Anti-CD123 binding molecules and methods of use thereof

Abstract

The present disclosure provides antibodies or antigen-binding fragments or derivatives thereof that specifically bind to CD123. Also provided are polynucleotides encoding the antibodies or antigen-binding fragments or derivatives thereof, as well as vectors and host cells comprising the polynucleotides. The present disclosure further provides methods for producing and / or using the antibodies or antigen-binding fragments or derivatives thereof that specifically bind to CD123. TIFF2024508709000019.tif102165

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 150,488, filed February 17, 2021, and U.S. Provisional Patent Application Serial No. 63 / 249,455, filed September 28, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, generated on February 17, 2022, is named 038WO1-Sequence-Listing.txt and is 84,588 bytes in size. [Background technology]

[0003] background Acute myeloid leukemia (AML) is the leading cause of leukemia deaths in the United States, with over 20,000 new cases per year and a 5-year survival rate of less than 30%, decreasing to 10% for patients over 60 years of age (National Cancer Institute Surveillance, Epidemiology and End-Result Program (SEER) data, Oran and Weisdorf 2012, Haematologica 97(12)1916). There has been little progress in treating AML patients over the past 40 years, and current treatment options consist primarily of intensive chemotherapy and stem cell transplantation (Luppi et al. 2018, Cancers 10,429). Multiple approaches have been taken to target cell surface molecules on AML cells to instruct T cells to bind and kill AML cells. One such surface molecule is CD123 (also known as IL-3 receptor alpha chain or IL-3Rα), which is expressed on leukemic cells and leukemic stem cells (the cell type often responsible for disease relapse after therapy) in over 90% of AML patients (Kovtun et al. 2018, Blood Advances 2(8)848; Xie et al 2017, Blood Cancer Journal 7, e567). In addition, CD123 is highly expressed in patients with genetic mutations (such as FLT3) that are associated with very poor prognosis (Xie et al 2017, Blood Cancer Journal 7, e567).The amino acid sequences of the two human isoforms of CD123 are presented as SEQ ID NO: 14 (isoform, mature protein 1: approximately amino acids 23-378 of SEQ ID NO: 14) and SEQ ID NO: 15 (isoform, mature protein 2: approximately amino acids 23-300 of SEQ ID NO: 15), the cynomolgus monkey CD123 amino acid sequence is presented as SEQ ID NO: 16 (approximately 87% identical to human isoform 1, mature protein: approximately amino acids 23-378 of SEQ ID NO: 16), and the mouse CD123 amino acid sequence is presented as SEQ ID NO: 17 (approximately 30% identical to human isoform 1, mature protein: approximately amino acids 17-396 of SEQ ID NO: 17).

[0004] CD123 is a clinically validated target for several hematological malignancies, as evidenced by the FDA approval of recombinant IL-3 cytokine conjugated with diphtheria toxin for the treatment of blastic plasmacytoid dendritic cell neoplasms (Pemmaraju et al 2019, NEJM 380:1628). This and other CD123 targeting agents are being tested in preclinical and clinical trials. Early Phase 1 clinical trials have been conducted with CD123xCD3 bispecific antibodies by Xencor (XmAb14045 - IgG-based), Macrogenics (Flotetuzumab - DART), and Jansen (JNJ-63709178 - duobody). Although early signs of clinical efficacy have been reported in some of these patients, severe cytokine release syndrome and some patient deaths have also been observed with this class of bispecific drugs (Ravandi et al. 2018 Blood 132:763; Jacobs et al. 2018, Blood 132:2738; Uy et al. 2018, Blood 132:764). Cytokine release syndrome (or CRS) is characterized by fever, hypotension, blood clotting abnormalities, and capillary leakage, which can be life-threatening, and such findings have also been associated with other T cell-mediated approaches, including CAR-T and BiTE (Teachley et al. 2016, Cancer Discovery 6(6)664; Hay et al. 2017, Blood 130(21)2295). These adverse safety events associated with cytokine release tend to be of the nature of dose-limiting toxicities of IgG-based bispecific antibodies that bind CD3 and present a challenge to the safe, efficient and tolerated administration of such agents and potentially to the ability to optimize the efficacy of these therapeutics due to the resulting dosing limitations.

