric and humanized antibodies.

Antigen binding fragments are any proteinaceous structures that can exhibit binding affinity for a specific antigen. Antigen binding fragments include those provided by any known technology such as enzymatic cleavage, peptide synthesis, and recombinant technology. Some antigen binding fragments are composed of portions of intact antibodies that retain the antigen-binding specificity of the parent antibody molecule. For example, an antigen binding fragment can comprise at least one variable region (heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind to a specific antigen. Examples of suitable antigen-binding fragments include, but are not limited to, diabodies and single-chain molecules, as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules; single-chain (Sc) antibodies; single antibody light chains; single antibody heavy chains; chimeric fusions between antibody chains or CDRs and other proteins; protein scaffolds; heavy chain monomers or dimers; light chain monomers or dimers; dimers consisting of one heavy chain and one light chain; monovalent fragments consisting of VL, VH, CL, and CH1 domains; or monovalent antibodies as described in WO2007059782; a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; an Fd fragment consisting essentially of the V.sub.H and C.sub.H1 domains; an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; a dAb fragment consisting essentially of a VH domain (Ward et al., Nature 341, 544-546 (1989)), also known as a domain antibody (Holt et al., Trends Biotechnol. 2003 Nov.; 21(11): 484-90); camelid or nanobodies (Revets et al., Expert Opin Biol Ther. 2005 Jan.; 5(1): 111-24); isolated complementarity determining regions (CDRs), etc. All antibody isotypes can be used to produce antigen-binding fragments. In addition, antigen-binding fragments can include non-antibody protein frameworks that can successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest (e.g., a protein scaffold). Antigen-binding fragments can be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase "antibody or antigen-binding fragment thereof" can be used to indicate that a given antigen-binding fragment is incorporated into one or more amino acid segments of the antibody mentioned in the phrase. When used herein in the context of two or more antibodies or antigen-binding fragments, the term "compete with" or "cross-compete with" means that two or more antibodies or antigen-binding fragments compete for binding to CD123, for example, competing for binding to CD123 in the assay described in Example 9. For certain antibody or antigen-binding fragment pairs, competition or blocking in the assays of the Examples is observed only when one antibody is coated on the plate and the other antibody in the pair is used for competition, but not when one antibody is coated on the plate. Unless otherwise defined or contradicted by context, the terms "compete with" or "cross-compete with" as used herein are also intended to encompass these antibody or antigen-binding fragment pairs.

The term "epitope" refers to a protein determinant that is capable of specific binding to an antibody. An epitope is typically composed of surface groups of a molecule, such as amino acids or sugar side chains, and typically has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that the binding to the former, but not the latter, is lost in the presence of a denaturing solvent. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by a specific antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specific antigen-binding peptide).

When used in the context of an antibody or antibody fragment, "specific binding" or "immunospecific binding" or its derivatives means binding to one or more epitopes of a protein of interest through a domain encoded by an immunoglobulin gene or immunoglobulin gene fragment, without preferentially binding to other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1 × ^10-8 M, as measured by a surface plasmon resonance assay or a cell binding assay. Phrases such as "[antigen] specific" antibody (e.g., CD123-specific antibody) are intended to express that the antibody specifically binds to the antigen.

As used herein, the term "kd" (sec-1) refers to the dissociation rate constant for a specific antibody-antigen interaction. This value is also known as the koff value.

As used herein, the term "ka" (M-1 sec-1) refers to the association rate constant for a specific antibody-antigen interaction.

As used herein, the term "KD" (M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

As used herein, the term "KA" (M-1) refers to the association equilibrium constant for a specific antibody-antigen interaction and is obtained by dividing ka by kd.

The term "subject" refers to humans and non-human animals, including all vertebrates, e.g., mammals and non-mammals such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians and reptiles. In many embodiments of the methods, the subject is a human.

As used herein, the term "sample" refers to a collection of similar fluids, cells or tissues (e.g., surgically removed tumor tissue, biopsy tissue, including fine needle aspiration tissue) separated from a subject and fluids, cells or tissues present in a subject. In some embodiments, a sample is a biological fluid. A biological fluid is typically a liquid at physiological temperature and can include a naturally occurring fluid present in a subject or a biological source, extracted, expressed or otherwise extracted from a subject or a biological source. Some biological fluids derive from specific tissues, organs or local areas, and some other biological fluids can be more systemically or systematically located in a subject or a biological source. The example of a biological fluid includes blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, the fluid of the pleura, pericardium, peritoneum, abdomen and other body cavities, the fluid collected by bronchial lavage, etc. Biological fluids may also include liquid solutions in contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media, lavage fluids, etc. As used herein, the term "sample" encompasses material removed from a subject or present in a subject.

A "known standard" can be a solution having a known amount or known concentration of CD123, wherein the solution can be a naturally occurring solution, such as a sample from a patient known to have early, moderate, advanced, progressive, or quiescent cancer; or the solution can be a synthetic solution, such as a buffered aqueous solution in which a known amount of CD123 is diluted. The known standards described herein can include CD123 isolated from a subject, recombinant or purified CD123 protein, or a CD123 concentration value associated with a disease condition.

The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex is composed of 6 different polypeptide chains. These polypeptide chains include CD3γ chain (SwissProt P09693), CD3β chain (SwissProt P04234), two CD3ε chains (SwissProt P07766) and a CD3ζ chain homodimer (SwissProt 20963), and the complex is associated with T cell receptor α and β chains. Unless otherwise indicated, the term "CD3" includes any CD3 variants, isoforms and species homologs that are naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNAs encoding those polypeptides.

As used herein, the terms "α subunit of the IL-3 receptor," "IL3Rα," "CD123," "IL3Rα chain," and "IL3Rα subunit" are interchangeable and refer to an antigenic determinant detectable on leukemic precursor cells that immunobinds to interleukin 3 (IL3). In a specific embodiment, CD123 is human CD123. In a specific embodiment, CD123 is cynomolgus monkey CD123. In a specific embodiment, CD123 is CD123 SP1. In a specific embodiment, CD123 is CD123SP2. Unless otherwise indicated, the term "CD123" includes any CD123 variants, isoforms, and species homologs.

"CD123 × CD3 antibody" is a multispecific antibody, optionally a bispecific antibody, comprising two different antigen binding regions, wherein one binding region specifically binds to the antigen CD123 and the other binding region specifically binds to CD3. Multispecific antibodies can be bispecific antibodies, diabodies or similar molecules (for a description of diabodies, see, for example, PNAS USA 90 (14), 6444-8 (1993)). In addition to a portion of CD123, the bispecific antibodies, diabodies, etc. provided herein can be combined with any suitable target. The term "bispecific antibody" should be understood as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as different target combinations, but also includes different epitopes bound to one target.

" reference sample " is a sample that can be compared with another sample such as a test sample to characterize the sample being compared. The reference sample has some characteristic attributes, as a basis for comparison with the test sample. For example, the reference sample can be used as a benchmark for indicating that a subject has CD123 levels of cancer. The reference sample does not necessarily have to be analyzed in parallel with the test sample, so in some cases, the reference sample can be a numerical value or range previously determined for characterizing a given condition, such as indicating that a subject has CD123 levels of cancer. The term also includes samples known to be related to physiological state or disease condition (such as CD123 expression type cancer) but with an unknown amount of CD123 for comparison purposes.

The term "progression" as used in the context of progression of CD123-expressing cancers includes a change in cancer from a less severe state to a more severe state. This may include an increase in the number or severity of tumors, the extent of metastasis, the rate at which the cancer grows or spreads, etc. For example, "progression of colon cancer" includes the progression of such cancer from a less severe state to a more severe state, such as from stage I to stage II, from stage II to stage III, etc.

The term "regression" as used in the context of regression of a CD123-expressing cancer includes a change in cancer from a more severe state to a less severe state. This may include a decrease in the number or severity of tumors, the extent of metastasis, the rate of cancer growth or spread, etc. For example, "regression of colon cancer" includes regression of such cancer from a more severe state to a less severe state, such as regression from stage III to stage II, from stage II to stage I, etc.

The term "stable" as used in the context of a stable CD123-expressing cancer is intended to describe a disease condition that has not or has not changed significantly over a clinically relevant period of time to be considered a progressive cancer or a regressing cancer.

The embodiments described herein are not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary.

CD123-specific antibodies and antigen-binding fragments

Described herein are isolated monoclonal antibodies or antigen-binding fragments that specifically bind to CD123. The general structure of an antibody molecule includes an antigen-binding domain, which includes heavy and light chains and an Fc domain, and performs multiple functions (including complement fixation and binding to antibody receptors).

The CD123-specific antibody or Fab includes all isotypes IgA, IgD, IgE, IgG and IgM, and a synthetic multimer of four-chain immunoglobulin structures. The antibody or Fab also includes the IgY isotype that is typically present in hen or turkey serum and hen or turkey egg yolk.

CD123-specific antibodies and antigen-binding fragments can be derived from any species by recombinant methods. For example, the antibody or antigen-binding fragment can be a mouse, rat, goat, horse, pig, cattle, chicken, rabbit, camel, donkey, human or a chimeric version thereof. To be suitable for administration to humans, non-human antibodies or antigen-binding fragments can be genetically or structurally altered to be less antigenic when administered to human patients.

In some embodiments, the antibody or antigen-binding fragment is chimeric. As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment thereof in which at least some portion of at least one variable domain is derived from an antibody amino acid sequence of a non-human mammal, rodent, or reptile, while the remainder of the antibody or antigen-binding fragment thereof is derived from a human.

In some embodiments, the antibody is a humanized antibody. A humanized antibody can be a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. To a large extent, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues in the complementary determining region (CDR) of the receptor are replaced by residues in the CDR of a non-human species (donor antibody) such as a mouse, rat, or rabbit with desired specificity, affinity, and ability. Generally speaking, a humanized antibody will comprise substantially all of at least one, and generally two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin.

The antibodies or antigen-binding fragments described herein may exist in a variety of forms, but will comprise one or more of the antibody CDRs shown in Table 1.

Described herein are antibodies and Fabs that immunospecifically bind to the separation of CD123. In some embodiments, the CD123-specific antibody or Fab is human IgG or a derivative thereof. Although the CD123-specific antibody or Fab illustrated herein are human, the illustrated antibody or Fab can also be chimeric.

In some embodiments, CD123-specific antibodies or antigen-binding fragments thereof are provided, which comprise a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1. In some embodiments, CD123-specific antibodies or antigen-binding fragments thereof are provided, which comprise a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1 and a light chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 006, a heavy chain CDR2 comprising SEQ ID NO: 007, and a heavy chain CDR3 comprising SEQ ID NO: 008. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 006, a heavy chain CDR2 comprising SEQ ID NO: 007, a heavy chain CDR3 comprising SEQ ID NO: 008, a light chain CDR1 comprising SEQ ID NO: 009, a light chain CDR2 comprising SEQ ID NO: 010, and a light chain CDR3 comprising SEQ ID NO: 011. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 119. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 119 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 164. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 012, a heavy chain CDR2 comprising SEQ ID NO: 013, and a heavy chain CDR3 comprising SEQ ID NO: 014. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 012, a heavy chain CDR2 comprising SEQ ID NO: 013, a heavy chain CDR3 comprising SEQ ID NO: 014, a light chain CDR1 comprising SEQ ID NO: 015, a light chain CDR2 comprising SEQ ID NO: 016, and a light chain CDR3 comprising SEQ ID NO: 017. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 120. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 120 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 165. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 018, a heavy chain CDR2 comprising SEQ ID NO: 019, and a heavy chain CDR3 comprising SEQ ID NO: 020. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 018, a heavy chain CDR2 comprising SEQ ID NO: 019, a heavy chain CDR3 comprising SEQ ID NO: 020, a light chain CDR1 comprising SEQ ID NO: 009, a light chain CDR2 comprising SEQ ID NO: 010, and a light chain CDR3 comprising SEQ ID NO: 011. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 121. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 121 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 164. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 021, a heavy chain CDR2 comprising SEQ ID NO: 022, and a heavy chain CDR3 comprising SEQ ID NO: 023. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 021, a heavy chain CDR2 comprising SEQ ID NO: 022, a heavy chain CDR3 comprising SEQ ID NO: 023, a light chain CDR1 comprising SEQ ID NO: 024, a light chain CDR2 comprising SEQ ID NO: 025, and a light chain CDR3 comprising SEQ ID NO: 026. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 122. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 122 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 166. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 027, a heavy chain CDR2 comprising SEQ ID NO: 028, and a heavy chain CDR3 comprising SEQ ID NO: 029. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 027, a heavy chain CDR2 comprising SEQ ID NO: 028, a heavy chain CDR3 comprising SEQ ID NO: 029, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 123. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 123 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 035. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 035, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 124. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 124 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 036. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 036, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 125. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 125 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 037. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 037, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 126. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 126 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 038. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 038, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 127. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 127 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 040, and a heavy chain CDR3 comprising SEQ ID NO: 041. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 040, a heavy chain CDR3 comprising SEQ ID NO: 041, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 128. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 128 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 042. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 042, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 129. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 129 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 043. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 034, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 043, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 130. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 130 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 044, and a heavy chain CDR3 comprising SEQ ID NO: 045. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 044, a heavy chain CDR3 comprising SEQ ID NO: 045, a light chain CDR1 comprising SEQ ID NO: 015, a light chain CDR2 comprising SEQ ID NO: 016, and a light chain CDR3 comprising SEQ ID NO: 017. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 131. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 131 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 165. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 046, and a heavy chain CDR3 comprising SEQ ID NO: 047. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 046, a heavy chain CDR3 comprising SEQ ID NO: 047, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 132. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 132 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 048. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 048, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 133. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 133 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 049. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 049, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 134. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 134 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 050. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 050, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 135. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 135 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 051, a heavy chain CDR2 comprising SEQ ID NO: 052, and a heavy chain CDR3 comprising SEQ ID NO: 053. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 051, a heavy chain CDR2 comprising SEQ ID NO: 052, a heavy chain CDR3 comprising SEQ ID NO: 053, a light chain CDR1 comprising SEQ ID NO: 024, a light chain CDR2 comprising SEQ ID NO: 025, and a light chain CDR3 comprising SEQ ID NO: 054. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 136. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 136 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 168. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 055, a heavy chain CDR2 comprising SEQ ID NO: 056, and a heavy chain CDR3 comprising SEQ ID NO: 057. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 055, a heavy chain CDR2 comprising SEQ ID NO: 056, a heavy chain CDR3 comprising SEQ ID NO: 057, a light chain CDR1 comprising SEQ ID NO: 058, a light chain CDR2 comprising SEQ ID NO: 059, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 137. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 137 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 169. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 060. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 060, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 138. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 138 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 061. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 061, a light chain CDR1 comprising SEQ ID NO: 062, a light chain CDR2 comprising SEQ ID NO: 063, and a light chain CDR3 comprising SEQ ID NO: 064. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 139. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 139 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 170. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 065. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 065, a light chain CDR1 comprising SEQ ID NO: 066, a light chain CDR2 comprising SEQ ID NO: 067, and a light chain CDR3 comprising SEQ ID NO: 068. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 140. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 140 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 171. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 069. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 069, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 070. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 141. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 141 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 172. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 071. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 071, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, ^{5×10-8 M} or less, 1× ^10-8 M or less, 5× ^{10-9 M or less, or 1×10-9 M} or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 142. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 142 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 072, and a heavy chain CDR3 comprising SEQ ID NO: 073. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 072, a heavy chain CDR3 comprising SEQ ID NO: 073, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 143. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 143 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 074. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 074, a light chain CDR1 comprising SEQ ID NO: 075, a light chain CDR2 comprising SEQ ID NO: 076, and a light chain CDR3 comprising SEQ ID NO: 077. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 144. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 144 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 173. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 078. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 078, a light chain CDR1 comprising SEQ ID NO: 079, a light chain CDR2 comprising SEQ ID NO: 063, and a light chain CDR3 comprising SEQ ID NO: 080. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 145. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 145 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 174. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 081. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 081, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 146. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 146 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 082. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 082, a light chain CDR1 comprising SEQ ID NO: 083, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 084. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 147. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 147 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 175. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 085, and a heavy chain CDR3 comprising SEQ ID NO: 086. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 085, a heavy chain CDR3 comprising SEQ ID NO: 086, a light chain CDR1 comprising SEQ ID NO: 087, a light chain CDR2 comprising SEQ ID NO: 067, and a light chain CDR3 comprising SEQ ID NO: 088. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 148. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 148 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 176. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 089, and a heavy chain CDR3 comprising SEQ ID NO: 090. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 089, a heavy chain CDR3 comprising SEQ ID NO: 090, a light chain CDR1 comprising SEQ ID NO: 091, a light chain CDR2 comprising SEQ ID NO: 076, and a light chain CDR3 comprising SEQ ID NO: 092. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 149. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 149 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 177. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 093. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 093, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 150. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 150 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 094. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 094, a light chain CDR1 comprising SEQ ID NO: 095, a light chain CDR2 comprising SEQ ID NO: 076, and a light chain CDR3 comprising SEQ ID NO: 096. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 151. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 151 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 178. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 097. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 097, a light chain CDR1 comprising SEQ ID NO: 098, a light chain CDR2 comprising SEQ ID NO: 067, and a light chain CDR3 comprising SEQ ID NO: 099. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 152. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 152 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 179. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 100. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 100, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 101. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 153. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 153 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 180. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 102. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 102, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 154. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 154 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 103. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 103, a light chain CDR1 comprising SEQ ID NO: 104, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 105. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 155. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 155 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 181. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 106. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 039, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 106, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 156. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 156 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 107. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 107, a light chain CDR1 comprising SEQ ID NO: 108, a light chain CDR2 comprising SEQ ID NO: 109, and a light chain CDR3 comprising SEQ ID NO: 110. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 157. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 157 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 182. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 111. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 111, a light chain CDR1 comprising SEQ ID NO: 112, a light chain CDR2 comprising SEQ ID NO: 076, and a light chain CDR3 comprising SEQ ID NO: 113. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 158. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 158 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 183. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 114, a heavy chain CDR2 comprising SEQ ID NO: 022, and a heavy chain CDR3 comprising SEQ ID NO: 115. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 114, a heavy chain CDR2 comprising SEQ ID NO: 022, a heavy chain CDR3 comprising SEQ ID NO: 115, a light chain CDR1 comprising SEQ ID NO: 024, a light chain CDR2 comprising SEQ ID NO: 025, and a light chain CDR3 comprising SEQ ID NO: 026. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 159. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 159 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 166. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 114, a heavy chain CDR2 comprising SEQ ID NO: 022, and a heavy chain CDR3 comprising SEQ ID NO: 116. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 114, a heavy chain CDR2 comprising SEQ ID NO: 022, a heavy chain CDR3 comprising SEQ ID NO: 116, a light chain CDR1 comprising SEQ ID NO: 015, a light chain CDR2 comprising SEQ ID NO: 016, and a light chain CDR3 comprising SEQ ID NO: 017. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 160. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 160 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 165. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 117, a heavy chain CDR2 comprising SEQ ID NO: 013, and a heavy chain CDR3 comprising SEQ ID NO: 118. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 117, a heavy chain CDR2 comprising SEQ ID NO: 013, a heavy chain CDR3 comprising SEQ ID NO: 118, a light chain CDR1 comprising SEQ ID NO: 015, a light chain CDR2 comprising SEQ ID NO: 016, and a light chain CDR3 comprising SEQ ID NO: 017. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 161. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 161 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 165. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 051, a heavy chain CDR2 comprising SEQ ID NO: 052, and a heavy chain CDR3 comprising SEQ ID NO: 053. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 051, a heavy chain CDR2 comprising SEQ ID NO: 052, a heavy chain CDR3 comprising SEQ ID NO: 053, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 162. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 162 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, and a heavy chain CDR3 comprising SEQ ID NO: 042. In some embodiments, the CD123-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 033, a heavy chain CDR2 comprising SEQ ID NO: 034, a heavy chain CDR3 comprising SEQ ID NO: 042, a light chain CDR1 comprising SEQ ID NO: 030, a light chain CDR2 comprising SEQ ID NO: 031, and a light chain CDR3 comprising SEQ ID NO: 032. The CD123-specific antibodies or antigen-binding fragments may comprise human framework sequences. The CD123-specific antibody or antigen-binding fragment can be bound to CD123 with an affinity of 5× ^10-7 M or less, such as 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 163. In some embodiments, the CD123-specific antibody and antigen-binding fragment comprise a heavy chain variable domain that is substantially the same or identical to SEQ ID NO: 163 and a light chain variable domain that is substantially the same or identical to SEQ ID NO: 167. The heavy chain variable domain and light chain variable domain of the antibodies discussed in this paragraph are suitable for inclusion in a bispecific construct, wherein one arm is an anti-CD123 arm.

Anti-CD123 antibodies and Fab provided by the present invention also include antibodies that compete with the above-mentioned antibodies for binding. Competition can be used in conjunction with competition ELISA, determined according to the technology described in Example 5 below. Competition binding can be determined by detecting that the combination of the first antibody is suppressed by the second antibody by at least 20%, and no matter how the antibody is bound to the order of CD123 (that is, if when antibody A is bound to CD123 before antibody B, only 10% inhibition is observed, but when antibody B is bound to CD123 before antibody A, 30% inhibition is observed, then because in one of these experiments, greater than 20% inhibition has been observed, so competition binding can be terminated).

In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, and IgG4 isotype. In some embodiments wherein the antibody has an IgG1 isotype, the antibody contains L234A, L235A, and K409R substitutions in its Fc region. In some embodiments wherein the antibody has an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region. Specific antibodies defined by the CDR and/or variable domain sequences discussed in the above paragraphs may include these modifications.

The present invention also discloses isolated polynucleotides encoding antibodies or antigen-binding fragments that immunospecifically bind to CD123. The isolated polynucleotides encoding the variable domain segments provided herein can be included in the same or different vectors to produce antibodies or antigen-binding fragments.

Polynucleotides encoding recombinant antigen-binding proteins are also within the scope of the present disclosure. In some embodiments, the polynucleotides (and their encoded peptides) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence may include, but is not limited to, a restriction site or a translation initiation site.