[0005] Antibodies and antibody-like molecules capable of multimerization (such as IgA and IgM antibodies) have emerged as promising drug candidates, allowing for improved specificity, improved avidity, and the ability to bind to multiple targets, for example in the fields of immuno-oncology and infectious diseases. See, for example, U.S. Patent Nos. 9,951,134 (Patent Document 1), 9,938,347 (Patent Document 2), 10,570,191 (Patent Document 3), 10,604,559 (Patent Document 4), 10,618,978 (Patent Document 5), 10,787,520 (Patent Document 6), and 10,899,835 (Patent Document 7), and U.S. Patent Application Publication Nos. 2003-2009023 and 2009-2011364. Please refer to Patent Publication Nos. 2019-0185570 (Patent Document 8), 2019-0330374 (Patent Document 9), 2019-0338041 (Patent Document 10), 2019-0330360 (Patent Document 11) and 2019-0338040 (Patent Document 12) (the contents of which are incorporated by reference in their entirety into this specification).

[0006] There remains a need to target AML cells expressing CD123 and induce T cell-mediated killing of those cells while minimizing CRS. We evaluated whether targeting CD123 with the CD3 bispecific IgM technology of the present invention would not only effectively target CD123-expressing AML tumor cells for T cell-mediated cytotoxicity, but also produce responses with a favorable safety profile against cytokine release syndrome, which is sometimes severe in patients treated with IgG-based CD123xCD3 bispecific antibodies. In addition, the high avidity binding of IgM antibodies may enable the CD123xCD3 bispecific IgM of the present invention to target tumor cells expressing CD123 at relatively low levels of cell surface expression compared to IgG-based bispecific antibodies. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 9,951,134 [Patent Document 2] U.S. Patent No. 9,938,347 [Patent Document 3] U.S. Patent No. 10,570,191 [Patent Document 4] U.S. Patent No. 10,604,559 [Patent Document 5] U.S. Patent No. 10,618,978 [Patent Document 6] U.S. Patent No. 10,787,520 [Patent Document 7] U.S. Patent No. 10,899,835 [Patent Document 8] U.S. Patent Application Publication No. 2019-0185570 [Patent Document 9] U.S. Patent Application Publication No. 2019-0330374 [Patent Document 10] U.S. Patent Application Publication No. 2019-0338041 [Patent Document 11] U.S. Patent Application Publication No. 2019-0330360 [Patent Document 12] U.S. Patent Application Publication No. 2019-0338040 [Non-patent literature]

[0008] [Non-Patent Document 1] Oran and Weisdorf 2012, Haematologica 97(12)1916 [Non-Patent Document 2] Luppi et al.2018,Cancers 10,429 [Non-Patent Document 3] Kovtun et al.2018,Blood Advances 2(8)848 [Non-Patent Document 4] Xie et al 2017,Blood Cancer Journal 7,e567 [Non-Patent Document 5] Pemmaraju et al 2019,NEJM 380:1628 [Non-Patent Document 6] Ravandi et al 2018 Blood 132:763 [Non-Patent Document 7] Jacobs et al 2018,Blood 132:2738 [Non-Patent Document 8] Uy et al 2018,Blood 132:764 [Non-Patent Document 9] Teachley et al 2016, Cancer Discovery 6(6)664 [Non-Patent Document 10] Hay et al 2017,Blood 130(21)2295 Summary of the Invention

[0009] overview Provided herein is an antibody or antigen-binding fragment or derivative thereof comprising an antigen-binding domain that specifically binds to CD123, the antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL comprise the amino acid sequences SEQ ID NO:76 and SEQ ID NO:79, SEQ ID NO:77 and SEQ ID NO:79, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:83, SEQ ID NO:81 and SEQ ID NO:83, and SEQ ID NO:82 and SEQ ID NO:83. In some embodiments, VH and VL comprise the amino acid sequences SEQ ID NO:76 and SEQ ID NO:79, respectively.

[0010] In some embodiments, the antibody or fragment or derivative thereof is an Fv fragment, a single chain Fv fragment (scFv) or a disulfide-linked Fv fragment (sdFv).