CD123 specific antibodies or antigen-binding fragments as described herein include such variants：It has a single or multiple amino acid replacement, deletion or addition of the biological properties (for example, binding affinity or immune effector activity) retaining the CD123 specific antibodies or antigen-binding fragments.In the context of the present invention, unless otherwise indicated, the following notation is used to describe mutations：i) the replacement of amino acids at a given position is written as, for example, K409R, which means that the lysine at position 409 is replaced by arginine；And ii) for a particular variant, a specific three-letter or single-letter code (including code Xaa and X) is used to indicate any amino acid residue. Therefore, arginine replaces the lysine at position 409 and is expressed as: K409R, or any amino acid residue replaces the lysine at position 409 and is expressed as K409X. In the case of lysine deletion at position 409, it is represented by K409*. Technicians can prepare variants with single or multiple amino acid replacements, deletions or additions.

These variants may include: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include substituents, and (d) variants in which the polypeptide is fused to another peptide or polypeptide (e.g., a fusion partner, a protein tag, or other chemical moiety) that can confer useful properties on the polypeptide, such as an epitope of an antibody, a polyhistidine sequence, a biotin moiety, and the like. The antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are replaced at conservative or non-conservative positions with corresponding residues in another species. In other embodiments, amino acid residues at non-conservative positions are replaced by conservative or non-conservative residues. The techniques for obtaining these variants (including genetic techniques (deletions, mutations, etc.), chemical techniques, and enzymatic techniques) are known to those of ordinary skill in the art.

CD123 specific antibodies or antigen-binding fragments described herein can include several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is IgG1, IgG2, IgG3 or IgG4 isotype, preferably IgG1 or IgG4 isotype. The specificity of an antibody or its antigen-binding fragment is mainly determined by the amino acid sequence and arrangement of CDR. Therefore, the CDR of a kind of isotype can be converted into another isotype without changing the antigen specificity. Alternatively, technology has been established to convert hybridomas from producing a kind of antibody isotype to producing another isotype (isotype conversion) without changing the antigen specificity. Therefore, this type of antibody isotype is within the scope of the antibody or antigen-binding fragment.

The CD123-specific antibodies or antigen-binding fragments described herein have binding affinity for CD123SP1, including a dissociation constant (KD) of less than about 5× ^10-7 M, preferably less than about 5× ^10-8 M. In some embodiments, the CD123-specific antibodies or antigen-binding fragments described herein have binding affinity for CD123SP2, including a dissociation constant (KD) of less than about 5× ^10-7 M, preferably less than about 5× ^10-8 M. The affinity of the CD123-specific antibodies or antigen-binding fragments can be determined by various methods known in the art (e.g., surface plasmon resonance or ELISA-based methods). Assays for measuring affinity by SPR include assays performed using a BIAcore 3000 machine, wherein the assay is performed at room temperature (e.g., at or near 25° C.), wherein antibodies capable of binding to CD123 are captured on a BIAcore sensor chip with an anti-Fc antibody (e.g., goat anti-human IgG Fc specific antibody, Jackson ImmunoResearch laboratories Prod#109-005-098) to a level of approximately 75 RU, and association and dissociation data are then collected at a flow rate of 40 μl/min.

Also provided are vectors comprising the polynucleotides described herein. The vector can be an expression vector. Thus, it is contemplated that recombinant expression vectors comprising sequences encoding a polypeptide of interest are also within the scope of the present disclosure. The expression vector may contain one or more additional sequences, such as, but not limited to, regulatory sequences (e.g., promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a variety of host cells are well known, including but not limited to plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

Recombinant expression vectors within the scope of this specification include synthetic, genomic or cDNA derived nucleic acid fragments that encode at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements can include transcriptional promoters, sequences encoding suitable mRNA ribosome binding sites, and sequences that control transcription and translation termination. Expression vectors, particularly mammalian expression vectors, can also include one or more non-transcriptional elements, such as an origin of replication, a suitable promoter and enhancer that are connected to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (such as necessary ribosome binding sites), polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences. An origin of replication that confers replication capability in the host can also be incorporated.

Transcriptional and translational control sequences in expression vectors used to transform vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody or antigen-binding fragment coding sequence is placed under the control of a strong constitutive promoter (e.g., a promoter for the following genes: hypoxanthine phosphorucleoside transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin, human myosin, human hemoglobin, human muscle creatine, etc.). In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the embodiments described. Such viral promoters include, but are not limited to, the immediate early promoter of cytomegalovirus (CMV), the early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeats (LTR) of Maroney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In one embodiment, the CD123-specific antibody or antigen-binding fragment thereof coding sequence is placed under the control of an inducible promoter, such as a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, or a promoter containing one or more interferon-stimulated response elements (ISREs) such as protein kinase R 2′, 5′-oligoadenylate synthetase, Mx gene, ADAR1, etc.

The vectors described herein may contain one or more internal ribosome entry sites (IRES). The inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing the expression of some proteins. In some embodiments, the vector system includes one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the above-mentioned nucleic acid sequences. The vector components may be connected continuously, or arranged in a manner that provides optimal spacing for expressing the gene product (i.e., by introducing "spacer" nucleotides between the ORFs), or positioned in another manner. Regulatory elements such as the IRES motif may also be arranged to provide optimal spacing for expression.

The vector may include a selection marker well known in the art. Selection markers include positive and negative selection markers, such as antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene), glutamate synthase gene, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al., 7Gene Ther. 1738-1743 (2000)). The nucleic acid sequence encoding the selection marker or cloning site can be upstream or downstream of the nucleic acid sequence encoding the polypeptide of interest or the cloning site.

The carriers described herein can be used to transform various cells with genes encoding the antibody or Fab. For example, the carrier can be used to generate CD123-specific antibodies or produce cells of Fab. Therefore, the feature on the other hand is a host cell transformed by a carrier comprising a nucleic acid sequence encoding an antibody or its Fab (such as the antibody or Fab described and exemplified herein) specifically binding to CD123.

The various techniques for introducing foreign genes into cells are known in the art, and for the purpose of implementing the method, these techniques can be used to construct recombinant cells according to the various embodiments described and illustrated herein. The technology used should allow the heterologous gene sequence to be stably transferred to the host cell so that the heterologous gene sequence is heritable and can be expressed by progeny, so that the necessary development and physiological functions of the recipient cell are not destroyed. Available technology includes but is not limited to chromosome transfer (such as cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (such as transfection, spheroplast fusion, microinjection, electroporation, liposome vectors), viral vector transfer (such as, recombinant DNA virus, recombinant RNA virus) etc. (described in Cline, 29Pharmac.Ther.69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced bacterial protoplasts and mammalian cell fusion can also be used to transform cells.

The cell suitable for expressing CD123 specific antibody as herein described or Fab is preferably a eukaryotic cell, more preferably a cell in plant, rodent or human origin, such as but not limited to NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cell, COS-7, T98G, CV-1/EBNA, L cell, C127, 3T3, HeLa, NS1, Sp2/0 myeloma cell and BHK cell line etc. In addition, hybridoma cell can also be used to complete the expression of antibody.The method for producing hybridoma is well known in the art.

Cells transformed with expression vectors as described herein can be selected or screened for recombinant expression of antibodies or antigen-binding fragments as described herein. Amplification and screening of recombinant positive cells are performed to screen subclones that exhibit a desired phenotype (e.g., high-level expression, enhanced growth characteristics, or, for example, the ability of a protein to produce a protein with desired biochemical characteristics due to post-translational modification of a protein modification or change). These phenotypes may be due to the inherent properties of a given subclone or due to mutation. Mutation can be achieved by using chemicals, UV wavelength light, radiation, viruses, insertion mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

Methods of treatment using CD123-specific antibodies

Provided herein is a CD123 specific antibody or its antigen binding fragment for use in treatment. Specifically, these antibodies or antigen binding fragments can be used to treat cancer, such as CD123 expression type cancer. Therefore, the present invention provides a method for treating cancer, the method including administering an antibody as described herein, such as CD123 specific antibody or antigen binding fragment. For example, this use can be to inhibit the biological effects of IL-3 by preventing IL-3 from being bound to IL-3R or wherein the antibody is conjugated to a toxin, thereby targeting the toxin to CD123 expression type cancer. In some embodiments, CD123 expression type cancer includes hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN). Antibodies used in these methods include those described above, such as CD123-specific antibodies or antigen-binding fragments that bind to an epitope comprising one or more residues from the following segments: the CD123 SP2 ECD segment comprising residues 195-202 (RARERVYE (SEQ ID NO: 234)) and/or the CD123 SP2 ECD segment comprising residues 156-161 (RKFRYE (SEQ ID NO: 232)) and/or the CD123 SP2 ECD segment comprising residues 173-178 (SEQ ID NO: 233). Also useful in these methods are antibodies having the characteristics described in Table 1, such as the CDR or variable domain sequences in the further discussion of these antibodies.

In some embodiments described herein, the immune effector properties of CD123 specific antibodies can be enhanced or silenced via Fc modifications by technology known to those skilled in the art. For example, Fc effector functions such as C1q combinations, complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), cell surface receptors (such as B cell receptors; BCR) reduction etc. can be provided and/or controlled by modifying the residues responsible for the following activities in Fc.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

The ability of monoclonal antibodies to induce ADCC can be enhanced by modifying their oligosaccharide composition. Human IgG1 or IgG3 is N-glycosylated at Asn297 by the well-known bibranched G0, G0F, G1, G1F, G2, or G2F forms of most glycans. Antibodies produced by unmodified CHO cells typically have a glycan fucose content of approximately at least 85%. Removing core fucose from bibranched complex-type oligosaccharides attached to the Fc region can enhance antibody ADCC through improved FcγRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be achieved using different approaches that have been reported to result in the successful expression of relatively highly defucosylated antibodies with bibranched complex-type Fc oligosaccharides, such as: controlling culture osmotic pressure (Konno et al., Cytotechnology 64:249-65, 2012), using the variant CHO cell line Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), using the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID: 20562582), using the rat hybridoma cell line YB2/0 as a host cell line (Shinkawa et al., J Biol Chem 277:26733-26740, 2002), and using the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID: 20562582). 278:3466-3473, 2003), introduction of small interfering RNA specific for the α-1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or the potent α-mannosidase I inhibitor kifunensine (Ferrara et al., J Biol Chem 281:5032-5036, 2006, Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

In some embodiments described herein, ADCC caused by CD123 antibodies can also be enhanced by some replacements in antibody Fc. Exemplary replacements are, for example, replacements at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residues are numbered according to the EU index), as described in U.S. Patent No. 6,737,056.

Method for detecting CD123

Provided herein is a method for detecting the CD123 in a biological sample by contacting a sample with an antibody as described herein or its antigen-binding fragment. As described herein, sample can be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspiration tissue), histological preparations, etc. In some embodiments, the method includes contacting a sample with any CD123-specific antibody as described herein or its antigen-binding fragment to detect the CD123 in a biological sample.

In some embodiments, sample can be contacted with more than one CD123-specific antibody or Fab as described herein.For example, sample can first be contacted with the first CD123-specific antibody or its Fab, and then contacted with the second CD123-specific antibody or its Fab, wherein the first antibody or Fab and the second antibody or Fab are not identical antibodies or Fab.In some embodiments, the first antibody or its Fab can be attached to a surface, such as a multiwell plate, a chip or a similar substrate before contacting the sample.In other embodiments, the first antibody or its Fab can not be attached to or connected to anything before contacting the sample.

The CD123-specific antibodies and Fabs can be detectably labeled. In some embodiments, by the methods described herein, labeled antibodies and Fabs can be beneficial for detecting CD123. Many such labels are readily known to those skilled in the art. For example, suitable labels include but should not be considered to be limited to radiolabels, fluorescent labels, epitope labels, biotin, chromophore labels, ECL labels or enzymes. More specifically, the labels include ruthenium, 111In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tags), acridine dyes, cyanine dyes, fluorescent dyes, oxazine dyes, phenanthridinium dyes, rhodamine dyes, Alexa dyes, etc.

The CD123-specific antibodies and Fabs can be used in multiple assays to detect CD123 in biological samples. Some suitable assays include but should not be considered to be limited to Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assays.

In some embodiments described herein, detection of CD123-expressing cancer cells in a subject can be used to determine whether the subject can be treated with a therapeutic agent directed against CD123.

CD123 is present in blood and serum samples at a detectable level. Therefore, provided herein is a method for detecting CD123 in a sample (such as a serum sample) derived from blood by contacting the sample with an antibody or its Fab that specifically binds CD123. Blood sample or its derivatives can be diluted, fractionated or otherwise processed to obtain a sample to which the method can be performed. In some embodiments, CD123 in a blood sample or its derivatives can be detected by any number of assays known in the art, such as but not limited to Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assays.

Methods for diagnosing cancer

Provided herein is a method for diagnosing CD123 expression type cancer in a subject. In some embodiments, CD123 expression type cancer includes hematological cancer, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell tumor (DPDCN). In some embodiments, as described above, the CD123 in the detection biological sample such as blood sample or serum sample provides the ability of diagnosing cancer in the subject from which the sample is obtained. Alternatively, in some embodiments, other samples such as histological samples, fine needle aspiration samples, excised tumor tissue, circulating cells, circulating tumor cells, etc. can also be used to assess whether the subject from which the sample is obtained suffers from cancer. In some embodiments, it may be known that the subject from which the sample is obtained suffers from cancer, but it may not be clear that the type of cancer suffered by the subject or the preliminary diagnosis result may not yet be diagnosed, therefore detection of the CD123 in the biological sample obtained from the subject can achieve or clearly diagnose cancer. For example, it may be known that a subject has cancer, but it may not be known or may not be clear whether the subject's cancer is CD123-expressing.

In some embodiments, the method is directed to assessing whether a subject suffers from CD123 expression type cancer by measuring the amount of CD123 present in a biological sample derived from a subject; and the amount of the observed CD123 is compared with the amount of CD123 in a control or reference sample, wherein the difference between the amount of CD123 in the sample derived from the subject and the amount of CD123 in the control or reference sample indicates that the subject suffers from CD123 expression type cancer. In another embodiment, the amount of CD123 observed in the biological sample obtained from the subject can be compared with the CD123 levels known to be associated with certain forms or stages of cancer, thereby determining the form or stage of the cancer suffered from the subject. In some embodiments, the amount of CD123 in a sample derived from a subject is assessed by contacting the sample with an antibody or its antigen-binding fragment (e.g., CD123-specific antibody as described herein) that immunospecifically binds to CD123. The sample in which the presence of CD123 is assessed can be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e. free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspiration tissue), histological preparations, etc. In some embodiments, CD123-expressing cancers include hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN). In some embodiments, the subject is a human.

In some embodiments, the method for diagnosing CD123 expression type cancer involves: contacting the biological sample of the subject with a CD123-specific antibody or its antigen-binding fragment (such as those of the antibodies and fragments provided in Table 1); quantitative analysis of the amount of CD123 present in the sample bound by the antibody or its antigen-binding fragment; comparing the amount of CD123 present in the sample with a known standard or a reference sample; and determining whether the CD123 level of the subject falls within the CD123 level associated with cancer. In another embodiment, the diagnostic method may be followed by an additional step of administering or giving a cancer-specific treatment. In another embodiment, the diagnostic method may be followed by an additional step of transmitting the assay result for the purpose of treating cancer. In some embodiments, cancer-specific treatment may be directed to CD123 expression type cancer, such as the CD123×CD3 multispecific antibodies described herein.

In some embodiments, the method involves assessing whether a subject has a CD123-expressing cancer by determining the amount of CD123 present in a blood or serum sample obtained from the subject; and comparing the observed amount of CD123 to the amount of CD123 in a control or reference sample, wherein a difference between the amount of CD123 in the sample derived from the subject and the amount of CD123 in the control or reference sample indicates that the subject has a CD123-expressing cancer.

In some embodiments, the control or reference sample may be derived from a subject not suffering from CD123 expression type cancer. In some embodiments, the control or reference sample may be derived from a subject suffering from CD123 expression type cancer. In some embodiments wherein the control sample or reference sample are derived from a subject not suffering from CD123 expression type cancer, the amount of CD123 observed in the test sample is increased relative to the amount of CD123 observed in the control sample or reference sample, indicating that the subject being assessed suffers from CD123 expression type cancer. In some embodiments wherein the control sample is derived from a subject not suffering from CD123 expression type cancer, the amount of CD123 observed in the test sample is reduced or approximate relative to the amount of CD123 observed in the control sample or reference sample, indicating that the subject being assessed does not suffer from CD123 expression type cancer. In some embodiments wherein the control sample or reference sample are derived from a subject suffering from CD123 expression type cancer, the amount of CD123 observed in the test sample is approximate relative to the amount of CD123 observed in the control sample or reference sample, indicating that the subject being assessed suffers from CD123 expression type cancer. In some embodiments where the control or reference sample is derived from a subject with a CD123-expressing cancer, observing a decrease in the amount of CD123 present in the test sample relative to the amount of CD123 observed in the control or reference sample indicates that the subject being evaluated does not have a CD123-expressing cancer.

In some embodiments, the amount of CD123 in a sample derived from a subject is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds CD123 (e.g., an antibody as described herein). The sample in which the presence of CD123 is assessed can be derived from a blood sample, a serum sample, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspiration tissue), histological preparations, etc.

In various aspects, the amount of CD123 is determined by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds CD123. In some embodiments, the sample may be contacted with one or more types of antibodies or antigen-binding fragments thereof that specifically bind CD123. In some embodiments, the sample may first be contacted with a first antibody or antigen-binding fragment thereof that specifically binds CD123, and then contacted with a second antibody or antigen-binding fragment thereof that specifically binds CD123. CD123-specific antibodies or antigen-binding fragments, such as those described herein, can be used in this function.

Various combinations of CD123-specific antibodies and Fabs can be used to provide "first" and "second" antibodies or Fabs to implement the diagnostic method. In some embodiments, CD123 expression type cancers include hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

In certain embodiments, the amount of CD123 is determined by Western blot analysis, radioimmunoassay, immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or ELISA assay.

In various embodiments of the diagnostic method, a control sample or a reference sample is used. The sample can be a positive or negative determination control sample to ensure that the assay works properly; For example, a determination control sample of this nature can generally be used in immunohistochemistry assays. Alternatively, the sample can be a standardized reference sample of CD123 amount in a biological sample from a healthy subject. In some embodiments, the observed CD123 levels of the test subject can be compared with the CD123 levels observed in a sample from a subject known to have CD123 expression type cancer. In some embodiments, the control subject may suffer from a specific cancer of interest. In some embodiments, it is known that the control subject suffers from early-stage cancer, which may or may not be CD123 expression type cancer. In some embodiments, it is known that the control subject suffers from mid-stage cancer, which may or may not be CD123 expression type cancer. In some embodiments, it is known that the control subject suffers from late-stage cancer, which may or may not be CD123 expression type cancer.

Methods for monitoring cancer

Provided herein is a method for monitoring CD123 expression type cancer in a subject. In some embodiments, CD123 expression type cancer includes hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell tumor (DPDCN). In some embodiments, the method is directed to assessing whether CD123 expression type cancer is progressing, disappearing or remaining stable by measuring the amount of CD123 present in the test sample derived from the subject；And the amount of observed CD123 is compared with the amount of CD123 obtained from the biological sample of the subject in a similar manner at an earlier time point, wherein the difference between the amount of CD123 in the test sample and the earlier sample provides an indication of whether cancer is progressing, disappearing or remaining stable. In this regard, an increase in the amount of CD123 in a test sample relative to the amount observed in an earlier sample can indicate progression of a CD123-expressing cancer. Conversely, a decrease in the amount of CD123 in a test sample relative to the amount observed in an earlier sample can indicate regression of a CD123-expressing cancer.

Therefore, the amount of CD123 in the test sample is not significantly different from the amount in the observed earlier sample and can indicate that CD123 expression type cancer is in a stable disease state. In some embodiments, the amount of CD123 in the biological sample derived from the subject is assessed by contacting the sample with an antibody or its antibody fragment (such as an antibody as described herein) that specifically binds CD123. The sample for assessing the presence of CD123 can be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (such as surgically resected tumor tissue, biopsy, including fine needle aspiration tissue), histological preparations, etc. In some embodiments, the subject is a person.

In some embodiments, the method for monitoring CD123 expression type cancer involves: contacting the biological sample of the subject with a CD123-specific antibody or its antigen-binding fragment (such as those that can be derived from the antibodies and fragments provided in Table 1); quantitatively analyzing the amount of CD123 present in the sample; comparing the amount of CD123 present in the sample with the amount of CD123 measured in a biological sample obtained from the same subject in a similar manner at an earlier time point; and determining whether the CD123 level of the subject changes over time. The amount of CD123 in the test sample increases relative to the amount in the observed earlier sample and can indicate the progression of cancer. On the contrary, the amount of CD123 in the test sample decreases relative to the amount in the observed earlier sample and can indicate the regression of CD123 expression type cancer. Therefore, the amount of CD123 in the test sample is not significantly different from the amount in the observed earlier sample and can indicate that CD123 expression type cancer is in a stable disease state. In some embodiments, the CD123 level of the sample can be compared with a known standard or reference sample alone, or the CD123 level of the sample can be compared with a known standard or reference sample in addition to being compared with the observed CD123 level in the sample assessed at an earlier time point. In another embodiment, the diagnostic method can be followed by an additional step of administering a cancer-specific treatment. In some embodiments, cancer-specific treatment can be directed against CD123-expressing cancers, such as the CD123×CD3 multispecific antibodies described herein.

In various aspects, the amount of CD123 is determined by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds to CD123. In some embodiments, the sample may be contacted with one or more types of antibodies or antigen-binding fragments thereof that specifically bind to CD123. In some embodiments, the sample may first be contacted with a first antibody or antigen-binding fragment thereof that specifically binds to CD123, and then contacted with a second antibody or antigen-binding fragment thereof that specifically binds to CD123. Antibodies such as those described herein can be used in this function.

Various combinations of antibodies and antigen-binding fragments described in Table 1 can be used to provide "first" and "second" antibodies or antigen-binding fragments to implement the monitoring methods described. In some embodiments, CD123-expressing cancers include hematological cancers such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

In certain embodiments, the amount of CD123 is determined by Western blot analysis, radioimmunoassay, immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or ELISA assay.

Kit for detecting CD123

Provided herein are kits for detecting CD123 in biological samples. These kits include one or more CD123-specific antibodies or their antigen-binding fragments as described herein, and the instructions for use of the kit.

The provided CD123-specific antibodies or antigen-binding fragments can be dissolved; lyophilized; attached to a substrate, support or plate; or detectably labeled.

The kits may also include additional components useful for practicing the methods described herein. For example, the kits may include a device for obtaining a sample from a subject, a control sample or reference sample (e.g., a sample from a subject with a slow-growing cancer and/or a subject without cancer), one or more sample chambers and/or instructional materials describing the performance of the methods of the invention, and tissue-specific controls or standards.

The device for determining CD123 levels may also include, for example, a buffer or other reagent used in an assay for determining CD123 levels. The instructions may be, for example, printed instructions for performing the assay and/or instructions for assessing CD123 expression levels.

The kit may also include a device for isolating a sample from a subject. These devices may include one or more devices or reagents that can be used to obtain fluids or tissues from a subject. The device for obtaining a sample from a subject may also include a device for isolating blood components, such as serum, from a blood sample. Preferably, the kit is designed to be suitable for use with human subjects.

Multispecific antibodies

The binding domains of anti-CD123 antibodies described herein identify cells expressing CD123 on their surfaces. As described above, CD123 expression can indicate cancer cells. More specifically targeting a specific cell subset can be achieved by preparing a bispecific molecule (such as an antibody or antibody fragment) that is bound to CD123 and another target. The example of such other targets includes CD3 and CD33. This is achieved by preparing a molecule comprising a first district that is bound to CD123 and a second binding district that is bound to another antigen. Antigen binding region can take any form that allows the specific recognition of the target, such as a binding region can be or can include a heavy chain variable domain or Fv (a combination of a heavy chain variable domain and a light chain variable domain). Therefore, there is provided a bispecific molecule comprising two different antigen binding regions that are respectively bound to CD123 and another antigen.

Some multispecific antibodies described herein include two different antigen binding regions that bind CD123 and CD3, respectively. In a preferred embodiment, a multispecific antibody (CD123×CD3 multispecific antibody) and a multispecific antigen binding fragment thereof that bind CD123 and CD3 are provided. In some embodiments, the CD123×CD3 multispecific antibody includes a first heavy chain (HC1) and a first light chain (LC1) that are paired to form a first antigen binding site that is immunospecifically bound to CD123, and a second heavy chain (HC2) and a second light chain (LC2) that are paired to form a second antigen binding site that is immunospecifically bound to CD3. In a preferred embodiment, the CD123×CD3 multispecific antibody is a bispecific antibody including a CD123-specific arm and a CD3-specific arm, wherein the CD123-specific arm includes a first heavy chain (HC1) and a first light chain (LC1) that are paired to form a first antigen binding site that is immunospecifically bound to CD123, and the CD3-specific arm includes a second heavy chain (HC2) and a second light chain (LC2) that are paired to form a second antigen binding site that is immunospecifically bound to CD3. In some embodiments, the bispecific antibodies of the present invention include antibodies with full-length antibody structures. As used herein, "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) includes a heavy chain variable domain VH and heavy chain constant domains CH1, CH2, and CH3. A full-length antibody light chain (LC) includes a light chain variable domain VL and a light chain constant domain and CL. A full-length antibody may lack a C-terminal lysine (K) in one or both heavy chains. The term "Fab arm" or "half molecule" refers to a heavy chain-light chain pair that specifically binds to an antigen.

The CD123-binding arm of the multispecific antibodies provided herein can be derived from any of the CD123-specific antibodies described above. In some embodiments, the CD123 binding arm binds to an epitope that includes one or more residues from: (i) the CD123 SP2 ECD segment comprising residues 195-202 (RARERVYE (SEQ ID NO: 234)) and/or the CD123 SP2 ECD segment comprising residues 156-161 (RKFRYE (SEQ ID NO: 232)) and/or the CD123 SP2 ECD segment comprising residues 173-178 (TEQVRD (SEQ ID NO: 233)), or (ii) the CD123 SP2 ECD segment comprising residues 164-175 (IQKRMQPVITEQ (SEQ ID NO: 228)) and/or the CD123 SP2 ECD segment comprising residues 184-189 (LLNPGT (SEQ ID NO: 229)). In some embodiments, the CD123 binding arm competes for binding to CD123 with a CD123-specific antibody or antigen-binding fragment that binds to an epitope that includes one or more residues from: (i) a CD123 SP2 ECD segment comprising residues 195-202 (RARERVYE (SEQ ID NO: 234)) and/or a CD123 SP2 ECD segment comprising residues 156-161 (RKFRYE (SEQ ID NO: 232)) and/or a CD123 SP2 ECD segment comprising residues 173-178 (TEQVRDR (SEQ ID NO: 233)), or (ii) a CD123 SP2 ECD segment comprising residues 164-175 (IQKRMQPVITEQ (SEQ ID NO: 228)) and/or a CD123 SP2 ECD segment comprising residues 184-189 ECD segment (LLNPGT (SEQ ID NO: 229)). A CD123 binding arm that binds to at least one residue in these epitopes may also bind to additional residues in the CD123 ECD. In some embodiments, the CD123 binding arm is neutralizing. Neutralizing CD123 binding arms include those that can inhibit IL-3 binding to CD123 as determined by measuring a decrease in STAT5 phosphorylation when TF-1 cells are stimulated with rhIL-3. In some embodiments of the bispecific antibody, the CD123 binding arm binds to human CD123 SP1, preferably its extracellular domain.

In some exemplary embodiments of such CD123 SP1-binding arms, the first antigen-binding region that binds CD123 comprises heavy chain CDR1, CDR2, and CDR3 derived from an antibody clone as described in Table 1. In some exemplary embodiments of such CD123 SP1-binding arms, the first antigen-binding region that binds CD123 comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 derived from an antibody clone as described in Table 1. In some exemplary embodiments of such CD123 SP1 binding arms, the first antigen-binding region that binds CD123 comprises the heavy chain CDR1, CDR2, and CDR3 of clone I3RB1, I3RB2, I3RB5, I3RB6, I3RB7, I3RB8, I3RB9, I3RB11, I3RB12, I3RB16, I3RB17, I3RB18, I3RB19, I3RB20, I3RB21, I3RB22, I3RB24, I3RB28, I3RB29, I3RB30, I3RB32, I3RB33, I3RB34, I3RB35, I3RB36, I3RB37, I3RB38, I3RB40, or I3RB47. In some exemplary embodiments of such CD123 SP1 binding arms, the first antigen-binding region that binds CD123 comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of clone I3RB1, I3RB2, I3RB5, I3RB6, I3RB7, I3RB8, I3RB9, I3RB11, I3RB12, I3RB16, I3RB17, I3RB18, I3RB19, I3RB20, I3RB21, I3RB22, I3RB24, I3RB28, I3RB29, I3RB30, I3RB32, I3RB33, I3RB34, I3RB35, I3RB36, I3RB37, I3RB38, I3RB40, or I3RB47. In some exemplary embodiments of such CD123 SP1-binding arms, the first antigen-binding region that binds CD123 comprises a heavy chain variable domain derived from an antibody clone as described in Table 1. In some exemplary embodiments of such CD123 SP1-binding arms, the first antigen-binding region that binds CD123 comprises a heavy chain variable domain and a light chain variable domain derived from an antibody clone as described in Table 1. In some exemplary embodiments of such CD123 SP1 binding arms, the first antigen binding region that binds CD123 comprises a heavy chain variable domain of clone I3RB1, I3RB2, I3RB5, I3RB6, I3RB7, I3RB8, I3RB9, I3RB11, I3RB12, I3RB16, I3RB17, I3RB18, I3RB19, I3RB20, I3RB21, I3RB22, I3RB24, I3RB28, I3RB29, I3RB30, I3RB32, I3RB33, I3RB34, I3RB35, I3RB36, I3RB37, I3RB38, I3RB40, or I3RB47. In some exemplary embodiments of such CD123SP1 binding arms, the first antigen binding region that binds CD123 comprises a heavy chain variable domain and a light chain variable domain of clone I3RB1, I3RB2, I3RB5, I3RB6, I3RB7, I3RB8, I3RB9, I3RB11, I3RB12, I3RB16, I3RB17, I3RB18, I3RB19, I3RB20, I3RB21, I3RB22, I3RB24, I3RB28, I3RB29, I3RB30, I3RB32, I3RB33, I3RB34, I3RB35, I3RB36, I3RB37, I3RB38, I3RB40, or I3RB47.

In some embodiments of the bispecific antibody, the CD123 binding arm binds to human CD123 SP2, preferably its extracellular domain. In a preferred embodiment of the bispecific antibody, the CD123 binding arm binds to human CD123 SP1 and human CD123 SP2, more preferably its extracellular domain. In some exemplary embodiments of such CD123 SP2 binding arms, the first antigen-binding region binding to CD123 includes heavy chain CDR1, CDR2 and CDR3 of clones I3RB1, I3RB2, I3RB5, I3RB18, I3RB19 or I3RB30. In some exemplary embodiments of such CD123 SP2 binding arms, the first antigen-binding region binding to CD123 includes heavy chain CDR1, CDR2 and CDR3 of clones I3RB1, I3RB2, I3RB5, I3RB18, I3RB19 or I3RB30 and light chain CDR1, CDR2 and CDR3. In some exemplary embodiments of such CD123 SP2-binding arms, the first antigen-binding region that binds CD123 comprises a heavy chain variable domain of clone I3RB1, I3RB2, I3RB5, I3RB18, I3RB19, or I3RB30. In some exemplary embodiments of such CD123 SP2-binding arms, the first antigen-binding region that binds CD123 comprises a heavy chain variable domain and a light chain variable domain of clone I3RB1, I3RB2, I3RB5, I3RB18, I3RB19, or I3RB30.

In some embodiments of the bispecific antibody, the CD123-binding arm also binds cynomolgus monkey CD123, preferably its extracellular domain.

In some embodiments of bispecific antibodies, the CD123 binding arm is derived from the CD123 specific antibody that is bound to CD123 with antibody clone I3RB2, I3RB60, I3RB70, I3RB79 or I3RB118 competition. In some embodiments of bispecific antibodies, the CD123 binding arm is derived from the CD123 specific antibody that is bound to CD123 with antibody clone I3RB18, I3RB49 or I3RB55 competition. Competition can be used in conjunction with competition ELISA, determined according to the technology described in Example 5 below. Competition binding can be determined by detecting that the binding of the first antibody is suppressed by the second antibody by at least 20%, and no matter how the antibody is bound to the order of CD123 (that is, if when antibody A is bound to CD123 before antibody B, only 10% inhibition is observed, but when antibody B is bound to CD123 before antibody A, 30% inhibition is observed, because in one of these experiments, greater than 20% inhibition has been observed, so competition binding can be ended).

In some embodiments, the CD123 binding arm of the multispecific antibody is IgG or derivatives thereof, such as IgG1, IgG2, IgG3 and IgG4 isotypes. In some embodiments in which the CD123 binding arm has an IgG1 isotype, the binding arm contains L234A, L235A and K409R displacements in its Fc region. In some embodiments in which the CD123 binding arm has an IgG4 isotype, the binding arm contains S228P, L234A and L235A displacements in its Fc region.

In some embodiments of the bispecific antibody, the second antigen-binding arm binds to human CD3. In some preferred embodiments, the CD3-specific arm of the CD123×CD3 bispecific antibody is derived from a CD3-specific antibody that binds to and activates human primary T cells and/or cynomolgus monkey primary T cells. In some embodiments, the CD3-binding arm binds to an epitope at the N-terminus of CD3ε. In some embodiments, the CD3-binding arm contacts an epitope comprising the six N-terminal amino acids of CD3ε. In some embodiments, the CD3-specific binding arm of the bispecific antibody is derived from the mouse monoclonal antibody SP34, a mouse IgG3/λ isotype. In some embodiments, the CD3-binding arm comprises the CDRs of the antibody SP34. Such CD3-binding arms can bind to CD3 with an affinity of 5× ^10-7 M or less, e.g., 1× ^10-7 M or less, 5× ^10-8 M or less, 1× ^10-8 M or less, 5× ^10-9 M or less, or 1× ^10-9 M or less. The CD3-specific binding arm can be a humanized version of the arm of the mouse monoclonal antibody SP34. Human framework adaptation (HFA) can be used to humanize the anti-CD3 antibody from which the CD3-specific arm is derived. In some embodiments of the bispecific antibody, the CD3-binding arm comprises a heavy chain and light chain pair selected from Table 2. In some embodiments, the CD3-binding arm of the CD123×CD3 bispecific antibody is from Table 3.

In some embodiments, the CD3 binding arm is an IgG or a derivative thereof. In some embodiments, the CD3 binding arm is an IgG1, IgG2, IgG3, or IgG4. In some embodiments wherein the CD3 binding arm has an IgG1 isotype, the binding arm contains L234A, L235A, and F405L substitutions in its Fc region. In some embodiments wherein the CD3 binding arm has an IgG4 isotype, the binding arm contains S228P, L234A, L235A, F405L, and R409K substitutions in its Fc region. In some embodiments, the antibody or antigen-binding fragment is an IgG-AA Fc. In some embodiments, the antibody or antigen-binding fragment is an IgG-AA Fc-L234A, L235A, and F405L. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary human T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary cynomolgus monkey T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary human and cynomolgus monkey T cells. In some embodiments, the antibody or antigen-binding fragment activates primary human CD4+ T cells. In some embodiments, the antibody or antigen-binding fragment activates primary cynomolgus monkey CD4+ T cells.

In some embodiments, a CD123×CD3 bispecific antibody is provided having a CD123 binding arm comprising the heavy chain of antibody clone I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, Ab 7959, Ab3978, Ab 7955, Ab 9958, Ab 8747, Ab8876, Ab 4435, or Ab 5466. In some embodiments, a CD123×CD3 bispecific antibody is provided having a CD123 binding arm comprising the heavy and light chains of antibody clones I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, Ab 7959, Ab3978, Ab 7955, Ab9958, Ab 8747, Ab 8876, Ab 4435, or Ab 5466. In some embodiments, a CD123×CD3 bispecific antibody is provided having a CD3 binding arm comprising the heavy chain of antibody clone I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, Ab 7959, Ab3978, Ab 7955, Ab 9958, Ab 8747, Ab 8876, Ab 4435, or Ab 5466. In some embodiments, a CD123×CD3 bispecific antibody is provided having a CD3 binding arm comprising the heavy and light chains of antibody clones I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, Ab 7959, Ab3978, Ab 7955, Ab 9958, Ab 8747, Ab 8876, Ab 4435, or Ab 5466. In some embodiments, a CD123×CD3 bispecific antibody is provided having a CD123 binding arm and a CD3 binding arm, wherein the CD123 binding arm comprises antibody clones I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, mAB 7959, Ab3978, Ab 7955, Ab 9958, Ab8747, Ab 8876, Ab 4435, or Ab In some embodiments, the CD3 binding arm comprises the heavy chain of antibody clone I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, AbB 7959, Ab3978, Ab 7955, Ab 9958, Ab 8747, Ab 8876, Ab 4435, or Ab 5466. In some embodiments, a CD123×CD3 bispecific antibody having a CD123 binding arm and a CD3 binding arm is provided, wherein the CD123 binding arm comprises antibody clones I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, Ab 7959, Ab3978, Ab 7955, Ab 9958, Ab 8747, Ab 8876, Ab In some embodiments, the CD3 binding arm comprises the heavy and light chains of antibody clones I3RB179, I3RB180, I3RB181, I3RB182, I3RB183, I3RB186, I3RB187, I3RB188, I3RB189, CD3B191, Ab 7959, Ab3978, Ab 7955, Ab9958, Ab 8747, Ab 8876, Ab 4435, or Ab 5466.

Preferred CD123×CD3 bispecific antibodies are provided in Tables 13 and 17.

Different formats of bispecific antibodies have been described and recently reviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276.

In some embodiments, the bispecific antibodies of the invention are diabodies, crossbodies, or bispecific antibodies obtained by controlled Fab arm exchange, such as those described herein.

In some embodiments, bispecific antibodies include IgG-like molecules with complementary CH3 domains to force heterodimerization; recombinant IgG-like dual-targeting molecules, wherein each side of the molecule contains a Fab fragment or a portion of a Fab fragment of at least two different antibodies; IgG fusion molecules, wherein a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; Fc fusion molecules, wherein a single-chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; Fab fusion molecules, wherein different Fab fragments are fused together; ScFv- and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies), wherein different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

In some embodiments, IgG-like molecules with complementary CH3 domain molecules include Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A/S).

In some embodiments, recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK/Domantis), two-in-one antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX bodies (CovX/Pfizer).

In some embodiments, IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), IgG-like bispecific antibodies (InnClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

In some embodiments, Fc fusion molecules include ScFv/Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), dual affinity retargeting molecule (Fc-DART) (MacroGenics), and Dual (ScFv).sub.2-Fab (China National Antibody Medical Research Center).

In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex/AMGEN), dual-action or dual Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-based, diabody-based domain antibodies include, but are not limited to, bispecific T cell engager (BITE) (Micromet), tandem diabody (Tandab) (Affimed), dual affinity retargeting molecule (DART) (MacroGenics), single-chain diabody (Academic), TCR-like antibody (AIT, ReceptorLogics), human serum albumin ScFv fusion (Merrimack) and COMBODY (Epigen Biotech), dual-targeting nanobody (Ablynx), dual-targeting heavy chain domain only antibodies.

The full-length bispecific antibodies of the present invention can be produced, for example, by Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies in the following manner: a substitution is introduced at the heavy chain CH3 junction in each half molecule to promote heterodimer formation in an in vitro cell-free environment or using co-expression of two antibody half molecules with different specificities. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and recombined by dissociation association. The CH3 domain of the Fab arm can be modified to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each of which binds to a different epitope, i.e., an epitope on CD123 (IL3-Rα) and an epitope on CD3.

As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains having the same CH3 amino acid sequence.

As used herein, "heterodimerization" refers to the interaction of two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody with two heavy chains that have different CH3 amino acid sequences.

The "button" technology (see, e.g., PCT International Publication No. WO 2006/028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the junction of the CH3 domains in human IgG can be mutated at positions that affect the interaction of the CH3 domains, thereby promoting heterodimer formation. Amino acids with small side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. Upon co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "button" with the heavy chain with the "knob." Exemplary CH3 substitution pairs that form a knob and buckle (represented as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain) are: T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S, and T366W/T366S_L368A_Y407V.

Other techniques may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and negatively charged residues on the other CH3 surface, as described in U.S. Patent Publication No. US2010/0015133, U.S. Patent Publication No. US2009/0182127, U.S. Patent Publication No. US2010/028637, or U.S. Patent Publication No. US2011/0123532. In other techniques, heterodimerization can be promoted by the following substitutions (indicated as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V K409F Y407A/T366A_K409F, or T350V_L351Y_F405A Y407V/T350V_T366L_K392L_T394W, as described in U.S. Patent Publication No. US2012/0149876 or U.S. Patent Publication No. US2013/0195849.

In addition to the above methods, the bispecific antibodies of the present invention can also be produced in an in vitro cell-free environment by the following manner: introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions, thereby performing disulfide bond isomerization according to the method described in Patent Publication No. WO2011/131746. In the method, a first monospecific bivalent antibody (e.g., an anti-CD123 (IL3-Rα) antibody) and a second monospecific bivalent antibody (e.g., an anti-CD3 antibody) are engineered to have certain substitutions in the CH3 domain that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of cysteines in the hinge region; thereby producing bispecific antibodies by Fab arm exchange. The incubation conditions can optimally be restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, and tris (2-carboxyethyl) phosphine. For example, at a temperature of at least 20° C., in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at pH 5-8 (e.g., at pH 7.0 or at pH 7.4), incubation for at least 90 min can be used.

In addition to the CD123×CD3 multispecific antibody, a polynucleotide sequence capable of encoding the CD123×CD3 multispecific antibody is also provided. A vector comprising the polynucleotide and a cell expressing the CD123×CD3 multispecific antibody provided herein are also provided. Cells capable of expressing the disclosed vectors are also described. These cells can be mammalian cells (e.g., 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The antibody can also be produced by hybridoma cells.

Therapeutic compositions and methods of treating with multispecific antibodies and multispecific antigen-binding fragments thereof

The CD123 bispecific antibodies described above, such as the CD123×CD3 bispecific antibodies described above, can be used in treatment. Specifically, the CD123 bispecific antibodies can be used to treat cancer. Also provided herein is a therapeutic composition for treating a hyperproliferative disease in a mammal, the composition comprising a therapeutically effective amount of a multispecific antibody or multispecific antigen-binding fragment as described herein and a pharmaceutically acceptable carrier. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a CD123×CD3 bispecific antibody or a CD123×CD3 bispecific antigen-binding fragment thereof as described herein. In one embodiment, the pharmaceutical composition is used to treat CD123-expressing cancers, including but not limited to the following cancers: CD123-expressing hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN); and other cancers in which CD123 is expressed that are yet to be determined. Specific bispecific antibodies that can be used to treat cancer (e.g., hematological cancers, including the specific cancers described above) include antibodies 7959, 3978, 7955, 9958, 8747, 4435 and 5466. An example of a bispecific antibody that can be used to treat cancer (e.g., hematological cancers, including these specific cancers) is antibody 9958. Another example of a bispecific antibody that can be used to treat cancer (e.g., hematological cancers, including these specific cancers) is antibody 3978. Another example of a bispecific antibody that can be used to treat cancer (e.g., hematological cancers, including these specific cancers) is antibody 8747. Another example of a bispecific antibody that can be used to treat cancer (e.g., hematological cancers, including these specific cancers) is antibody 7959.

The pharmaceutical compositions provided herein comprise: a) an effective amount of a multispecific antibody or antibody fragment of the present invention, and b) a pharmaceutically acceptable carrier, which may be an inert or physiologically active carrier. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a CD123×CD3 bispecific antibody or a CD123×CD3 bispecific antigen-binding fragment thereof as described herein. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents that are physiologically compatible. Examples of suitable carriers, diluents, and/or excipients include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases, it is preferred that an isotonic agent, such as a sugar, a polyol, or sodium chloride, be included in the composition. Specifically, relevant examples of suitable carriers include: (1) Dulbecco's phosphate-buffered saline at a pH of about 7.4 with or without about 1 mg/mL to 25 mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain antioxidants (such as tryptamine) and stabilizers (such as Tween).

The compositions herein may also contain additional therapeutic agents as necessary for the specific disease being treated. Preferably, the multispecific antibody or antibody fragment and the supplemental active compound have complementary activities that do not adversely affect each other. In a preferred embodiment, the additional therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin-2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent.

Compositions of the present invention can have various forms. These forms include, for example, liquid, semisolid and solid dosage forms, but preferred form depends on the mode of administration and therapeutic application of expectation. Typical preferred compositions are in the form of injectable or infusible solutions. Preferred modes of administration are parenteral administration (for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection). In a preferred embodiment, compositions of the present invention are administered intravenously or by continuous infusion over a period of time. In another preferred embodiment, these compositions are injected by intramuscular, subcutaneous, intraarticular, synovial, intratumor, around tumor, in lesion or around lesion approach, to give play to local and systemic therapeutic effect.