[0011] In some embodiments, the antibody or fragment or derivative thereof is a multimeric antibody comprising 5, 6 or 2 bivalent binding units and 10, 12 or 4 antigen binding domains, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 antigen binding domains specifically bind to CD123, each binding unit comprising two heavy chains, each of the two heavy chains comprising an IgM or IgA constant region, or a multimerized fragment or variant thereof, and wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 heavy chain constant regions of the multimeric antibody are associated with a copy of a VH. In some embodiments, the antibody or fragment or derivative thereof comprises a bivalent binding unit comprising two antigen binding domains, at least one antigen binding domain specifically binds to CD123, the binding unit comprises two heavy chains, each of the two heavy chains comprising a heavy chain constant region, or a fragment or variant thereof, and at least one heavy chain constant region, or a fragment or variant thereof, of the binding unit is fused to a copy of the VH.

[0012] In some embodiments, the antibody or fragment or derivative thereof comprises a single bivalent binding unit comprising two antigen binding domains, at least one antigen binding domain specifically binds to CD123, the binding unit comprises two heavy chains, each of the two heavy chains comprising a heavy chain constant region or a fragment or variant thereof, and at least one heavy chain constant region or a fragment or variant thereof of the binding unit is associated with a copy of a VH. In some embodiments, the heavy chain comprises an IgG heavy chain constant region or a fragment or variant thereof.

[0013] In some embodiments, the antibody or fragment or derivative thereof is a multimeric antibody comprising 2, 5 or 6 bivalent binding units and 4, 10 or 12 antigen binding domains, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 antigen binding domains specifically bind to CD123, each binding unit comprising two heavy chains, each of the two heavy chains comprising an IgA or IgM constant region, or a multimerized fragment or variant thereof, and wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 heavy chain constant regions of the multimeric antibody are fused to a copy of the VH. In some embodiments, the antibody or fragment or derivative thereof is a multimeric antibody comprising 2, 5 or 6 bivalent binding units and 4, 10 or 12 antigen binding domains, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 antigen binding domains specifically bind to CD123, each binding unit comprising two heavy chains, each of the two heavy chains comprising an IgA or IgM constant region, or a multimerized fragment or variant thereof, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 heavy chain constant regions of the multimeric antibody are fused to a copy of VH. In some embodiments, each heavy chain constant region, or a multimerized fragment or variant thereof, is associated with a copy of VH. In some embodiments, each heavy chain constant region, or a fragment or variant thereof, is fused to a copy of VH.

[0014] In some embodiments, each binding unit further comprises two light chains, each of the two light chains comprising a light chain constant region or a fragment or variant thereof, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 light chain constant regions or fragments or variants thereof are associated with a copy of the VL. In some embodiments, each binding unit further comprises two light chains, each of the two light chains comprising a light chain constant region or a fragment or variant thereof, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 light chain constant regions or fragments or variants thereof are fused to a copy of the VL. In some embodiments, each light chain constant region or fragment or variant thereof is associated with a copy of the VL. In some embodiments, each light chain constant region or fragment or variant thereof is fused to a copy of the VL.

[0015] In some embodiments, the antibody or fragment or derivative thereof comprises a complete antibody, a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0016] In some embodiments, the antibody or fragment or derivative thereof is a dimer and comprises two bivalent IgA binding units, and a J chain or fragment or variant thereof, each binding unit comprising a Cα3 domain and an α tailpiece (αtp) domain. In some embodiments, each of the IgA heavy chain constant regions or fragments or variants thereof further comprises a Cα1 domain, a Cα2 domain, an IgA hinge region, or any combination thereof.

[0017] In some embodiments, the antibody or fragment or derivative thereof is hexamer or pentamer and comprises 5 or 6 bivalent IgM binding units, each binding unit comprising a Cμ4 domain and a μ tailpiece (μtp) domain, or a fragment or variant thereof. In some embodiments, each of the IgM heavy chain constant regions or multimerized fragments or variants thereof further comprises a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof. In some embodiments, the IgA heavy chain constant region or fragment or variant thereof is an IgA1 heavy chain constant region or fragment or variant thereof. In some embodiments, the IgA heavy chain constant region or fragment or variant thereof comprises SEQ ID NO: 3. In some embodiments, the IgA heavy chain constant region or fragment or variant thereof is an IgA2 heavy chain constant region or fragment or variant thereof. In some embodiments, the IgA heavy chain constant region or fragment or variant thereof comprises SEQ ID NO: 4.