The sterile composition for parenteral administration can be prepared in the following manner: the antibody of the present invention, antibody fragment or antibody conjugate of the desired amount are mixed in a suitable solvent and then sterilized by microfiltration technology. Water, saline, phosphate buffered saline, dextrose, glycerol, ethanol etc. and their combination can be used as solvent or carrier. In many cases, it is preferred to comprise isotonic agents, such as sugar, polyols or sodium chloride in the composition. These compositions can also contain adjuvants, especially wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. The sterile composition for parenteral administration can also be prepared into the form of sterile solid compositions, which can be dissolved in sterile water or any other injectable sterile medium when in use.

Multispecific antibodies or antibody fragments can also be taken orally. As solid compositions for oral administration, tablets, pills, powders (gelatin capsules, sachets) or granules can be used. In these compositions, the active ingredient according to the present invention is mixed with one or more inert diluents (such as starch, cellulose, sucrose, lactose or silicon dioxide) under an argon stream. These compositions may also contain substances other than the diluent, such as one or more lubricants (such as magnesium stearate or talc), colorants, coatings (sugar-coated tablets) or glazes.

As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing an inert diluent (e.g., water, ethanol, glycerol, vegetable oil or paraffin oil) can be used. These compositions may contain substances other than diluents, such as to moisten, sweeten, thicken, flavor or stabilize the product.

The dosage of these substances depends on the desired effect, duration of treatment, and route of administration used; for adults, daily oral doses of between 5 mg and 1000 mg of these substances are generally taken, with unit doses ranging from 1 mg to 250 mg of active substance. In general, the physician will determine the appropriate dosage based on age, weight, and any other factors specific to the subject being treated.

Also provided herein is a method for killing CD123+ cells by administering to a patient in need thereof a multispecific antibody that binds to the CD123 and can recruit T cells to kill the CD123+ cells (i.e., T cell redirection). Any multispecific antibody or antibody fragment of the present invention can be used therapeutically. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or its multispecific antigen-binding fragment as described herein, more preferably a CD123×CD3 bispecific antibody or its CD123×CD3 bispecific antigen-binding fragment as described herein.

In a preferred embodiment, the multispecific antibodies or antibody fragments of the present invention are used to treat hyperproliferative diseases in mammals. In a more preferred embodiment, one of the pharmaceutical compositions disclosed above containing the multispecific antibodies or antibody fragments of the present invention is used to treat hyperproliferative diseases in mammals. In one embodiment, the disease is cancer. Specifically, the disease is CD123-expressing cancer, including but not limited to the following cancers: CD123-expressing hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN); and other cancers in which CD123 is expressed that have yet to be determined. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a CD123×CD3 bispecific antibody or a CD123×CD3 bispecific antigen-binding fragment thereof as described herein.

Thus, the pharmaceutical compositions of the present invention can be used to treat or prevent a variety of cancers, including but not limited to the following CD123-expressing cancers, including but not limited to the following cancers: CD123-expressing hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN); and other cancers in which CD123 is expressed that have yet to be determined.

Similarly, there is also provided herein a method for suppressing the growth of selected cell groups, the method including contacting CD123 expression type target cells or tissues containing such target cells with an effective amount of multispecific antibodies or antibody fragments of the present invention alone in the presence of peripheral blood mononuclear cells (PBMC), or contacting CD123 expression type target cells or tissues containing such target cells with an effective amount of multispecific antibodies or antibody fragments of the present invention and a combination of other cytotoxic agents or therapeutic agents. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or its multispecific antigen-binding fragment as described herein, more preferably a CD123×CD3 bispecific antibody or its CD123×CD3 bispecific antigen-binding fragment as described herein. In a preferred embodiment, another therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride or interleukin 2. In a preferred embodiment, another therapeutic agent is a chemotherapeutic drug. The method for suppressing the growth of selected cell groups can be performed in vitro, in vivo or ex vivo.

Examples of in vitro use include treating autologous bone marrow to kill diseased or malignant cells before transplantation into the same patient; treating bone marrow to kill competent T cells and prevent graft-versus-host disease (GVHD) before transplantation; treating cell cultures to kill all cells except the desired variant that does not express the target antigen; or killing variants that express an undesirable antigen. One of ordinary skill in the art can readily determine the conditions for non-clinical in vitro use.

An example of clinical ex vivo use is the removal of tumor cells from the bone marrow prior to autologous transplantation in cancer treatment. The treatment can be carried out according to the following steps. Bone marrow is collected from the patient or other individual and then incubated in a culture medium containing serum to which the cytotoxic agent of the present invention is added. The concentration range is about 10 μM to 1 μM, and the incubation is about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and incubation time, i.e., the dosage, are easily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with a culture medium containing serum, and these bone marrow cells are returned to the patient by intravenous infusion according to known methods. In the case where the patient receives other treatment (e.g., a course of ablative chemotherapy or whole-body radiotherapy between the time of bone marrow collection and the time of re-infusion of the treated cells), the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

For clinical in vivo use, a therapeutically effective amount of multispecific antibodies or antigen-binding fragments are administered to a subject in need thereof. For example, CD123×CD3 multispecific antibodies and multispecific antigen-binding fragments thereof can be used to treat CD123 expression-type cancers in a subject in need thereof. In some embodiments, CD123 expression-type cancers are hematological cancers, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN). In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or its multispecific antigen-binding fragment as described herein, more preferably a CD123×CD3 bispecific antibody or its CD123×CD3 bispecific antigen-binding fragment as described herein. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the multispecific antibody or antigen-binding fragment is administered as a solution that has been tested for sterility.

The above-described treatment methods and dosage regimens used are adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus injection may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

The effective dose and dosage regimen of the multispecific antibodies and fragments depends on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the invention is about 0.001-10 mg/kg, such as about 0.001-5 mg/kg (e.g., about 0.001-2 mg/kg), such as about 0.001-1 mg/kg (e.g., about 0.001 mg/kg, about 0.01 mg/kg, about 0.1 mg/kg, about 1 mg/kg, or about 10 mg/kg).

A physician or veterinarian with ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may start the multispecific antibody or fragment used in the pharmaceutical composition at a dose lower than that required to achieve the desired therapeutic effect, and then gradually increase the dose until the desired therapeutic effect is achieved. Generally, a suitable daily dose of the bispecific antibody of the present invention is the amount of the compound at the lowest dose effective to produce a therapeutic effect. The administration route may be, for example, parenteral administration, such as intravenous, intramuscular, or subcutaneous injection. In one embodiment, the multispecific antibody or fragment may be administered by infusion at a weekly dose in mg/m ^2. Such a dose may be, for example, based on the mg/kg dose provided above, according to the formula: dose (mg/kg) × 70: 1.8. Such administration may be repeated, for example, 1 to 8 times, for example 3 to 5 times. Administration may be performed by continuous infusion over a period of 2 to 24 hours (e.g., 2 to 12 hours). In one embodiment, the multispecific antibody or fragment may be administered by slow continuous infusion over a long period of time (e.g., more than 24 hours) to reduce toxic side effects.

In one embodiment, the multispecific antibody or fragment can be administered up to 8 times in a weekly dosage manner calculated as a fixed dose, for example 4 to 6 times when administered once a week. Such a regimen can be repeated one or more times, for example, after six months or twelve months, as needed. Such a fixed dose can be, for example, based on the mg/kg dosage provided above, where body weight is estimated to be 70 kg. The dosage can be determined or adjusted by measuring the amount of the bispecific antibody of the present invention in the blood when administered by, for example, taking out a biological sample and using an anti-idiotypic antibody targeting the CD123 antigen binding region of the multispecific antibody of the present invention.

In one embodiment, the multispecific antibody or fragment can be administered via maintenance therapy, eg, once a week for 6 months or longer.

Multispecific antibodies or fragments can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of an event in cancer progression, and/or reduce the risk of recurrence after cancer is in remission.

The multispecific antibodies and fragments thereof described herein can also be administered in combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition to be treated. Thus, in one embodiment, the antibody-containing medicament is used in combination with one or more additional therapeutic agents (e.g., chemotherapeutic agents). In some embodiments, the other therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin-2. Such combined administration can be performed simultaneously, separately, or sequentially in any order. For simultaneous administration, the therapeutic agents can be administered as a single composition or as separate compositions, as appropriate.

In one embodiment, there is provided a method for treating a disease involving cells expressing CD123 in a subject, the method comprising administering a therapeutically effective amount of a multispecific antibody or fragment (such as CD123×CD3 bispecific antibodies provided herein) and radiotherapy to a subject in need thereof. In one embodiment, there is provided a method for treating or preventing cancer, the method comprising administering a therapeutically effective amount of a multispecific antibody or fragment (such as CD123×CD3 antibodies as described herein) and radiotherapy to a subject in need thereof. Radiotherapy may include radiation or administering a related radiopharmaceutical to the patient. A radiation source may be external or internal (radiation therapy may be in the form of, for example, external beam radiation therapy (EBRT) or short-range radiation therapy (BT)) to the patient being treated. Radioactive elements that can be used for implementing such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111.

Reagent test kit

Also provided herein is a kit comprising, for example, a multispecific antibody or antigen-binding fragment thereof as described herein and instructions for using the antibody or fragment to kill a specific type of cell. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a CD123×CD3 bispecific antibody or a CD123×CD3 bispecific antigen-binding fragment thereof as described herein. The instructions may include instructions for using the multispecific antibody or antigen-binding fragment thereof in vitro, in vivo, or ex vivo.

Typically, the kit has a compartment containing the multispecific antibody or antigen-binding fragment thereof. The multispecific antibody or antigen-binding fragment thereof can be in lyophilized form, liquid form, or other form suitable for inclusion in the kit. The kit may also include other elements necessary to carry out the methods described in the instructions for use in the kit, such as a sterile solution for reconstitution of the lyophilized powder, other reagents for combining with the multispecific antibody or antigen-binding fragment thereof prior to administration to a patient, and tools to facilitate administration of the multispecific antibody or antigen-binding fragment thereof to a patient.

Diagnostic uses

The multispecific antibodies and fragments described herein can also be used for diagnostic purposes. Therefore, diagnostic compositions comprising multispecific antibodies or fragments as defined herein and their uses are also provided. In a preferred embodiment, the multispecific antibody is a CD123×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a CD123×CD3 bispecific antibody or a CD123×CD3 bispecific antigen-binding fragment thereof as described herein. In one embodiment, the present invention provides a kit for diagnosing cancer, the kit comprising a container containing a bispecific CD123×CD3 antibody and one or more reagents for detecting the binding of the antibody to CD123. These reagents may include, for example, fluorescent labels, enzyme labels or other detectable labels. These reagents may also include secondary or tertiary antibodies or reagents for enzyme reactions, wherein the enzyme reaction generates a product that can be visualized. For example, the multispecific antibodies or antigen-binding fragments thereof described herein can be labeled with a radiolabel, a fluorescent label, an epitope tag, biotin, a chromophore label, an ECL label, an enzyme, ruthenium, 111In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β-galactosidase, or polyhistidine or similar such labels known in the art.

The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be construed as limiting the subject matter described. Rather, it should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or alterations thereto will be apparent to those skilled in the art and may be made without departing from the true scope of the invention.

Example 1: Materials

Generation of CD123 cell lines

Generate a group of pDisplay™ vectors representing human CD123 SP1 ECD (amino acids 20-305) (SEQ ID NO: 1), human CD123 SP2 ECD (amino acids 19-227 of SEQ ID NO: 2) and cynomolgus monkey CD123 ECD (amino acids 19-305 of SEQ ID NO: 3) and use them as screening tools to evaluate anti-CD123 leads. Use the mammalian expression vector ^pDisplay (Invitrogen) (Figure 1) that allows protein to be displayed on the cell surface. The protein expressed by pDisplay ^™ is fused to the mouse Ig κ chain leader sequence at the N-terminus, which directs the protein to the secretory pathway and to the platelet-derived growth factor receptor (PDGFR) transmembrane domain at the C-terminus, which anchors the protein to the plasma membrane and displays it on the outside of the cell. The recombinant protein expressed by pDisplay ^™ contains hemagglutinin A and myc epitopes for detection by Western blot or immunofluorescence. CMV promoter drives expression.

The vector is transiently transfected into HEK293T cells using standard methods. The 293F adherent cells selected for transfection are then subjected to stable plasmid integration, followed by single cell and CD123 surface receptor expression by FACS quantitative sorting using BangsLabs Quantum FITC-5 test kit (catalog number 855, BangsLaboratories company). A group of 10 single cell clones of each cell line are selected for screening, and quantitative CD123 ECD expression is performed. The cell line screened for subsequent hits has a surface expression of approximately 500,000 CD123 ECD copies per cell.

Generation of soluble CD123 ECD protein

Recombinant human CD123 SP1 ECD-HIS tag protein (lot number LV081110A) corresponding to amino acids 20 to 305 of CD123 SP1 (SEQ ID NO: 1) was purchased from R&D Systems (#301-R3/CF) for phage panning and hit screening. The protein was tested for endotoxin before use and biotinylated for phage panning studies. This material was also used for binding and affinity measurements.

Purify the recombinant human CD123 SP2ECD protein corresponding to the 18-225 amino acids of human CD123 SP2 (SEQ ID NO: 2) for binding and affinity measurement. Gene synthesis technology is used to prepare cDNA (U.S. Patent No. 6,670,127; U.S. Patent No. 6,521,427). Standard molecular biology techniques are used to prepare the plasmid for expressing synthetic soluble CD123 ECDSP2. The CD123 ECD SP2 gene fragment with N-terminal gp67 signal sequence and c-terminal 6-HIS tag is cloned into the Eco RI site and Not I site of pFastbacl (Invitrogen) and expressed in High Five cells (Invitrogen) with Bac to Bac system (Invitrogen). Secreted protein (SEQ ID NO: 226) is purified by HisTrap (GE) column and Superdex 75 (GE) column. This material is used for binding and affinity measurement and epitope mapping.

Soluble CD123 ECD protein was biotinylated using the SureLink Biotinylation Kit (KPL#86-00-01) according to the manufacturer's instructions. Proteins were subjected to SDS/PAGE electrophoresis to confirm monomeric state.

Anti-CD3 antibodies for X-ray crystallography

Mouse IgG3/λ isotype SP34 mAb was purchased from BD Biosciences Pharmingen (San Diego, CA) under catalog number 556611 and comprises the light and heavy chains represented by SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

Example 2: Identification of anti-human CD123 mAb

In the following four rounds of panning, solution panning of a human Fab-pIX de novo library [Shi, L. et al., J Mol Biol, 2010, 397(2), pp. 385-396. WO 2009/085462] consisting of a VH1-69, 3-23, and 5-51 heavy chain library paired with a VK1-39, 3-11, 3-20, and 4-1 light chain library was performed using a biotinylated antigen-streptavidin magnetic bead capture method as described in (Rothe et al., J. Mol. Biol. 376: 1182-1200, 2008; Steidl et al., Mol. Immunol. 46: 135-144, 2008).

After the fourth round of panning, the pIX gene was cut out from the phagemid DNA to generate the Fab coding region of the soluble HIS tag. Fab was expressed in Escherichia coli and screened for Fab bound to recombinant human CD123 SP1 ECD-HIS tag protein in ELISA. In brief, 96-well Nunc Maxisorp plates (Nunc#437111) were coated with a PBS solution of sheep anti-human Fd (binding site #PC075) at a rate of 1 μg/mL at 4 ° C and stored overnight. Bacterial colonies containing Fab expression vectors were grown in 450 μL 2xYT (carbenicillin) in deep well culture plates until turbidity (OD600≈0.6). Fab expression was induced by adding IPTG to a concentration of 1 mM. The culture was grown overnight at 30 ° C and then clarified by centrifugation. The anti-Fd coated Maxisorp plate was washed once with TBS, 0.5% Tween-20 (Sigma #79039-10PAK) and blocked with 200 μL PBS-Tween (0.5%) + skim milk powder (3%) per well for one hour at room temperature. In this step and all subsequent steps, the plate was washed three times with TBS, 0.5% Tween-20 (Sigma #79039-10PAK). 50 μL of Fab supernatant was added to each well and incubated at room temperature for 1 hour. After washing, 50 μL of biotinylated CD123 was added and incubated at room temperature for 1 hour. After washing, 50 μL of streptavidin: HRP (Pierce #21130) was added at a dilution of 1:5000 and the plate was incubated at room temperature for 1 hour. The plates were washed and 50 μL of the chemiluminescent substrate PoD (Roche #121-5829500001) was added according to the manufacturer's instructions. The luminescence values of the plates were then read on an EnVision (Perkin Elmer) plate reader. Wells showing a signal >5 times background were considered hits.

The clones showing binding to recombinant human CD123 SP1 ECD-HIS tag proteins were sequenced in heavy chain (HC) and light chain (LC) variable regions. A total of 52 unique Fab sequences were identified by phage panning, of which 45 sequences were ultimately converted into IgG1 isotypes by in-fusion cloning. (Table 1) In-fusion cloning was performed using PCR SuperMix high-fidelity kit (LifeTechnologies#10790-020) by PCR amplification of HC variable regions and LC variable regions, and cloned into the Esp3I site in the vDR149 of HC and the vDR157 of LC using In-HD Cloning Plus kit (Clontech#638909). The VH and VL hits are shown in Table 4 below.

Table 1. CDR sequences of mAbs generated by phage panning against recombinant human CD123 SP1 ECD-HIS tagged protein. Column (the corresponding SEQ ID NO is listed in brackets)

Table 4: VH and VL sequences of mAbs generated by phage panning against CD123

Example 3: MSD cells binding to hCD123 SP1, hCD123 SP2, and cynoCD123 SP1

The binding of CD123 antibodies to engineered pDisplay cells was assessed using the MSD (Mesoscale) cell binding assay. The purpose of the screening assay was to identify antibodies that bind to cells expressing hCD123 SP1 and SP2, as well as cross-reactivity with cells expressing cynoCD123 SP1.

Fix cells and measure phage in triplicate. In short, expression supernatant or purified CD123 antibody is normalized to 10 μ g/mL. 5000 cells per well are seeded into 384-well plates (MA6000, catalog number L21XB, MSD) and allowed to adhere for 2 hours. The cells are then sealed with 20% FBS PBS solution (Gibco) for 15 minutes. Antibody supernatant is then added and placed at room temperature for 1 hour. Cells are washed 3 times with PBS, then ruthenium-labeled secondary antibodies (Jackson Immuno Research) are added at a rate of 1 μ g/mL, and incubated at room temperature for 1 hour. Additional washing steps are then carried out, followed by addition of 35 μ L/ well MSD reading buffer T (surfactant-free), and incubated for 30 minutes for detection. These plates are subsequently read using Sector imager 2400 (MSD). Data are normalized to control and mapped using GraphPad Prism version 5. Positive binders with a signal 3 times greater than background were identified as hits (Figures 2A, 2B, and 2C). The assay process was repeated to ensure data consistency, and excellent binders were selected for further expanded assays. The following hits were positive for binding to all three cell lines: I3RB2, I3RB5, I3RB8, I3RB18, I3RB20, I3RB21, and I3RB35.

Example 4: Affinity measurement by SPR .

ProteOn affinity measurement

The affinity of 29 anti-CD123 candidates to recombinant human CD123 SP1 ECD and CD123 SP2 ECD was measured by surface plasmon resonance (SPR) using the ProteOn XPR36 protein interaction array system (BioRad).

The association rate and dissociation rate of CD123 SP1 ECD or CD123 SP2 ECD of each variant are measured. Using the manufacturer's instructions for amine coupling chemistry, the biosensor surface is prepared by covalently coupling goat anti-human IgG (Fc) to the surface of a GLC chip (BioRad). Immobilize approximately 8800RU (response units) of goat anti-human IgG (Fc) antibody (Jackson ImmunoResearch laboratories Prod#109-005-098). The fixed RU also includes goat anti-mouse Fc antibody, which is added to capture other antibodies not included in the antibodies recorded herein. Because the mixture is 1: 1 mixing, it is expected that about 50% of these fixed RUs are goat anti-human Fc. At 25°C, kinetic experiments were carried out in running buffer (PBS pH 7.4, 0.005% P20, 3mM EDTA). A 4-fold (1:3) serial dilution of human CD123 SP1 ECD and CD123 SP2 ECD starting at 400 nM was prepared in running buffer. An average of 300 RU of mAb (174-600) was captured on each channel of the sensor chip. A reference point (goat anti-human IgG (Fc) modified surface) without captured candidates was used as the reference surface. After capturing the mAb, the antigen was injected at a rate of 40 μL/min for 3 minutes (association phase), and then the buffer was allowed to flow for 10 minutes (dissociation phase). The chip surface was regenerated by injecting 0.85% phosphoric acid at 100 μL/min. The data was processed on the instrument software. Double reference subtraction of the data was performed by subtracting the curve generated by buffer injection from the reference-subtracted curve of the analyte injection. Kinetic analysis of the data was performed using a 1:1 Langmuir binding model with group fitting. The results for each mAb were recorded in the form of Ka (k binding or association rate), Kd (k dissociation or dissociation rate), and KD (equilibrium dissociation constant) (Table 5).

The results showed that all 29 mAbs bound to the CD123 SP1 ECD, but only six showed binding to the CD123 SP2 ECD. To ensure data reproducibility, the four antibodies were assayed in at least duplicate. Generally, the results showed good reproducibility between replicates, with the exception of I3RB1, which had a slow on-rate.

Table 5. Affinity assessment of phage set 1 hits by SPR

¹ NBO = No binding observed

Biacore affinity measurement

In addition, using Biacore instrument, the affinity of several antibodies to CD123 SP1 ECD and CD123 SP2 ECD of two forms of mAb and Fab is measured by surface plasmon resonance (SPR).Biacore 3000 (BIAcore company, now a branch of GE Healthcare) is used to carry out kinetic studies at 25 DEG C.Goat anti-human IgG (Fc) specific antibody (Jackson ImmunoResearch laboratories, Prod#109-005-098) is covalently attached to two flow cells (usually 1 and 2) of the gold surface (CM-5 chip, Biacore) coated with carboxymethyl dextran.Sheep anti-human Fd specific antibody (binding site, Prod#PC075) is covalently attached to two flow cells (usually 3 and 4) of the gold surface (CM-5 chip, Biacore) coated with carboxymethyl dextran. The carboxymethyl groups of dextran were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). At pH 4.5, the antibody was connected in 10mM sodium acetate. Any remaining active sites on the surface were blocked by reaction with ethanolamine. For kinetic binding measurements, anti-CD123 antibodies were captured onto anti-human Fcγ specific antibodies, while anti-CD123 molecules were injected at a flow rate of 5 or 6 μL/min, and Fab was captured onto anti-Fd specific antibodies. Approximately 75RU of antibody and approximately 50RU of Fab were captured respectively. After capturing Ab and Fab, human CD123 SP1 or human CD123 SP2 at a concentration between 1.6nM and 400nM was injected at a speed of 40 μL/min. 2 minutes of association data were collected, followed by 10 minutes of dissociation. 30 μL of 100 mM H3PO4 was added at a rate of 100 μL/min to regenerate the surface. All samples were prepared with D-PBS containing 3 mM EDTA and 0.005% surfactant P20. The reported data are the difference in SPR signals between the flow cell containing the capture antibody or Fab and the reference cell without the capture antibody or Fab. The additional contribution of the device to the signal was eliminated by subtracting the data from the blank injection from the signal after subtracting the reference value. The data were obtained by repeating three times and analyzed using BIA evaluation software (BIAcore) by fitting the association and dissociation phases of all concentrations (global fit) with a 1:1 binding model. Repeated experiments were performed and showed good consistency. The data shown are average values.

The results showed that the affinity of CD123 SP1 ECD and CD123 SP2 ECD binding to mAbs (I3RB2, I3RB18, I3RB35, I3RB37) was consistent with that of their corresponding Fabs (I3RB120, I3RB119, I3RB121, I3RB122) (Table 6). The results of all anti-CD123 analyzed also showed that the affinity of Fab binding to CD123 SP1 ECD and CD123 SP2 ECD ranged from 1.8-46.9 nM and 0.4-12.5 nM, respectively; while the affinity of mAb binding ranged from 1.2-52 nM and 0.3-11.7 nM, respectively.

Table 6. Affinity and on/off rate values of anti-CD123 phage 1 hits by SPR (Biacore) .

**Assay response is lower than expected

ND: Apparent binding, but signal exceeds acceptance criteria; (<5RU, poor data quality or irregular sensorgram)

Example 5: Competing with 7G3

CD123 competition assay by ELISA

The CD123 antibody panel was screened in a 7G3 binding competition ELISA. 7G3 is a neutralizing monoclonal antibody whose epitope is located within the first 50 amino acids of the CD123 SP1 antigen (US6177078B1). 7G3 mAb was purchased from BD Biosciences Pharmingen (San Diego, CA, catalog number 554526) and labeled with MSD Sulfo-Tag ^™ NHS ester according to the manufacturer's instructions (Meso Scale Discovery).

For CD123 competitive ELISA, 2 μg/mL anti-6x histidine (R&D Systems, catalog number: MAB050) prepared in bicarbonate buffer (Pierce#: 28382) at pH 9.4 was treated with 100 μL/well 96-well transparent maxisorb plates and incubated overnight at 4°C. The plates were then washed three times with ELISA wash buffer (PBS, 0.01% Tween-20) and then blocked with 300 μL/well StartBlock (Thermo Scientific#: 37539) containing Tween-20, PBST. All wells were treated with 1 ng recombinant huCD123 ECD SP1, and the plates were incubated at room temperature for 1 hour. Unbound huCD123 ECD SP1 was washed with ELISA wash buffer. 20 μg/mL of 7G3 or mouse IgG2A (mIgG2A) was prepared in expression medium (FreeStyle ^™ Expression Medium, Gibco #: 12338-018) and added to the corresponding wells of the plate at 50 μL/well in duplicate, while 2 μg/mL or pure test anti-CD123 mAb was added to the remaining wells at 50 μL/well, and the plate was incubated for 1 hour at room temperature with gentle shaking. Biotinylated 7G3 was then added to all wells to a final concentration of 100 ng/mL, and the plate was incubated for an additional 1 hour. The plate was then washed three times with ELISA wash buffer, and bound biotinylated 7G3 was detected using a SA-HRP conjugate at an optical density of 450 nm.

Anti-CD123 mAbs that inhibit 7G3:CD123 binding were defined as having 20% inhibition of activity. That is, an antibody was considered an inhibitor if it inhibited at least 20% of the binding of biotinylated 7G3 to the human CD123 ECD. Based on this selection criterion, three inhibitors were identified: 13RB18, 13RB34, and 13RB44 (Figure 3).

Example 6: Functional pSTAT5 Assay

To evaluate the agonist or antagonist activity of the antibodies, the panel was screened in a cell-based IL-3-induced STAT5 phosphorylation assay using TF-1 cells (used as purchased). The presence of the anti-CD123 mAb inhibitor resulted in a decrease in STAT5 phosphorylation after stimulation with rhIL-3. A 20% inhibition standard (20% inhibition of rhIL-3 activity) was used in the STAT5 functional assay.

Approximately 50,000 TF-1 (human erythroleukemia) cells were seeded in 60 μL of RPMI containing 10% FBS in each well of a 96-well plate and incubated overnight at 37°C with a 5% CO2 incubator. All samples were prepared in expression medium (FreeStyle™ expression medium, Gibco#: 12338-018). 70 μL/well of 20 μg/mL 7G3 or mIgG2A isotype control was added to the control samples. 70 μL/well of 2 μg/mL or pure anti-human CD123 mAb samples were added to the remaining wells. All samples were incubated for 1 hour at 37°C with a 5% CO2 incubator. Cells were then treated with recombinant human IL-3, rhIL-3 (PeproTech, catalog number: 200-03), at a final concentration of 10 ng/mL in RPMI containing 10% FBS, except for cells that were zero-treated, 7G3-treated, or isotype-only treated. Samples were then incubated for an additional 15 min at 37°C in a 5% CO2 incubator. Cells were lysed with 46.7 μL of ice-cold complete lysis buffer per well and samples were incubated on ice for 30 minutes. The lysate was mixed 10 times by pipetting up and down. Phosphorylated STAT5 (pSTAT5a, b) was then measured using the Phospho (Tyr694)/Total STAT5a, b Kit (MSD#: K15163D-2) purchased from Meso Scale Discovery according to the manufacturer's instructions.

The inhibition of STAT5 phosphorylation by rhIL-3 by anti-CD123 mAbs was defined as 20% inhibition of activity. That is, an antibody was considered an inhibitor if it was able to inhibit STAT5 phosphorylation by rhIL-3 by at least 20%.

Five mAbs demonstrated the ability to block IL-3 stimulation of STAT5 ( FIG. 4A ). These five mAbs included 13RB18 as well as 13RB19, 13RB30, 13RB34, and 13RB44. However, when tested at 1 μg/mL, only one antibody, 13RB18, blocked IL-3 stimulation of STAT5 phosphorylation in TF-1 cells ( FIG. 4B ). Furthermore, 13RB18 (B18) demonstrated a dose-dependent effect in this assay ( FIG. 4C ). Based on these data, it is inferred that 13RB18 was the only antagonistic antibody.

Example 7: Confirmation of monovalent affinity on hCD123

Two anti-CD123 hits (I3RB120 (I3RB2 Fab), I3RB119 (I3RB18 Fab)) were analyzed in duplicate by MSD cell affinity technology for binding to Fabs of human or cynomolgus monkey CD123 SP1 expressed on the cell surface to obtain a measure of monovalent binding to cell surface CD123.

The monovalent affinity of selected anti-CD123 leaders for hCD123 or cynoCD123 expressed on the cell surface is determined using MSD cell affinity technology (MSD-CAT) method.MSD-CAT is developed internally and is used to determine affinity in a high-throughput format using intact cells as a label-free method.These experiments are carried out to assess the binding affinity and specificity of anti-CD123 candidates to cell surface people or cynomolgus monkey (cyno) CD123 SP1.The analysis can compare the affinity of anti-CD123 candidates to human and cynomolgus monkey antigens in the absence of recombinant soluble cyno CD123.The cell lines used are people pDisplayCD123SP1 and cynomolgus monkey pDisplay CD123SP1. In order to measure the affinity of these interactions using the MSD-CAT method, a series of mixtures containing fixed concentrations of anti-CD123 (1000, 200, 40 and/or 8 pM) and cells of varying concentrations (1.5 × 107-0 762 × 107 cells/mL) were prepared and the plate was allowed to reach equilibrium by rotating it for 24 h at 4 ° C. These samples were prepared in DMEM Glutamax culture medium containing 0.05% azide, 1% BSA, 3mM EDTA. According to reaction volume, cell density (cell number/L) and Avogadro's constant, the number of receptors for (3.15-4.18) × 106 hCD123/cell and (4.78-9.24) × 106 cyCD123/cell was converted to the M receptor concentration in the mixture. In this way, a concentration range of 104nM to 5.3pM is obtained for human CD123, and a concentration range of 12nM to 0.6pM is obtained for cynomolgus monkey CD123. After equilibrium, the plate is centrifuged for 5 minutes at about 1000rpm, and free anti-CD3 is detected in the supernatant. Using Mesoscale Discovery (MSD) plate reader, the free anti-CD123 in the mixture is detected by electrochemiluminescence (ECL). In order to detect the free anti-CD123 in the equilibrium mixture by electrochemiluminescence immunoassay (ECL), an assay plate is prepared. In order to prepare an assay plate (plate-bound antigen on SA-MSD plates), MSD streptavidin standard plates are blocked for 5min with 50 μL/well assay buffer (PBS (Life Sciences GIBCO 14190-136), 0.05% Tween 20, 0.2% BSA). The assay buffer was removed without washing, and 0.7 μg/mL biotinylated antigen in assay buffer was added to the MSD plate at 50 μL/well and incubated overnight (approximately 16 hours at 4°C). After overnight incubation, the plate was blocked by adding 150 μL/well of assay buffer without removing the coated antigen, incubated at ambient temperature for approximately 1 hour, and washed five times with wash buffer (assay buffer without BSA). 50 μL/well of the supernatant from the sample plate was transferred to the antigen-coated plate, incubated for 60 minutes, and then washed three times with wash buffer. Thereafter, 50 μL/well of ruthenium-labeled detection antibody (anti-human H+L) was added and incubated for 1 hour. After 1 hour, the plate was washed, and 150 μL of MSD read buffer (prepared by diluting the stock solution 1:4 in distilled water) was added to each well. The plate was immediately placed on an MSD SectorImager 6000 plate reader, and the luminescence level was read. The ECL signal detected by the MSD is expressed as % free antibody in the mixture, and the data are analyzed using a user-defined equation (derived from the law of mass action) introduced in the Prism software to determine affinity. The data show that I3RB18 and its Fab (I3RB119) are the tightest binders with pM affinity (or apparent affinity for mAb) for cell surface CD123 SP1, but the binding to cynomolgus monkey CD123 SP1 is more than 10 times weaker. For I3RB18 and its Fab (I3RB119), affinity values for mAb or Fab for cynoSP1-expressing cells cannot be obtained. However, it can be said that the affinity is >12nM. However, although I3RB120 binds to both antigens with nM affinity, it binds to human and cynomolgus monkey CD123 SP1 with the same or less than 5 times the affinity. The affinity for hCD123 SP1 obtained by SPR is weaker than the affinity observed on cells. This difference is most likely due to the presentation of the antigen on the cell surface and the location of the antibody epitope. The results are shown in Table 7.

Table 7. Affinity values of Fabs obtained by MSD-CAT for CD123 cells

^aThis K _D is greater than the listed value, but the actual value cannot be determined.

^bIn this fit, a parameter called Bo is constrained to produce an exact number rather than an approximation. When there are fluctuations in the curve, the fitting algorithm sometimes produces an approximation.

^c This is the apparent _KD , as it may be affected by the affinity due to bivalent binding.

The affinity measured for I3RB2 Fab was consistent with the mAb data obtained using Proteon. In addition, cynoCD123 cells had good binding to the Fab, clearly indicating that I3RB2 was a cross-reactive hit. Evaluation of the I3RB18 mAb and its corresponding Fab (I3RB119) showed that the affinity of recombinant CD123 SP1 obtained using Proteon was weaker than the affinity observed on cells; the affinity of the recombinant protein was 1 nM, while the affinity observed on cells was 55-300 pM. This difference is most likely due to the presentation of the antigen on the cell surface and the location of the antibody epitope. Affinity values for mAb or Fab were not available (affinity > 12 nM). This indicates that the monovalent form of the antibody has no cross-reactivity. Previous cell binding data indicated that the antibody has cross-reactivity, which is most likely due to bivalent binding to the cell surface.

Example 8: Endogenous Cell Binding

Confirm the combination of I3RB2 and I3RB18 with endogenous CD123 on AML cells. In dose-dependent MSD cell binding assay, OCI-AML5 cells (DSMZ) expressing about 75,000 CD123 copies on the cell surface were used. The combination of CD123 antibodies and AML cells was assessed using MSD (Mesoscale) cell binding assay. In brief, expression supernatant or purified CD123 antibodies were used with a dose range of 40 μg/mL to 0.039 μg/mL. 50,000 cells per well were seeded into 96-well plates (Mesoscale high binding plates) and allowed to adhere for 2h. The cells were then blocked for 15min with 20% FBS PBS solution plus Fc blockers (Fc blockers are the purified Fc portions (SEQ ID NO 209) of the antibodies cleaved by papain). Antibody supernatant was then added and placed at room temperature for 1h. Cells were washed 3 times with PBS, then ruthenium-labeled secondary antibodies (Jackson Immuno Research) were added at a rate of 1 μg/mL, and incubated for 1 h at room temperature. A further washing step was then performed, followed by addition of 150 μL/well MSD read buffer T (surfactant-free), and incubated for 30 minutes for detection. Plates were subsequently read using a SectorImager 2400 (MSD). Data were normalized to controls and mapped using GraphPad Prism version 5.

The results showed that I3RB2 and I3RB18 bound to endogenous CD123 expressed on OCI-AML5 cells in a dose-dependent manner (Figures 5A and 5B). A positive control mAb 7G3 was also included in the assay as a comparison (Figure 5C).

Example 9: Competitive Binding Analysis of CD123mAB with 13RB2 and 13RB18

Competition studies of 13RB2 and 13RB18 against other cross-reactive CD123 SP1/SP2 hits and a 7G3 control were performed to define groups or "epitope bins" of anti-CD123 antibody competitors.

For competitive ELISA, 5 μ L (20 μ g/mL) of purified human CD123 ECD proteins per well generated as described in Example 1 were coated on MSD high binding plates (Meso Scale Discovery, Gaithersburg, MD) for 2 h at room temperature. 150 μ L aliquots of 5% MSD Blocker A buffer (Meso Scale Discovery) were added to each well and incubated for 2 h at room temperature. The plate was washed three times with 0.1 M HEPES buffer (pH 7.4), followed by a mixture of labeled anti-CD123 mAb and the anti-CD123 mAb of different competitors. Labeled antibody (20 nM) was incubated with 2 μ M unlabeled anti-CD123 competitor antibodies and then added to designated wells with a volume of 25 μ L mixture. After incubation for 2 h at room temperature, the plate was washed 3 times with 0.1 M HEPES buffer (pH 7.4). MSD Read Buffer T was diluted with distilled water (4-fold dilution), dispensed at a volume of 150 μL/well, and analyzed using a SECTOR Imager 6000. Antibodies were labeled with MSD Sulfo-Tag ^™ NHS Ester according to the manufacturer's instructions (Meso Scale Discovery).

Competition ELISA results showed that I3RB2 competed with 13RB60, 13RB70, 13RB79, and 13RB118, but not with other antibodies, including I3RB18 (Figure 6A). It should be noted that when I3RB2 was labeled, competition with I3RB60 was observed; however, when I3RB60 was labeled, no competition was observed. One possible reason is that this is due to some nonspecific binding interactions. When I3RB18 was evaluated, it was found to compete with 13RB49 and 13RB55, but not with 13RB2 (Figure 6B).

Competitive binding analysis defines two competitive groups of cross-reactive CD123 SP1/SP2 antibodies (Table 8). Monoclonal antibody I3RB2 does not compete with I3RB18, and they belong to different epitope groups. Group 1 (dark grey) includes mAb 13RB2, 13RB60, I3RB70, I3RB79, and I3R118. Group 2 (light grey) consists of mAb I3RB18, I3RB49, and I3RB55. Commercial mAb 7G3 does not compete with any internal anti-CD123 antibodies.

Table 8. Competitive binding results of Ru-labeled I3RB2 and I3RB18 with anti-CD123 Ab

Example 10: Epitope mapping of I3RB2 and I3RB18

H/D exchange research

In order to identify the epitopes of I3RB2 and I3RB18 on human CD123, solution hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis was performed using the corresponding Fab. For H/D exchange, the method for analyzing Fab perturbations was similar to that described previously (Hamuro et al., J. Biomol. Techniques 14: 171-182, 2003; Horn et al., Bioehemistry 45: 8488-8498, 2006), but with some modifications. The CD123 SP2 ECD antigen was used for these studies because it has a reduced number of glycosylation sites compared to the SP1 molecule and is simpler than the SP1 molecule. Recombinant CD123 SP2 ECD (SEQ ID NO: 226) was incubated in a deuterated aqueous solution for a predetermined time to allow deuterium to be incorporated at exchangeable hydrogen atoms. At 4 ° C, deuterated CD123 SP2 ECD was complexed with I3RB119 (Fab of I3RB18) or I3RB120 (Fab of I3RB2) in 43 μL of deuterium oxide (DO) for 30 seconds, 2 minutes, 10 minutes and 60 minutes. The exchange reaction was quenched at low pH and the protein was digested with pepsin. The deuterium level at the identified peptide was monitored based on the mass shift on LC-MS. As a reference control, the CD123 SP2 ECD sample was treated in a similar manner, except that it was not complexed with the Fab molecule. It is inferred that the regions bound to the Fab are those sites that are relatively protected from exchange and therefore contain a higher ratio of deuterium than the reference CD123 SP2 ECD sample. Approximately 94% of the protein can be mapped to a specific peptide.

The solution HDX-MS perturbation diagrams of CD123 ECD SP2 with I3RB119 and T3RB120 are shown in Figures 7A and 7B, respectively. I3RB119 advantageously protects a fragment corresponding to the 195-202 amino acid residues of CD123 sp2, i.e., residues 176-184 (RARERVYEF (SEQ ID NO: 227)). I3RB120 recognizes two different regions corresponding to residues 164-175 and 184-189 of CD123 sp2, i.e., residues 145-156 (IQKRMQPVITEQ (SEQ ID NO: 228)) and residues 165-170 (LLNPGT (SEQ ID NO: 229)). These HDX-MS results show peptide-level epitopes of I3RB119 and I3RB120. There is no overlapping epitope region between the two antibodies. These results are consistent with the previous competition binding data showing that I3RB2 and I3RB18 do not compete with each other.

Example 11: Epitope mapping of anti-CD123 antibody I3RB18 by crystal structure

The binding epitope of antibody I3RB18 was determined by X-ray crystallography.

The single-chain Fv fragment of anti-CD123 mAb I3RB18 was produced in the form of VL-(Gly4Ser)4-VH-Gly-His6 (SEQ ID NO: 230). It was expressed in HEK293Expi cells and purified by affinity chromatography (HisTrap) and ion exchange (Source 15S and Mono S) chromatography.

The sp2 isoform of human CD123 ECD (SEQ ID NO: 231) with a C-terminal 8xHis tag was expressed in baculovirus-infected insect cells and purified by affinity (HisTrap) and size exclusion (Superdex 75) chromatography.

The CD123: I3RB18 scFv complex was prepared by mixing 1.8 mg CD123 (1.1 mg/mL) with 2.4 mg scFv (1.6 mg/mL) at a molar ratio of approximately 1:1.2 (excess scFv) and incubating overnight at 4°C. Small-scale (150 μg) SEC indicated the formation of the complex. The protein was concentrated to 18 mg/mL in 20 mM HEPES (pH 7.5), 100 mM NaCl.

Crystallization was performed at 20°C by vapor diffusion in a hanging drop format in MRC 2-well crystallization plates (Swissci). Crystals of the complex suitable for X-ray experiments were obtained under the following conditions: 2.0 M (NH₄)₂SO₄, 0.1 M MES buffer, pH 6.5. Crystallographic data are shown in Table 9. One crystal was transferred to a mother liquor supplemented with 24% glycerol, frozen in liquid nitrogen, and subsequently used for X-ray diffraction data collection. Its structure was determined at 100 nm resolution.

Table 9. Crystallographic data, X-ray data and refinement statistics .

The values in brackets are for the highest resolution layer.

I3RB18 binds to CD123 sp2 at the C-terminal (proximal to the cell surface) domain of the ECD. The epitope is a conformational epitope and includes three segments of the CD123 sp2 chain: residues 156-161 (RKFRYE, (SEQ ID NO: 232)), residues 173-178 (TEQVRD, (SEQ ID NO: 233)), and residues 195-202 (RARERVYE, (SEQ ID NO: 234)), which correspond to residues 234-239, 251-256, and 273-280 of CD123 sp1. The antibody-antigen interaction is primarily electrostatic. The epitope on CD123 sp2 contains a large number of basic residues, while the CDRs of I3RB18 are composed of acidic residues. The antibody residues involved in CD123 binding include 7 residues from the light chain and 9 residues from the heavy chain ( FIG8 ). All CDRs except LCDR2 are involved in binding.

The binding of I3RB18 to CD123 sp2 ( FIG9A ) distinguishes it from another anti-CD123 antibody, 7G3, in complex with CD123 sp1, which binds to the N-terminal domain 1 of the CD123 sp1 ECD, as shown in the crystal structure of the humanized 7G3 Fab CSL362 ( FIG9B ) ( pdb: 4JZJ , Broughton et al., Cell Rep. 2014; 8: 410-419 ).

Example 11: Crystal structure of anti-CD3 Fab

The crystal structure of SP34 Fab was determined at 100 nm resolution, revealing the complete amino acid sequence and identifying the likely mouse germline from which the SP34 mAb was derived.

Material

Mouse IgG3/λ isotype SP34 mAb was purchased from BD Biosciences Pharmingen (San Diego, CA) under catalog number 556611. SP34 mAb was purified from tissue culture supernatants by affinity chromatography according to the technical data sheet and stored at 4°C. Fab fragments were prepared by papain digestion of the mAb (Pierce, catalog number 44985, Thermofisher) and separated from the Fc using a Nab Protein A Plus Spin column (Pierce, catalog number 44985, Thermofisher) according to the manufacturer's protocol. Fab was further purified on a MonoS column (GE Healthcare) equilibrated with 20 mM MES (buffer A) at pH 6.5. Elution was performed with 50 column volumes of buffer A (13%-28% gradient in 1 M NaCl). Fractions corresponding to the main peak were combined, concentrated to 9.2 mg/mL, and used for crystallization.

crystallization

Crystallization was performed in a hanging drop format in a 96-well Corning 3550 plate by vapor diffusion at 20°C. Fab crystals for X-ray analysis were obtained from 12% PEG 3350, 0.2 M potassium tartrate/sodium tartrate (pH 7.4), 3% isopropanol, and 3% dioxane. Crystallization data are shown in Table 10.

Table 10 Crystallographic data, X-ray data and refinement statistics

*Numbers in brackets are for the highest resolution tier.

X-ray data acquisition and structure determination

For X-ray data collection, one crystal was immersed in a mother liquor supplemented with 20% glycerol for a few seconds and then rapidly frozen in liquid nitrogen. Diffraction data were collected on the Advanced Photon Source (Argonne, IL) IMCA beamline using a Pilatus CCD detector. X-ray data statistics are shown in Table 10.

The structure was solved by molecular replacement using a Fab model constructed from the mouse anti-Thomsen-Friedenreich antigen antibody Jaa-F11 (PDB 3gnm) to be of the IgG3/κ isotype. All crystallographic calculations were performed using the CCP4 program suite [CCP4.1994, Acta Crystallogr. D50:760-763]. Model adjustments were performed using the program COOT [Emsley P and Cowtan K. 2004. Acta Crystallogr. D60:2126-2132]. Refinement statistics are given in Table 10.

The sequence of SP34 is shown in Figure 10, where residues 1-215 of the light chain and residues 1-230 of the heavy chain were directly derived from the electron density map, while residues 231-455 were derived from IGHG3_MOUSE (mouse IgG3, isotype 2).

Example 12: Human framework adaptation of anti-CD3 antibody SP34

The anti-CD3 murine antibody SP34 was humanized by the human framework adaptation method (Fransson et al., JMB, 2010 398(2):214-31). Four different heavy chains were combined with three different light chains to generate 12 humanized variants.

SP34 humanization and affinity maturation

Selection of human lineage

The matrix of four human heavy chain v district sequences and three light chain v district sequences is selected for testing. The selection of human germline is based only on the overall sequence similarity with SP34 in framework region (FR). CDR sequences are not considered in this selection, nor are their length or canonical structure.

The closest matches to the heavy chain were the human GLs IGHV3-72 and IGHV3-73. Another GL, IGHV3-23, was chosen because of its high frequency in the human B cell repertoire.

The closest matches to the light chain are human lambda GL IGLV7-43 (aka 7a), IGLV7-46 (aka 7b), and IGLV1-51 (aka 1b). IGLV7-46 is virtually identical to IGLV7-43, but has the advantage of having Ala at position 2, as in SP34.

The selected J regions were as follows: IGHJ1 for the heavy chain and IGLJ3 for the lambda light chain.

Reversion

In order to maintain the conformation of CDR-H3, residues in several framework positions in VL must be retained, most notably Val38, Gly48 and Gly51 (Figure 11). These "back mutations" were added to the humanization plan.

The Asn at position 57 of the heavy chain does not have good side chain density in the structure. It is also located in the middle of CDR-H2 and away from the typical binding site. Based on this analysis, it is unlikely to significantly affect binding. Furthermore, the main chain geometry is located in a region that is most favorable for Gly residues in the Raman map. Therefore, it was truncated to Gly in the maturation plan to achieve the necessary flexibility and potentially improve stability (by reducing non-glycine-related local structural strain) without affecting binding.

Several other factors need to be considered in the humanization design. First, the human GLs IGLV7-46 and IGLV7-43 introduce a Trp with an undesirable oxidation potential at position 59. The other two GLs have a Gly corresponding to the mouse sequence at this position. Therefore, Gly59 was retained in both the IGLV7-46 and IGLV7-43 variants. Finally, the Ala at position 49 in VH may be necessary. In addition, the residue at position 99 (Val in SP34) may affect antigen binding. To test these positions, back mutations were introduced in some variants (Figure 12).

HFA Matrix

The HFA matrix (Table 11) consists of four variants of VH and three variants of VL (Figure 12). For the purposes of HFA, the AbM CDR definitions were used (K.R. Abhinandan and A.C. Martin, 2008. Mol. Immunol. 45, 3832-3839).

Variants of VH :

CD3H141 (SEQ ID NO: 184): IGHV3-72*01+Gly49Ala containing mouse CDRs

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CD3H142 (SEQ ID NO: 185): IGHV3-23*01+Ser49Ala containing mouse CDRs

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGNFGNSYVSWFAYWGQGTLVTVSS

CD3H143 (SEQ ID NO: 186): IGHV3-23*01 containing mouse CDRs + Ser49Ala, Ala99Val

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYVSWFAYWGQGTLVTVSS

CD3H144 (SEQ ID NO: 187): IGHV3-73*01+Asn57Gly containing mouse CDRs

EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNGYATYYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS

Variants of VL :

CD3L63 (SEO ID NO: 188): IGLV7-46*01+F38V, A48G, Y51G, W59G containing mouse CDRs

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL

CD3L64 (SEQ ID NO: 189): IGLV1-51*01+Y38V, L48G, Y51G containing mouse CDRs

QSVLTQPPSVSAAPGQKVTISCRSTGAVTTSNYANWVQQLPGTAPKGLIGGTNKRAPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCALWYSNLWVFGGGTKLTVL

CD3L66 (SEQ ID NO: 190): IGLV7-43*01+F38V, A48G, Y51G, W59G containing mouse CDRs

QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

Table 11 Matrix of CD3 heavy and light chains

(All prepared with IgG1-AA Fc containing L234A, L235A, and F405L)

The amino acid sequence was reverse translated into DNA and cDNA was prepared using gene synthesis technology (U.S. Patent No. 6,670,127; U.S. Patent No. 6,521,427). Using standard molecular biology techniques, the heavy chain (HC) variable region was subcloned into a human IgG1-AA Fc containing L234A, L235A and F405L mutations using an internal expression vector with a CMV promoter. Using standard molecular biology techniques, the light chain (LC) variable region was subcloned into a human lambda constant region using an internal expression vector with a CMV promoter. The resulting plasmid was transfected into Expi293F cells (Invitrogen) and mAb was expressed. Purification was performed by standard methods using a protein A column (hiTrap MAbSelect SuRe column). After elution, the harvested material was dialyzed into D-PBS (pH 7.2). The VH and VL sequences of the antibodies are shown in Table 12.

Table 12. VH and VL sequences of anti-CD3 antibodies

The monospecific anti-CD3 antibody CD3B143 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 184 and a VL of SEQ ID NO: 188, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B144 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 184 and a VL of SEQ ID NO: 189, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B146 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 184 and a VL of SEQ ID NO: 190, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B147 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 185 and a VL of SEQ ID NO: 188, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B148 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 185 and a VL of SEQ ID NO: 189, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B150 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 185 and a VL of SEQ ID NO: 190, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B151 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 186 and a VL of SEQ ID NO: 188, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B152 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 186 and a VL of SEQ ID NO: 189, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B154 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 186 and a VL of SEQ ID NO: 190, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B155 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 187 and a VL of SEQ ID NO: 188, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B156 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 187 and a VL of SEQ ID NO: 189, and an IgG1 constant region having L234A, L235A, and F405L substitutions. The monospecific anti-CD3 antibody CD3B158 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 187 and a VL of SEQ ID NO: 190, and an IgG1 constant region having L234A, L235A, and F405L substitutions.

Example 13: Endogenous Cell Binding of Humanized Anti-CD3 Hits to Primary T Cells

The obtained anti-CD3 antibody group was tested for binding to cell surface CD3ε on primary human T cells. To this end, polyclonal anti-human secondary antibodies were used to visualize the binding of antibodies in the expression supernatant and analyzed by flow cytometry. In brief, the binding of anti-CD3 antibodies to cell surface CD3ε was assessed by flow cytometry using primary human T lymphocytes purified by negative selection (Biological Specialty, Colmar, USA). The expression supernatant or purified antibody in culture medium or FACS buffer (BD BioSciences) was normalized to 10 μg/ml, respectively. 2×105 cells were aliquoted into each well of a 96-well round-bottom plate (CoStar) for labeling. The antibodies in the expression supernatant were added to the cells and incubated at 4°C for 45 min. At 1300rpm, centrifuge for 3min and remove the supernatant, followed by incubation of 50 μL anti-human IgG (H+L) Alexa Fluor 647 secondary antibodies (Lifetechnologies) at a final concentration of 10 μg/mL with cells at 4°C away from direct light for 30min. The cells are then washed and resuspended in 30 μL FAC buffer (BD BioSciences). Sample collection is performed on the Intellicyt HTFC system using ForeCyt software. Before binding analysis using green or red fixable survival/death dyes (Life Technologies), forward/side scatter area, and height parameters, live single cells are sorted. Graphs are generated using mean fluorescence intensity values in GraphPad Prism Version 5.

Although a titration series was run, intermediate concentrations are shown in Figure 13 for clarity. Two in-house phage-derived antibodies with the same Fc region as the therapeutic antibody were used as controls: the non-cynomolgus monkey cross-reactive agonist antibody G11 (HC SEQ ID NO: 222, LC SEQ ID NO: 223) was used as a positive control, and the non-binding/non-agonist antibody CD3B94 (HC-SEQ ID NO: 224, LC-SEQ ID NO: 225) was used to assess nonspecific binding. The commercially available SP34 antibody was not used as a comparator in this assay because it is a mouse antibody and the use of different secondary detection reagents would prevent direct comparison with the tested variants.

The data show a range of binding potentials within the humanized anti-CD3 hit group, with two antibodies (CD3B144, CD3B152) showing complete loss of binding to human T cells. The remaining antibodies showed some degree of binding potential and can be roughly divided into strong and weak binders using G11 binding as an arbitrary threshold. Using these parameters, 7 strong binders and 7 weak binders were identified from the variant group (Figure 13).

Then test the binding analysis of anti-CD3 hits and primary cynomolgus monkey CD4+T cells to evaluate the maintenance of cross-reactivity. CD4+T cells purified from the peripheral blood of cynomolgus monkeys (Zen Bio, Triangle Research Park, USA) were used. The assay protocol is similar to those described above. Since G11 does not cross-react with cynomolgus monkey CD3ε CD3B124, an internal chimeric SP34-derived antibody with VH and VL of SP34 (with mouse framework and human IgG1 Fc) is used in this assay as a positive control (Figure 14). Interestingly, compared with the binding potential of human cells, several variants show reduced binding potential. This includes strong binders CD3B150, CD3B151 and CD3B154, which are reduced in binding, and several weak binders in which binding cannot be detected on background. The loss of this binding is unrelated to the specific immunoglobulin chain, indicating that the combination of heavy chain and light chain plays an important role in the process of losing cross-reactivity. In short, these assays can identify variants that maintain species cross-reactivity between humans and cynomolgus monkey CD3g.

Example 14: Functional analysis of humanized anti-CD3 hits in primary T cells

Binding analysis showed that the humanized anti-CD3 hit group had a certain degree of binding potential for human and cynomolgus monkey T cells. In order to study the ability of each variant to cause activation by CD3ε cross-linking, primary T cells were cultured overnight in the presence of bead-conjugated antibodies. The next day, cells were harvested and labeled with anti-CD69 antibodies to measure activation capacity (Figure 15). Humanized anti-CD3 antibodies were combined with protein A-coated magnetic beads (SpheroTech, Lake Forest, USA) by incubation with 10 μg/mL of antibody overnight. The next day, 2×105 primary human T cells were seeded in triplicate in round-bottom cell culture plates and 2×105 coated beads were added. After overnight culture at 37°C, cells were harvested and labeled with anti-CD69 Alexa488 antibodies (clone FN50; Biolegend) to assess the upregulation of this activation marker. Sample collection and analysis were performed as described above for binding. Several negative control samples were run, including T cells alone, T cells with uncoated beads, and T cells with isotype control (CD3B94) coated beads. All of these samples showed similar mean fluorescence intensity values comparable to unstained T cells, indicating that the background value in this assay was very low. Several positive control samples were run for comparison, including OKT3 (US5929212) and commercially available SP34-2 antibody.

The humanized anti-CD3 hits were then tested for their ability to activate primary cynomolgus CD4+ T cells in the same assay (ZenBio, Triangle Research Park, USA) (Figure 16). The FN50 anti-CD69 antibody has been shown to cross-react with non-human proteins, so this antibody can be used to test activation of these cells.

Human and cynomolgus monkey activation data are associated with binding data because hit group shows a certain degree of activation potential.When compared with commercially available SP34-2, multiple strong binders demonstrate the ability of human T cell activation to equal or greater degree.Several variants demonstrate lower activation potential compared with SP34-2, yet some binding substances do not illustrate the evidence of CD69 stimulation.Only in the variant that does not show combination or shows weak combination, observe and can not activate, all strong binders all demonstrate a certain degree of activation, which shows that there is correlation between the combination and activation potential of people (Figure 17 A) and cynomolgus monkey (Figure 17 B).

Example 15: Preparation of IgG1 L234A, L235A bispecific antibody

Several monospecific CD123 antibodies are expressed as IgG1, and the Fc regions of these antibodies have Fc replacements L234A, L235A and K409R (on anti-CD123) (numbered according to the EU index). Monospecific antibodies are expressed in HEK cell lines. Monospecific CD3 antibodies are IgG1 with Fc replacements L234A, L235A and F405L.

A monospecific anti-CD123 antibody, I3RB135-K409R, was generated comprising the VH and VL regions of the anti-CD123 antibody I3RB2 having a VH of SEQ ID NO: 120 and a VL of SEQ ID NO: 165, and an IgG1 constant region having L234A, L235A, and K409R substitutions.

A monospecific anti-CD123 antibody, I3RB125-K409R, was generated comprising the VH and VL regions of the anti-CD123 antibody I3RB18 having a VH of SEQ ID NO: 136 and a VL of SEQ ID NO: 168, and an IgG1 constant region having L234A, L235A, and K409R substitutions.

As a control, the monospecific anti-RSV antibody B21M was generated, which comprises VH and VL regions with VH of SEQ ID NO: 191 and VL of SEQ ID NO: 192 and an IgG1 constant region with L234A, L235A and K409R or F405L (as an empty arm to pair with the CD3 or CD123 arm of the bispecific antibody).

The monospecific antibodies were purified by standard methods using a Protein A column (HiTrap MabSelect SuRe column). After elution, the pool was dialyzed into D-PBS (pH 7.2).

Monospecific anti-CD123 antibodies in in vitro Fab arm exchange were combined in a matrix format to generate bispecific antibodies that were subsequently further characterized (Table 13).

Table 13. CD123×CD3 mAb Matrix for Forming Bispecific Antibodies

Bispecific CD123×CD3 antibodies (as described in WO2011/131746) were generated by combining monospecific CD3 mAb and monospecific CD123 mAb in an in vitro Fab arm exchange. Briefly, a PBS solution of anti-CD123/anti-CD3 antibodies (pH 7-7.4) at a molar ratio of about 1-20 mg/mL was mixed together with 75 mM 2-mercaptoethanolamine (2-MEA) and incubated at 25-37°C for 2-6 h. 2-MEA was then removed by dialysis, diafiltration, tangential flow filtration, and/or spin cell filtration using standard methods. A control bispecific antibody with an anti-RSV-(B21M) arm was generated in a similar manner.

The generated monospecific anti-CD3 and CD123 antibodies were mixed in a matrix manner for in vitro Fab arm exchange and characterized in various assays. The bispecific antibody I3RB179-Ab comprises the CD3 binding arm of mAb CD3B146-F405L and the CD123 binding arm of mAb I3RB135-K409R. The bispecific antibody I3RB186-Ab comprises the CD3 binding arm of mAb CD3B146-F405L and the CD123 binding arm of mAb I3RB125-K409R. The bispecific antibody I3RB180-Ab comprises the CD3 binding arm of mAb CD3B147-F405L and the CD123 binding arm of mAb I3RB135-K409R. The bispecific antibody I3RB187-Ab comprises the CD3 binding arm of mAb CD3B147-F405L and the CD123 binding arm of mAb I3RB125-K409R. The bispecific antibody I3RB181-Ab comprises the CD3 binding arm of mAb CD3B151-F405L and the CD123 binding arm of mAb I3RB135-K409R. The bispecific antibody I3RB188-Ab comprises the CD3 binding arm of mAb CD3B155-F405L and the CD123 binding arm of mAb I3RB125-K409R. The bispecific antibody I3RB182-Ab comprises the CD3 binding arm of mAb CD3B154-F405L and the CD123 binding arm of mAb I3RB135-K409R. The bispecific antibody I3RB189-Ab comprises the CD3-binding arm of mAb CD3B154-F405L and the CD123-binding arm of mAb I3RB125-K409R. The bispecific antibody I3RB183-Ab comprises the CD3-binding arm of mAb CD3B155-F405L and the CD123-binding arm of mAb I3RB135-K409R. The bispecific antibody CD3B191-Ab comprises the CD3-binding arm of mAb CD3B155-F405L and the CD123-binding arm of mAb I3RB125-K409R.

For control bispecific antibodies, anti-RSV antibody, B21M (HC SEQ ID NO: 207-shown to have F405L mutation, LC SEQ ID NO: 208) and CD3 arm or CD123 arm are combined as follows. Bispecific antibody 13RB185-Ab comprises the anti-RSV binding arm of mAb B21M-F405L and the CD123 binding arm of mAb 13RB135-K409R. Bispecific antibody 13RB191-Ab comprises the anti-RSV binding arm of mAb B21M-F405L and the CD123 binding arm of mAb 13RB125-K409R. Bispecific antibody 13RB192-Ab comprises the anti-RSV binding arm of mAb B21M-K409R and the CD3 binding arm of mAb CD3B146-F405L. The bispecific antibody I3RB193-Ab comprises the RSV binding arm of mAb B2M-F409R and the CD3 binding arm of mAb CD3B147-F405L. The bispecific antibody I3RB194-Ab comprises the anti-RSV binding arm of mAb B2M-F409R and the CD3 binding arm of mAb CD3B151-F405L. The bispecific antibody I3RB195-Ab comprises the anti-RSV binding arm of mAb B21M-K409R and the CD3 binding arm of mAbCD3B154-F405L. The bispecific antibody I3RB196-Ab comprises the RSV binding arm of mAb B21M-K409R and the CD3 binding arm of mAb CD3B155-F405L.

The heavy and light chains of the CD123×CD3 bispecific Abs are shown in Table 14 below.

Table 14. Heavy and light chain sequences for bispecific IgG1 antibodies

Example 16: Evaluation of bispecific antibodies in functional cell killing assays

The T cell-mediated cytotoxicity assay is a functional assay that uses T cells from healthy donors to evaluate the ability of CD123×CD3 bispecific antibodies to lyse cells.

The protocol described by Laszlo et al. (Laszlo, G. et al., 2014 BLOOD 123:4, 554-561) was followed. Briefly, effector cells were harvested, counted, washed, and resuspended to 1×10∧6 cells/mL in RPMI (10% FBS) cell culture medium. Target cells were labeled with CFSE (Invitrogen #C34554) and resuspended to 2×105 cells/mL in RPMI (Invitrogen #61870-036) containing 10% FBS (Invitrogen #10082-147). Effector cells and CFSE-labeled target cells were mixed in a sterile 96-well round-bottom plate at a ratio of 5:1. A 5 μL aliquot of each bispecific antibody was added to each well at different concentrations. The cultures were incubated at 37°C, 5% CO2 for 48 h. After 48 h, LIVE/fixable near-infrared dead cell staining buffer (life technologies, product catalog number L10119) was added to the sample and the culture was incubated in the dark for 20 min at room temperature, then washed and resuspended in 170 μL FAC buffer. Drug-induced cytotoxicity was determined using a CANTO II flow cytometer (BD Biosciences) and analyzed using FlowJo software or Dive software (BD Biosciences). The population of interest was double-positive CFSE+/live/dead+ cells.

Shown are the results of T cell-mediated cell lysis of AML cell lines MV4-11 (Figures 18A and 18B), OCI-AML5 (Figures 19A and 19B), and OCI-M2 (Figures 20A and 20B) after incubation for 48 hours at 37°C and 5% CO2. MV4-11 and OCI-AML5 are CD123-expressing cell lines, and OCI-M2 has significantly low CD123 expression. The ratio is 5:1. A 2 mg/mL aliquot of Fc blocker was added to block Fc function.

When combined with anti-CD3 antibodies in a bispecific format, both I3RB2 and I3RB18 antibodies are very effective in specifically killing CD123+ cells. In addition, the data show that there is a clear hierarchy between I3RB135 (based on I3RB2) and I3RB125 (based on I3RB18) bispecific antibodies, where I3RB125×CD3 bispecific antibodies are more effective than I3RB135×CD3 bispecific antibodies. Within each family, bispecific antibodies based on CD3B146 and CD3B155 (higher affinity mAbs) are more effective than bispecific antibodies based on CD3B151 and CD3B154. Low levels of dose-dependent background cytotoxicity were observed in the low CD123 expressing cell line OCI-M2.

Example 17: Evaluation of the bispecific antibody I3RB186 in a disease tumor model

Materials and methods

Cell line. In order to determine the in vivo efficacy of the bispecific antibody I3RB186, commercially available tumor cell lines with high CD123 expression were selected for efficacy studies. At 37 ° C, in an atmosphere of 5% CO2 in air, KG-1 (DSMZ, product catalog number ACC 14) human acute myeloid leukemia (AML) tumor cells were maintained in RPMI culture medium supplemented with heat-inactivated fetal bovine serum (10% v/v) in vitro. The cells were routinely subcultured twice to three times a week. Cells grown in the exponential growth phase were harvested and counted for tumor cell inoculation.

Preparation of human PBMC for transplantation. Human mononuclear enriched cells (Cat. No. 213-15-04) purchased from Biological Specialty, Inc. (Colmar, PA) were used for hIgG1-AA molecule testing. PBMCs were separated by Ficoll density gradient separation apparatus (Ficoll-Paque ^™ Plus, GE Healthcare Bio-Sciences AB, Cat. No. 17-1440-03) and dispensed in freezing medium (Recovery cell freezing medium, Gibco, Cat. No. 12648-010) at 50 × 106 cells/vial. The vials were stored at -80°C for approximately 24 hours and then transferred to liquid nitrogen for long-term storage. IgG4 molecule testing was performed using frozen isolated peripheral blood mononuclear cell vials (100 × 106 cells/vial, Cat. No. PB009-3) purchased from HemaCare (Van Nuys, CA). To thaw PBMCs, the frozen vials were placed in a 37°C water bath. Transfer the cells to a conical tube containing cold thaw medium. Centrifuge the conical tube and resuspend the cells in sterile PBS. Assess cell viability using trypan blue exclusion. Resuspend the cells in sterile PBS to a cell concentration of 50 × 106 cells/mL for injection.

Peripheral blood was collected for FACS analysis. To this end, 50 μL of blood was collected from the retro-orbital sinus of each animal into a lithium heparin-coated tube. A 25 μL aliquot of blood from each sample was placed in 175 μL of culture medium (RPMI containing 10% FBS) in each of two 96-well plates. The plate was centrifuged and treated with three ACK lysis buffers to lyse the red blood cells. The remaining cells of each sample were combined and stained for CD45, CD3, CD8 and CD4 to quantify circulating human T lymphocytes (see Mouse Peripheral Blood Harvesting/Staining: Protocol for Leukocyte Isolation and FACS analysis).

Leukocyte FACS analysis protocol. Leukocyte FACS analysis protocol. Peripheral blood was collected up to twice during the fluorescence-activated cell sorting (FACS) analysis of circulating human PBMCs. Whole blood (25 μL) was diluted in 175 μL RPMI culture medium in a 96-well plate. The plate was centrifuged at 1400 rpm for 4 minutes and the supernatant was decanted. The cells were resuspended in 200 μL ACK lysis buffer and incubated on ice for 5 min. After centrifugation at 1300 rpm for 5 minutes, the supernatant was aspirated. The cells were treated twice more with ACK lysis buffer, washed once in 200 μL PBS, and centrifuged again at 1500 rpm for 5 min. The cell pellet was resuspended in 50 μL/well of an antibody mixture in PBS containing Live/Dead staining solution (Invitrogen, catalog number L10119, 0.25 μL/well stock solution. The stock solution is 1 vial diluted in 150 μL DMSO) and incubated in the dark at room temperature for 30 min. Cells were labeled with the following antibodies: CD4 (Becton Dickinson, catalog number 557922, 0.5 μL/well), CD8 (Invitrogen, Q010055, 0.5 μL of 1:10 dilution in PBS/well), CD3 (Becton Dickinson, catalog number 558117, 0.5 μL/well), CD45 (BioLegend, catalog number 304006, 0.5 μL/well). Cells were washed three times with FACS buffer (200 μL/well) and resuspended in 170 μL of FACS buffer. Sample acquisition was performed on a BD LSR Fortessa flow cytometer. Live single cells were sorted prior to analysis using near-infrared live/dead dye (Life Technologies) and forward/side scatter area and height parameters, respectively. Data were analyzed using BD FACS Diva software, version 7.

In vivo design. KG-1 cells (5×106 cells in 200 μL of phosphate-buffered saline) were subcutaneously inoculated on the dorsal flank of each female NSG (NOD.Cg-Prkdcscid Il2rgtm1 Wjl/SzJ) mouse. The date of tumor cell inoculation was designated as day 0. Tumor measurements were monitored twice weekly starting on day 7 post-implantation until the tumor volume was within the range of 100-150 ^mm3 (day 14 post-implantation), at which time the mice were randomly divided into treatment groups according to tumor volume. The mice were then transplanted intravenously (lateral tail vein) with human peripheral blood mononuclear cells (PBMCs) (10×106 cells in 200 μL of phosphate-buffered saline). After transplantation of PBMCs, the mice immediately received intravenous treatment with bispecific Ab I3RB186 (bispecific Ab I3RB186 was diluted in PBS and administered in a volume of 100 μL). Treatments were administered approximately every other day for a total of five doses (see Table 15 for exact dosing days). Tumor measurements and body weights were recorded twice weekly.

The endpoints of the study were tumor growth inhibition, maximum tumor burden (group mean greater than 1500 mm ³ ) and weight loss greater than 20% of treatment start weight. Tumor dimensions were measured twice weekly in two dimensions using calipers, and volume was expressed in mm ³ using the following formula: V = 0.5 a ^{x b 2} , where a and b are the major and minor diameters of the tumor, respectively. Complete tumor regression (CR) was defined as a tumor that had decreased to below the palpation limit (50 mm ³ ). Partial tumor regression (PR) was defined as a tumor that had decreased in size compared to the initial tumor volume. A minimum duration of CR or PR in three or more consecutive tumor measurements was required for CR or PR to be considered durable.

Transplantation of human PBMCs results in mice eventually developing graft-versus-host disease (GVHD), a condition in which transplanted donor T cells become activated and infiltrate host tissues, leading to organ failure, extreme weight loss, and inevitable death. To monitor the onset and severity of GVHD in this model, body weight is recorded twice weekly in grams (g). Percent body weight change is calculated using the following formula: Body weight change = [(C-I)/I]*100, where C is current body weight and I is body weight at the start of treatment.

Summary statistics (including mean and standard error of the mean (SEM)) are provided for tumor volume in each group at each time point, and differences in tumor volume are shown in the corresponding study tables. Statistical analysis of differences in tumor volume between groups was assessed using a two-way ANOVA repeated measures test followed by a Bonferroni post hoc test using GraphPad Prism version 5.01. P < 0.05 was considered statistically significant.

Efficacy of CD123×CD3IgG1, F234A, L235A bispecific Ab

NSG mice were subcutaneously inoculated with KG-1 cells and then intravenously transplanted with previously described human PBMCs. When tumors were established as described above (mean tumor volume = 102 +/- 5.9 ^mm3 ), they were dosed with the CD123 x CD3 bispecific Ab I3RB186 at doses of 0.01, 0.1, 1, and 10 μg per animal. A subset of tumor-bearing mice was not transplanted with PBMCs but was dosed to control for bispecific mechanism in the absence of the control bispecific Ab. Additionally, a subset of non-tumor-bearing mice was transplanted with PBMCs and dosed to serve as a control for peripheral blood FACS analysis (see study design in Table 15).

Table 15. Dosing regimen for in vivo efficacy of I3RB186

Results of in vivo efficacy studies

Figure 21 shows the efficacy of CD123×CD3IgG1-AA bispecific I3RB186-IgG1, F234A, L235A in KG-1 human AML xenografts at two doses of 0.1 μg and 1 μg (p < 0.001) per animal in the presence of human PBMCs. The bispecific antibody of 1 μg per animal (grey closed square) showed a more direct anti-tumor efficacy than the antibody at a dose of 0.1 μg, with complete tumor regression in 3/8 animals and partial tumor regression in 3/8 animals. However, tumor regrowth was observed in 6/8 mice starting on day 55 after tumor implantation. The bispecific antibody of 0.1 μg per animal (grey closed diamond) showed a delayed but stronger efficacy, with complete and partial tumor regression in all animals. The data indicate that the presence of effector T lymphocytes is necessary for killing target cells with bispecific antibodies.

Figure 22 shows FACS analysis of peripheral blood collected from mice on day 30 after tumor implantation. An increase in CD45+ cells, driven by an increase in CD8+ T lymphocytes, was evident in tumor-bearing animals treated with 0.1 μg and 1 μg of the bispecific antibody. This increase in CD8+ T lymphocytes occurred only in the presence of target cells (KG-1) in the group where anti-tumor efficacy was observed. Alternatively, 10 μg of the bispecific antibody may have depleted CD45+ PBMCs from the peripheral blood. This depletion of effector cells may explain the low efficacy observed at this dose.

Figure 23 shows FACS analysis of peripheral blood collected from mice on day 53 after tumor implantation. Tumor-bearing mice treated with 0.1 μg and 1 μg of bispecific antibody showed similar levels of CD45+, CD8+, and CD4+ cells, as did non-tumor-bearing mice treated with PBS and 0.01 μg and 0.1 μg of bispecific antibody. Non-tumor-bearing mice treated with 1 μg and 10 μg of bispecific antibody showed very low levels of CD45+, CD8+, and CD4+ cells; the reason for this phenomenon is unclear.

Figure 24 shows the change in the average body weight of the treatment group over time. As previously mentioned, weight loss is related to the onset and severity of GVHD caused by activated T cells. In both tumor-bearing mice and non-tumor-bearing mice, weight loss was the most serious when treated with 0.1 μg of bispecific antibody. Tumor-bearing mice treated with 1 μg of bispecific antibody did not experience severe weight loss. T lymphocytes appeared on the 53rd day after tumor implantation (analyzed by FACS, Figure 23), but there was no weight loss and GVHD onset indication loss of activated T cells, which may explain the tumor regrowth observed on the 55th day after tumor implantation in this group (Figure 21).

Example 18. In vivo evaluation of I3RB186 and control bispecific Abs (I3RB191 and I3RB192)

In the second in vivo experiment, bispecific Ab controls I3RB191 (CD3 empty arm) and I3RB192 (CD123 empty arm Ab) were added. This protocol was identical to that of Example 16. KG-1 human AML tumor xenografts were subcutaneously implanted into female NSG mice. On day 14 after implantation, mice were randomly divided into treatment groups based on tumor volume. Human PBMCs were implanted intravenously, followed by intravenous treatment with 1 μg of I3RB186 and I3RB191 and I3RB192 control bispecific Abs per animal (see Table 16 for dosing schedule). Treatment occurred on days 14, 16, 18, 21, and 23 after tumor implantation. The arrows in the figure indicate the days on which the bispecific Abs were administered.

Table 16 Dosage regimen for the second in vivo experiment

The antitumor activity of the bispecific Ab was expressed as tumor size (mm ³ ) over time ( FIG. 25 ). Treatment with 1 μg I3RB186 significantly inhibited tumor growth compared to animals treated with PBS and the control bispecific Ab (p<0.001).

At day 36 after tumor implantation, peripheral blood was collected for FACS analysis of circulating human PBMCs. Unlike the first study, there was no difference in the frequency of human CD45+ PBMCs (a) or CD8+ and CD4+ T lymphocytes (b) in animals treated with I3RB186 compared to those treated with PBS and I3RB191 (Figure 26). CD45+, CD8+, and CD4+ cells were at lower frequencies in tumor-bearing and non-tumor-bearing animals treated with the 13RB192CD123 empty-arm control bispecific Ab.

At day 63 after tumor implantation, peripheral blood was collected for FACS analysis of circulating human PBMCs. Among tumor-bearing animals, only animals treated with 1 μg I3RB186 were retained (Figure 27). Compared with non-tumor-bearing animals treated with PBS or 1 μg I3RB186, the frequencies of CD45+ human PBMCs (a) and CD8+ T lymphocytes (b) in tumor-bearing animals treated with 1 μg I3RB186 increased (Figure 25). In all other groups, CD4+ T lymphocytes were at similar frequencies. Non-tumor-bearing mice treated with PBS and 1 μg I3RB186 had very low frequencies of CD45+, CD8+, and CD4+ cells.

Figure 28 shows the change in the mean body weight of the treatment groups over time. As previously mentioned, weight loss is associated with the onset and severity of GVHD caused by activated T cells. Compared to all other groups, tumor-bearing mice lost more weight when treated with 1 μg of bispecific antibody. This contradicts the first study, in which tumor-bearing mice treated with 1 μg of bispecific antibody did not experience severe weight loss. T lymphocytes appeared on day 63 after tumor implantation (Figure 27), but the efficacy of the 1 μg dose was not as significant as in the first study (Figure 21, Figure 25).

Example 19. Preparation of IgG4 S228P, F234A, L235A bispecific antibody

Several monospecific CD3 and CD123 antibodies are expressed as IgG4 with Fc substitutions S228P, F234A and L235Ax (CD123 arm) or S228P, F234A, L235A, F405L and R409K (CD3 arm) in their Fc region (numbered according to the EU index). Monospecific antibodies were expressed in a CHO cell line under the influence of a CMV promoter.

The monospecific anti-CD3 antibody CD3B219 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 184 and a VL of SEQ ID NO: 190, and an IgG4 constant region having substitutions of S228P, F234A, L235A, F405L, and R409K. The monospecific anti-CD3 antibody CD3B217 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 186 and a VL of SEQ ID NO: 188, and an IgG4 constant region having substitutions of S228P, F234A, L235A, F405L, and R409K. The monospecific anti-CD3 antibody CD3B218 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 186 and a VL of SEQ ID NO: 190, and an IgG4 constant region having substitutions of S228P, F234A, L235A, F405L, and R409K. The monospecific anti-CD3 antibody CD3B220 was generated, comprising VH and VL regions having a VH of SEQ ID NO: 187 and a VL of SEQ ID NO: 188, and an IgG4 constant region having substitutions of S228P, F234A, L235A, F405L, and R409K.

Generate monospecific anti-CD123 antibody I3RB218, it comprises the VH and VL region of anti-CD123 antibody I3RB2 with SEQ ID NO: 120 VH and SEQ ID NO: 165 VL, and IgG4 constant region with S228P, F234A and L235A substitution.Generate monospecific anti-CD123 antibody I3RB217, it comprises the VH and VL region of anti-CD123 antibody I3RB18 with SEQ ID NO: 136 VH and SEQ ID NO: 168 VL, and IgG4 constant region with S228P, F234A and L235A substitution.

As a control, a monospecific anti-RSV antibody B21M derived from B21M was generated, which contained VH and VL regions with VH of SEQ ID NO: 191 and VL of SEQ ID NO: 192, and an IgG4 constant region with S228P, F234A, L235A or F234A, L235A, R409K, F405L (as an empty arm to pair with the CD3 or CD123 arm of the bispecific antibody).

Monospecific antibodies are purified, and as previously described in Example 15, generated monospecific anti-CD3 and CD123 antibodies are mixed in a matrix manner (table 12), exchanged for external Fab arms, and characterized in various assays. Bispecific antibody Ab 7959 comprises the CD3 binding arms of mAb CD3B219-F405L, R409K and the CD123 binding arms of mAb I3RB217-R409. Bispecific antibody Ab 3978 comprises the CD3 binding arms of mAb CD3B217-F405L, R409K and the CD123 binding arms of mAb I3RB217-R409. Bispecific antibody Ab 7955 comprises the CD3 binding arms of mAb CD3B218-F405L, R409K and the CD123 binding arms of mAb I3RB217-R409. Bispecific antibody Ab 9958 comprises the CD3 binding arm of mAb CD3B220-F405L, R409K and the CD123 binding arm of mAb I3RB217-R409. Bispecific antibody Ab 8747 comprises the CD3 binding arm of mAb CD3B219-F405L, R409K and the CD123 binding arm of mAb I3RB218-R409. Bispecific antibody Ab 8876 comprises the CD3 binding arm of mAb CD3B217-F405L, R409K and the CD123 binding arm of mAb I3RB218-R409. Bispecific antibody Ab 4435 comprises the CD3 binding arm of mAb CD3B218-F405L, R409K and the CD123 binding arm of mAb I3RB218-R409. The bispecific antibody Ab 5466 comprises the CD3 binding arm of mAb CD3B220-F405L, R409K and the CD123 binding arm of mAb I3RB218-R409.

For control bispecific antibodies, B2M1 in IgG4 PAA format was generated, purified, and combined with either a CD3 arm or a CD123 arm according to the following matrix in Table 17 below.

Table 17 IgG4 bispecific antibody matrix

The heavy and light chains of the CD123×CD3 bispecific antibodies are shown in Table 18.

Table 18. Heavy and light chain sequences of the bispecific Ab IgG4-PAA

Example 20. CD123 monovalent affinity of bispecific antibodies in IgG4-PAA format using recombinant antigens

Surface plasmon resonance (SPR) experiments were performed to determine the kinetics and affinity of CD3×CD123 bispecific antibody binding to human CD123 SP1 ECD and CD123 SP2 ECD.

Biacore instrument is used to measure the affinity of anti-CD123 × CD3 bispecific Ab3978,7955,7959,9958,8876,8747,5466 to recombinant human CD123 SP1 and recombinant human CD123 SP2 ECD by surface plasmon resonance (SPR).Biacore T200 (Biacore company, now a branch of GE Healthcare) is used to carry out kinetic study at 25 DEG C.Goat anti-human IgG (Fc) specific antibody (Jackson ImmunoResearch laboratories Prod#109-005-098) is covalently attached to the gold surface of the carboxymethyl dextran coating of CM-5 sensor chip (GE Healthcare).The carboxymethyl group of dextran is activated with N- ethyl -N '- (3- dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS). At pH 4.5, anti-Fc antibodies were connected in 10mM sodium acetate. Any remaining reactive sites on the surface were blocked by reaction with ethanolamine. For kinetic binding measurements, anti-CD123 antibodies were captured onto anti-human Fcγ specific antibodies. 40-70RU of antibodies were captured. After capturing Ab, human CD123 SP1 or human CD123 SP2 at a concentration between 0.4nM and 400nM was injected at a speed of 40 μL/min. 2 minutes of association data were collected, followed by 10 minutes of dissociation. 30 μL of 100mM H3PO4 was added at a speed of 100 μL/min, followed by surface regeneration with 50mM NaOH. Samples for kinetic analysis were prepared in PBS-based buffer (D-PBS containing 3mM EDTA and 0.005% surfactant P20). The reported data are the SPR signal differences between the flow cell containing the capture antibody and the reference cell without the capture antibody. The additional contribution of the device to the signal was eliminated by subtracting the data from the blank injection from the signal subtracted from the reference value. The data were analyzed using BIA evaluation software (BIAcore) by fitting the association and dissociation phases of all concentrations to a 1:1 binding model (global fit). Tables 20 and 21 summarize the kinetic and affinity results obtained by Biacore. These two tables represent data obtained during three or more independent experiments.

Biacore data show that within the same family, I3RB18-derived bispecific Ab and I3RB2-derived bispecific Ab bind to CD123 SP1 (Table 19) with similar affinity and bind to CD123 SP2 (Table 20) with similar affinity. The binding of I3RB18-derived bispecific Ab to recombinant CD123 SP1 is more than 10 times tighter than that of I3RB2-derived bispecific Ab, with affinities of ˜1 nM and 14 nM, respectively. When bound to recombinant CD123 SP2, the binding of I3RB18-derived bispecific Ab is more than 5 times tighter than that of I3RB2-derived bispecific Ab, with affinities of ˜0.3 nM and 1.7 nM, respectively. The standard deviations in Tables 19 and 20 indicate that the data are reproducible.

Table 19 Biacore kinetics and affinity data of bispecific antibodies binding to recombinant human CD123 SP1 .

NB - Not bound

Table 20. Biacore kinetic and affinity data for binding of anti-CD123 bispecific antibodies to recombinant human CD123 SP2 .

NB - Not bound

Example 21. Determination of the effect of bispecific antibodies in the form of IgG4-PAA on cell surface expression using MSD-CAT Original CD123 monovalent affinity

The monovalent affinity of the selected anti-CD123 bispecific antibodies for hCD123 SP1 and SP2 expressed on the cell surface was determined using MSD cell affinity technology (MSD-CAT) method. MSD-CAT was developed internally as a label-free method to determine affinity using intact cells in a high-throughput format. These experiments were conducted to assess the binding affinity and specificity of anti-CD123 candidates to cell surface human CD123 SP1 and CD123 SP2. The cell lines used were human pDisplay CD123SP1 and pDisplayCD123SP2. Negative control antibodies were used to test whether the bispecific Ab scaffold non-specifically binds to cells and distinguish between non-specific and specific binding to CD123. In order to measure the affinity of these interactions using the MSD-CAT method, a series of mixtures of anti-CD123 (800, 160, 32, and 6 pM) with fixed concentrations and cells (20 million to 1016 cells/mL) at different concentrations were prepared and the plate was allowed to reach equilibrium by rotating it at 4°C for 24 hours. These samples were prepared in DMEM Glutamax culture medium containing 0.05% azide, 1% BSA, 3mM EDTA. According to the reaction volume, cell density (cells/L) and Avogadro's constant, the number of receptors of (0.29-1.08)×106hCD123 SP1/cell and (0.57-1.5)×106hCD123 SP2/cell was converted to the M receptor concentration in the mixture. This resulted in a concentration range of 35nM to 0.5M for human CD123 SP1; and a concentration range of 49nM to 0.97pM for human CD123 SP2. After equilibrium, the plate was centrifuged at approximately 1000rpm for 5 minutes, and free anti-CD3 was detected in the supernatant. Free anti-CD123 in the mixture was detected by electrochemiluminescence (ECL) using a mesoscale discovery (MSD) reader. In order to detect the free anti-CD123 in the equilibrium mixture by electrochemiluminescence immunoassay (ECL), a detection plate was prepared. In order to prepare the detection plate (plate-bound antigen on SA-MSD plate), the MSD streptavidin standard plate was blocked with 50 μL/ wells of assay buffer (PBS (Life Sciences GIBCO 14190-136), 0.05% Tween 20, 0.2% BSA) for 5 minutes. The assay buffer was removed without washing, and the 0.7 μg/mL biotinylated antigen in the assay buffer of 50 μL/ wells was added to the MSD flat plate, and incubated overnight (about 16 hours at 4 ° C). After incubation overnight, the plate was blocked by adding 150 μL/ wells of assay buffer but without removing the coated antigen, and it was incubated for about 1 hour at ambient temperature, and washed 5 times with wash buffer (without the assay buffer of BSA). The supernatant of 50 μL/ wells in the sample plate was transferred to the plate coated with antigen, incubated for 60 minutes, then washed 3 times with wash buffer. The ruthenium-labeled detection antibody (anti-human H+L) of adding 50 μ L/ holes is incubated for 1 hour.After this, use wash buffer solution to wash plate, and add 150 μ L MSD to every hole and read buffer solution (reading buffer solution T 4X, R92TD-2, MSD). Immediately plate is placed in MSDSector Imager and reads luminescence level on plate reader. The ECL signal detected by MSD is represented with the free antibody % in the mixture, and uses the user-defined equation (derived from the law of mass action) analytical data introduced in Prism software to determine affinity. The result of MSD-CAT experiment is shown in Table 21.

Table 21. MSD-CAT affinity data showing binding of anti-CD123 molecules to cell surface human CD123 SP1 and human CD123 SP2. The data were fitted by nonlinear least squares analysis using a 1:1 binding model .

NA = Not Applicable; assay not performed

The MSD-CAT affinity of the bispecific Ab for cell surface CD123 SP1 was more than 6 times tighter than the SPR data for recombinant CD123 SP1; however, the affinity for cell surface CD123 SP2 was similar to that of recombinant CD123 SP2 (difference <2-fold). The difference in SPR and MSD-CAT affinity for CD123 SP1 is most likely due to the fact that its antigen is displayed on the cell surface compared to the recombinant antigen. MSD-CAT showed that the I3RB18-derived bispecific Ab (3978, 7955, 7959, 9958) was the tightest binder to cell surface human CD123 SP1 and human CD123 SP2 with pM affinity. The affinity of the I3RB18-derived Ab for cell surface CD123 SP1 and CD123 SP2 was approximately 10 times and approximately 5 times tighter than that of the I3RB2-derived bispecific Ab, respectively. The affinities of bispecific Abs in the same family are similar.

In summary, molecular interaction analyses using Biacore and MSD-CAT consistently showed that I3RB18-derived bispecific Abs bound more tightly to recombinant and cell surface human CD123 (SP1 and SP2) than I3RB2-derived bispecific Abs.

Example 22. Determination of the effect of bispecific antibodies in the form of IgG4-PAA on cell surface expression by flow cytometry CD123 monovalent affinity for antigen

Flow cytometry is used to measure the affinity values of several CD123×CD3 bispecific Abs to human T cells (BiologicalSpecialty, Colmar, USA) and cynomolgus monkey T cells (Zen Bio, Triangle Research Park, USA). This format involves the competitive binding of labeled anti-CD3mAbs of known affinity using a fixed concentration and increasing concentrations of unlabeled test Abs (Ashkenazi A et al., PNAS: 88: 10535, 1991). The anti-CD3mAb used is the CD3B146hu IgG1-AlaAla F405L antibody with an affinity value similar to that of SP34-2. The Kd of SP34-2 was determined using saturation binding, and examples of human and cynomolgus monkey T cell binding curves are shown in Figure 29. Figure 30 shows the competitive binding of labeled B146 and various concentrations of unlabeled CD123×CD3 bispecific antibodies obtained for human (Figure 30A) and cynomolgus monkey (Figure 30B) T cells. Comparable values were obtained for human and cynomolgus monkey T cells. Three CD3 affinity groups could be present in the samples analyzed: high (9-15 nM), medium (25-50 nM) and low (110-270 nM), which are summarized in Table 22.

Table 22. Affinity values (Kd) of CD123×CD3 bispecific antibodies for human or cynomolgus monkey T cells - using labeled Competition binding of B146 with increasing concentrations of unlabeled antibody

Example 23 Evaluation of IgG4-PAA CD123×CD3 Bispecific Ab in a Functional Cell Killing Assay

T cell-mediated cytotoxicity assays as described in Example 16 were used to evaluate the ability of CD123×CD3 bispecific Abs to lyse cells using T cells from two healthy donors. For these experiments, OCI-AML5, KG-1, and JIM3 cells were used. JIM3 is a myeloma tumor line that does not express CD123 and was used as a control. Cells were treated with the bispecific Abs for 48 hours. The effector cell: CFSE-labeled target cell ratio for this study was 5:1, and 2 mg/mL Fc blocker was added to block Fc function.

Shown are the results of T cell-mediated cell lysis of AML cell lines OCI-AML ( FIG. 31 ), KG-1 ( FIG. 32 ), and JIM3 ( FIG. 33 ) after incubation for 48 h at 37° C., 5% CO . MV4-11 and OCI-AML5 are CD123 expressing cell lines, and JIM3 has very low or no CD123 expression. The effector cell/target cell ratio for this study was 5:1. A 2 mg/mL aliquot of Fc blocker was added to block Fc function.

The results are similar to the cell killing experiments of CD123×CD3 bispecific Abs previously used in the form of IgG1-AA. When combined with anti-CD3 antibodies in a bispecific format, I3RB217 (I3RB18) and I3RB218 (I3RB2) antibodies are very effective in specifically killing CD123+ cells. Cell killing is specific to cells containing CD123, as demonstrated by the absence of an effect on JIM3 cells. In addition, the data show that there is a clear level between I3RB218 (based on I3RB2) and I3RB217 (based on I3RB18) bispecific antibodies, with I3RB217×CD3 bispecific Ab being more effective than I3RB218×CD3 bispecific Ab, consistent with previous cell killing data.

Example 24. Evaluation of bispecific antibodies in receptor heterodimerization assays

The ability of CD123 antibodies to prevent IL3-induced IL3Rα (CD123)/IL3Rβ heteromerization was assessed using the DiscoveRx receptor dimerization assay for IL3RA/CD131 (DiscoveRx 93-0969-C1). CD123 and CD131 were labeled with ProLink ^™ (PK) or enzyme receptor (EA). Upon IL3-induced activation, protein dimerization forms the IL3 receptor, forcing the two β-galactosidase components to complement each other and produce active enzymes. Active β-galactosidase generates a chemiluminescent signal in the presence of a substrate. As the antibody concentration increases, anti-CD123 antibodies or bispecific antibodies that show a reduced signal are positive for preventing heterodimerization.

Cells were tested for increased enzyme activity in the presence of IL-3 ligand using a detection reagent (DiscoveRx) according to the manufacturer's protocol. HEK293 IL3RA-PK/CSF2RB-EA cell lines were seeded in quadruplicate in 20 μL of assay medium on 384-well plates with 5,000 cells/well. Antibody stock solutions were serially diluted in 0.1% BSA/PBS to a high concentration of 10 μg/mL compound. High-dose antibody was serially diluted 1:3 with each of the 11 doses tested. 5 μL of the diluted antibody was added to the wells. The cells were incubated at 37°C for 1 hour. A 100 μg/mL recombinant human IL-3 stock solution was diluted so that 5 μL of a 60 ng/mL IL-3 dilution was added to each well. The final concentration of IL-3 used was 10 ng/mL. The cells were incubated at 37°C for an additional 6 hours. PathHunter rapid detection reagent containing lysis buffer and enzyme substrate was added to the cells, incubated at room temperature for 30 minutes, and read on an Envision luminometer. Data were analyzed using GraphPad Prism 6. Curves were fitted using a sigmoidal dose response (four parameters) with a variable slope and no constraints; fitting method = least squares (normal fit).

IgG4 PAA bispecific antibodies 8747 and 7959 and parental antibodies I3RB218 and I3RB217 were run in the assay. Two independent assays were performed in the presence of 10 ng/ml IL-3, and positive control CD123 antibody 7G3 was used as a comparison sample in the assay. Antibodies containing anti-CD123 arm I3RB18 sequences I3RB217 and 7959 (Figure 34 C and Figure 34 D) were able to prevent the formation of functional IL-3 receptors in the presence of IL-3 ligands. In this assay, antibodies containing anti-CD123 arms I3RB2, I3RB218 and 8747 (Figure 34 A and Figure 34 B) did not prevent the formation of functional IL-3 receptors. This is related to previous data, indicating that I3RB18 can inhibit downstream signaling associated with functional IL-3 receptors.

Example 24. Evaluation of several bispecific antibodies in the KG-1 tumor model

The efficacy of several CD123×CD3 bispecific Abs was evaluated in the KG-1 AML mouse model as previously described. The protocol for this study was identical to that of Examples 16 and 17, except that frozen, isolated peripheral blood mononuclear cell vials (100×10 cells per vial, catalog number PB009-3) purchased from HemaCare (Van Nuys, CA) were used to test the IgG4 bispecific antibodies. NSG mice were subcutaneously inoculated with KG-1 cells and then, when tumors were established (mean tumor volume = 135.7 +/- 4.7 mm ³ ), were transplanted intravenously with human PBMCs. Mice were then administered IgG4 PAA CD123×CD3 bispecific Abs with various affinities and corresponding control bispecific Abs at a dose range as described in Table 23.

Table 23. Dosing regimen for the third in vivo study

The results of the in vivo efficacy studies of various CD123×CD3 bispecific Abs are shown in Figures 35-42. Figures 35-38 show the efficacy of CD123×CD3IgG4-PAA bispecific Abs with various affinities and various doses in KG-1 human AML xenografts. In Figure 35, bispecific Abs with high affinity CD123 and CD3 arms had significant efficacy compared to PBS and control bispecific Abs from day 25 to day 36 after tumor implantation (p < 0.001). Before day 36 after tumor implantation (p < 0.01), 1 μg dose of bispecific Ab 9958 was significantly more effective than 0.1 μg dose of bispecific Ab 9958, as well as compared to 1 μg and 0.1 μg doses of bispecific Ab 7959. This suggests that high affinity CD123 and CD3 arms are necessary for significant efficacy in this model.

In Figure 36, a 10 μg dose of bispecific Ab 3978 had significant efficacy compared to PBS and control bispecific Ab at days 28 (p < 0.05) to 36 (p < 0.001) after tumor implantation, compared to a 1 μg dose of bispecific Ab 3978 at days 32 (p < 0.05) to 36 (p < 0.01), and compared to a 0.1 μg dose of bispecific Ab 3978 at days 32 (p < 0.01) to 36 (p < 0.001). There was a dose-dependent response for this bispecific Ab, indicating that high doses of the high-affinity CD123 arm can produce efficacy in this model.

In Figure 37, a 0.1 μg dose of bispecific Ab 8747 had significant efficacy compared to PBS and control bispecific Ab at days 32 to 36 post-tumor implantation (p < 0.001), and compared to 1 μg and 10 μg doses of bispecific Ab 8747 before day 36 post-tumor implantation (p < 0.001). This suggests that low doses of the high-affinity CD3 arm can produce efficacy in this model.

In Figure 38, bispecific Ab 8876 had no significant efficacy at any dose compared to PBS and the control bispecific Ab.

Figures 39-42 show the mean body weight changes over time for the treatment groups. As previously mentioned, weight loss is associated with the onset and severity of GVHD caused by activated T cells.

Animals treated with 0.1 μg of bispecific Ab 7959 and 1 μg of bispecific Ab 9958 lost more weight and experienced earlier onset of weight loss compared to animals treated with PBS, a control bispecific Ab, and other doses of bispecific Ab 7959 and bispecific Ab 9958 ( FIG. 39 ). This correlates with the significant anti-tumor efficacy observed at 1 μg of bispecific Ab 9958 ( FIG. 35 ).

Animals treated with the 10 μg dose of bispecific Ab 3978 lost more weight and experienced earlier onset of weight loss compared to those treated with PBS and the control bispecific Ab ( FIG40 ). Mice treated with the 1 μg and 0.1 μg doses experienced weight loss in a dose-dependent manner. The dose-dependent weight loss correlated with the dose-dependent anti-tumor efficacy shown in FIG36 .

Animals treated with the 0.1 μg dose of bispecific Ab 8747 experienced similar weight loss as the PBS-treated group, however, these mice began to regain weight on day 39 after tumor implantation ( FIG. 41 ). Animals treated with the 1 μg or 10 μg doses did not experience weight loss. The weight loss observed at the 0.1 μg dose correlated with the antitumor efficacy observed at this dose ( FIG. 37 ).

Animals treated with bispecific Ab 8876 experienced weight loss that was indistinguishable from that of mice treated with PBS or a control bispecific antibody ( FIG. 42 ), corresponding to the low anti-tumor efficacy exhibited by this bispecific antibody ( FIG. 38 ).

In summary, only in the presence of effector cells (T lymphocytes) does the CD123×CD3 bispecific Ab show consistent efficacy in the CD123-expressing human AML cell line KG-1. Only in the case of disease (KG-1 xenografts) is T cell expansion observed shortly after the dosing period. In addition, the bispecific efficacy is associated with the onset of GVHD as measured by weight loss, indicating the presence of activated T lymphocytes. In summary, these data show that the CD123×CD3 bispecific antibody has anti-tumor efficacy through the proposed mechanism of target cell and effector cell binding and T cell killing.

Example 26. In vivo PK study in mice

The test Ab preparation was prepared in phosphate buffered saline at 0.2 mg/mL. The concentration was confirmed using a Nanodrop spectrophotometer and then sterile filtered using a 0.2 micron syringe filter.

The transgenic animals used in these studies were derived from C57BL/6 mice. The endogenous mouse FcRnα gene of Tg32, licensed from the Jackson Laboratory (Bar Harbor), was knocked out and transgenic with the human FcRnα gene under the control of the native human gene promoter. Tg32 hemizygous mice refer to mice with hemizygous FcRn transgenes, which are obtained by mating homozygous transgenic mice with FcRnα knockout mice. A significant correlation was observed between the PK of human antibodies and the PK of primates with the Tg32 hemizygous mouse model, so this correlation was used in the following PK studies to assess Ab half-life. All mouse breeding was performed at SAGE Research Labs Boyertown, PA.

For this study, 48 female Tg32 hemizygous mice were used at 6 weeks of age and injected intravenously with the hIgG4-PAA bispecific Ab, with 5 mice per group. Retroorbital bleeding was performed at the same time points.

After collecting the samples, serum analysis was performed. The concentration of human IgG in the serum samples was determined by electrochemiluminescence immunoassay using a MESO Scale Discovery (MSD) format. Streptavidin MSD plates were coated overnight with 50 μL/well of a 2 μg/mL biotinylated F(ab')2 goat anti-hu IgG (H+L, Jackson batch number 109-066-08) Starting Block T20 (Thermo) solution. The plates were washed with PBS buffer and the samples were diluted in a Starting Block T20 solution of 10% mouse serum (Bioreclamations, NY). A standard curve for each test product was drawn on each plate, starting with 0.1 mg/mL and performing a 2-fold serial dilution. The plates were incubated on a shaker for 2-3 h at room temperature, washed, and then incubated on a shaker for 1 hour at room temperature with 2 μg/mL MSD-TAG (ruthenium-labeled anti-human IgG mAb, i.e., R10Z8E9, MSD). The plates were washed, 200 μL MSD read buffer (MSD) was added and read on an MSD Sector Imager 6000.

To determine whether the PK serum samples had significant immune titers that could affect the PK of the test samples, ELISA was performed on Maxisorb plates (Nunc) coated with 10 μg/mL of the corresponding test article and incubated overnight at 4°C. The serum samples were diluted in 1% BSA-PBS, shaken and incubated on the plates at room temperature for 2-3 hours. The captured antibodies were detected using horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch); 3,3',5,5'-tetramethylbenzidine (Fitzgerald) was then added for substrate development. The plates were read and spectrophotometer readings three times greater than the buffer or control serum values were considered positive. The immune titers were expressed as 1/serum dilution. No immune titers were observed (data not shown).

Finally, the pharmacokinetics of the molecule were determined. The terminal half-life (t1/2) calculation of the elimination phase of the PK study was determined using a 1-phase exponential decay model that was fitted by linear regression of the natural logarithm concentration versus time using Prism 5.01 version software (GraphPad Software). A two-phase model was excluded because the best fitting model was a 1-phase exponential decay model for each test article, as determined by the non-significance (p>0.05) of the additional sum of squares F test for most animals. The least squares nonlinear decay model was weighted with 1/fitting concentration. The half-life calculation of the elimination phase was determined using the formula t1/2=ln2/β, where β is the slope of the line fitted by the least squares regression analysis starting after the first dose.

In the PK studies described herein, the terminal half-life values of the antibodies were determined by averaging the t1/2 values calculated for each animal in the test group. Outliers in the study were identified as animals that exhibited a mouse anti-human IgG titer greater than 1 to 1000 approximately 7 days after dosing or exhibited an initial serum value that was more than 2-fold lower than the initial serum values of other mice in the group, likely due to incomplete dosing.

The human PK prediction from mouse data is based on the half-life difference observed for a group of 8 human IgG antibodies in huFcRn transgenic mice and humans. It is believed that the clearance of the IgG antibody is not significantly affected by target binding in mice or humans. Based on these analyses, it is assumed that the extrapolation of the effect of target binding on clearance is comparable in mice and humans, and it is estimated that the terminal half-life of human CD123×CD3 bispecific Ab is 2-4 times longer than the terminal half-life observed in huFcRn transgenic mice. Table 24 summarizes the mouse half-life values observed for various bispecific antibody variants and the corresponding predicted human values reflecting this hypothesis. Because the well-known human PK prediction method based on cross-species allometric changes has not been validated using mouse PK data, allometric changes are not used for prediction. The PK results are shown in Figure 43 as serum concentration changes over time. The PK curve shows that the serum concentration decreases linearly over the course of 28 days. The mouse half-life values estimated for all CD123×CD3 bispecific antibody Abs are similar between 5.2-6.6 days. Minimal immune titers (<1:40) were observed in all groups. Mouse PK data (including means +/- standard deviations) are summarized in Table 24 along with predicted human clearance and human half-life values. The human half-life prediction method assumes that target binding in humans is no greater than that in mice.

IgG4-PAA bispecific antibody Abs showed similar values between the mouse I3RB2 and I3RB18 groups. Mouse half-life calculations for the elimination phase were determined using a 1-phase exponential decay model fitted by linear regression of the natural log concentration versus time. Calculated half-life values for the eight bispecific Abs in Tg32 hemizygous mice were: 3978, 6.6 +/- 0.7 days; 7955, 5.2 +/- 0.4 days; 7959, 6.6 +/- 0.6 days; 9958, 6.4 +/- 0.7 days; 8876, 4.1 +/- 0.7 days; 4435, 5.4 +/- 1.0 days; 8747, 6.4 +/- 0.4 days; and 5466, 5.6 +/- 0.1 days. Human PK predictions from mouse data were based on observed half-life differences between huFcRn transgenic mice and humans. Based on these analyses, assuming that the effect of target binding on clearance is comparable in mice and humans, the terminal half-life of human CD123×CD3 bispecific antibodies is estimated to be 2- to 4-fold longer than that observed in huFcRn transgenic mice. Table 24 summarizes the observed mouse half-life values for the bispecific antibody variants and the corresponding predicted human values reflecting this assumption.

Table 24. Summary of PK of CD123×CD3 IgG4-PAA bispecific Abs

Results from mouse PK studies with the CD123×CD3 bispecific antibody demonstrated that the t1/2 values observed in Tg32 hemizygous mice were favorable compared to eight clinical antibodies analyzed in the same manner (Tam et al., MAb (2013) 5(3): 3987-405).

Claims (16)

1. A separated antibody or antigen-binding fragment thereof binding to CD123 SP2 and CD123 SP1, comprising a heavy chain and a light chain, having: a. Heavy chain CDR1 composed of the amino acid sequence of SEQ ID NO: 012, heavy chain CDR2 composed of the amino acid sequence of SEQ ID NO: 013, and heavy chain CDR3 composed of the amino acid sequence of SEQ ID NO: 014; light chain CDR1 composed of the amino acid sequence of SEQ ID NO: 015, light chain CDR2 composed of the amino acid sequence of SEQ ID NO: 016, and light chain CDR3 composed of the amino acid sequence of SEQ ID NO: 017; or b. Heavy chain CDR1 composed of the amino acid sequence of SEQ ID NO: 051, heavy chain CDR2 composed of the amino acid sequence of SEQ ID NO: 052, and heavy chain CDR3 composed of the amino acid sequence of SEQ ID NO: 053; light chain CDR1 composed of the amino acid sequence of SEQ ID NO: 024, light chain CDR2 composed of the amino acid sequence of SEQ ID NO: 025, and light chain CDR3 composed of the amino acid sequence of SEQ ID NO: 054. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 120, and the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 165. 3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 136, and the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 168. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment is an IgG1 or IgG4 isotype. 5. An isolated CD123 (IL3-Rα)×CD3 bispecific antibody or its antigen-binding fragment thereof, comprising a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second light chain (LC2), such that HC1 and LC1 pair to form an immune-specific binding site for CD123 (IL3-Rα), and HC2 and LC2 pair to form an immune-specific binding site for CD3 or its CD123 (IL3-Rα)×CD3 bispecific binding fragment, wherein: i) HC1 and LC1 each contain any of the following pairs: They are respectively a. SEQ ID NO:203 and SEQ ID NO:204, or b. SEQ ID NO:205 and SEQ ID NO:206, and ii) HC2 and LC2 each contain any of the following pairs: They are respectively a.SEQ ID NO:193 and SEQ ID NO:194, b.SEQ ID NO:195 and SEQ ID NO:196, c. SEQ ID NO:197 and SEQ ID NO:198, d. SEQ ID NO:199 and SEQ ID NO:200, or e. SEQ ID NO:201 and SEQ ID NO:202. 6. The bispecific antibody or its antigen-binding fragment according to claim 5, wherein HC1 comprises SEQ ID NO:203 and LC1 comprises SEQ ID NO:204, and HC2 comprises SEQ ID NO:193 and LC2 comprises SEQ ID NO:194. 7. The bispecific antibody or its antigen-binding fragment according to claim 5, wherein HC1 comprises SEQ ID NO:205 and LC1 comprises SEQ ID NO:206, and HC2 comprises SEQ ID NO:193 and LC2 comprises SEQ ID NO:194. 8. An isolated CD123 (IL3-Rα)×CD3 bispecific antibody or a CD123 (IL3-Rα)×CD3 bispecific binding fragment, comprising: a) First heavy chain (HC1); b) Second chain (HC2); c) The first light chain (LC1); and d) Second light chain (LC2), The first heavy chain (HC1) and the first light chain (LC1) pair to form a first antigen-binding site that specifically binds to CD123 (IL3-Rα), and the second heavy chain (HC2) and the second light chain (LC2) pair to form a second antigen-binding site that specifically binds to CD3. a. In the paired heavy and light chains that specifically bind to CD3, the second heavy chain (HC2) comprises SEQ ID NO: 184 and the second light chain (LC2) comprises SEQ ID NO: 190, and b. In the paired heavy and light chains that specifically bind to CD123, i. The first heavy chain (HC1) comprises SEQ ID NO:120 and the first light chain (LC1) comprises SEQ ID NO:165, or ii. The first heavy chain (HC1) contains SEQ ID NO:136 and the first light chain (LC1) contains SEQ ID NO:168. 9. An isolated cell expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8. 10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a medicament for treating cancer. 11. Use of the CD123 (IL3-Rα)×CD3 bispecific antibody or bispecific binding fragment according to any one of claims 5 to 8 in the preparation of a medicament for inhibiting the growth or proliferation of cancer cells. 12. Use of the CD123 (IL3-Rα)×CD3 bispecific antibody or bispecific binding fragment of any one of claims 5 to 8 in the preparation of a medicament for redirecting T cells to CD123-expressing cancer cells. 13. A pharmaceutical composition comprising a CD123 (IL3-Rα)×CD3 bispecific antibody or bispecific binding fragment according to any one of claims 5 to 8 and a pharmaceutically acceptable carrier. 14. An isolated synthetic polynucleotide encoding an antibody or antibody fragment according to any one of claims 5 to 8. 15. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8 and its packaging. 16. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, wherein the bispecific antibody or antigen-binding fragment thereof is immune-specifically bound to CD123 SP2 and CD123 SP1.