[0018] In some embodiments, each IgM heavy chain constant region is a human IgM constant region or a multimerized variant or fragment thereof comprising the amino acid sequence SEQ ID NO:1, SEQ ID NO:2, or a variant or fragment thereof. In some embodiments, the antibody or fragment or derivative thereof comprises a variant human IgM constant region, and the antibody or fragment or derivative thereof has reduced CDC activity compared to an antibody or antigen-binding fragment or derivative thereof comprising an IgM heavy chain constant region comprising the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, each variant human IgM constant region comprises an amino acid substitution corresponding to position P311 in SEQ ID NO:1 or SEQ ID NO:2, an amino acid substitution corresponding to position P313 in SEQ ID NO:1 or SEQ ID NO:2, or an amino acid substitution corresponding to positions P311 and P313 in SEQ ID NO:1 or SEQ ID NO:2.

[0019] In some embodiments, each IgM heavy chain constant region or multimerized variant or fragment thereof is a variant human IgM constant region having one or more half-life-altering single amino acid substitutions, deletions or insertions compared to a reference IgM heavy chain constant region that is identical to the variant IgM heavy chain constant region except for the one or more half-life-altering single amino acid substitutions, deletions or insertions; the antibody or fragment or derivative thereof exhibits increased serum half-life upon administration to a subject animal compared to an antibody or antigen-binding fragment or derivative thereof comprising the reference IgM heavy chain constant region administered in the same manner to the same animal species. In some embodiments, the variant IgM heavy chain constant region comprises a half-life-altering substitution of an amino acid at one or more amino acid positions corresponding to amino acids E345A, S401A, E402A or E403A of SEQ ID NO: 1 or SEQ ID NO: 2 of the wild-type human IgM constant region.

[0020] In some embodiments, each of the IgM heavy chain constant regions or multimerized variants or fragments thereof comprises one or more glycosylation substitutions corresponding to N46, N209, N272 or N440 of SEQ ID NO:1 or SEQ ID NO:2, wherein the one or more glycosylation substitutions prevent asparagine (N)-linked glycosylation.

[0021] In some embodiments, the antibody or fragment or derivative thereof is a pentamer and further comprises a J chain or a fragment or variant thereof.

[0022] In some embodiments, the J chain or fragment or variant thereof is a mature human J chain comprising the amino acid sequence SEQ ID NO:7, or a fragment or variant thereof. In some embodiments, the antibody or fragment or derivative thereof comprises a variant J chain or fragment thereof, wherein the variant J chain comprises an amino acid substitution at an amino acid position corresponding to amino acid Y102 of the wild-type mature human J chain of SEQ ID NO:7, and an IgM antibody comprising the variant J chain exhibits increased serum half-life upon administration to an animal compared to a reference IgM antibody that is identical except for one or more single amino acid substitutions, deletions or insertions in the J chain and administered in the same manner to the same animal species. In some embodiments, the amino acid corresponding to Y102 of SEQ ID NO:7 is substituted with alanine (A). In some embodiments, the J chain comprises the amino acid sequence SEQ ID NO:8.

[0023] In some embodiments, the J chain or fragment or variant thereof is a modified J chain further comprising a heterologous moiety, which is fused or conjugated to the J chain or fragment or variant thereof. In some embodiments, the heterologous moiety is a polypeptide fused to the J chain or fragment or variant thereof. In some embodiments, the heterologous polypeptide is fused to the J chain or fragment or variant thereof via a peptide linker comprising at least 5 amino acids and no more than 25 amino acids. In some embodiments, the heterologous polypeptide is fused to the N-terminus of the J chain or fragment or variant thereof, to the C-terminus of the J chain or fragment or variant thereof, or to both the N-terminus and C-terminus of the J chain or fragment or variant thereof, and the heterologous polypeptides fused to both the N-terminus and C-terminus can be the same or different. In some embodiments, the polypeptide fused to the J chain or fragment or variant thereof is an antigen-binding domain of an antibody or a subunit thereof. In some embodiments, the antigen-binding domain of the antibody comprises an scFv fragment.

[0024] In some embodiments, the heterologous polypeptide binds to CD3. In some embodiments, the antibody or fragment or derivative thereof may bind to CD3. In some embodiments, the antibody or fragment or derivative thereof comprises a CD3-binding VH and VL sequence disclosed herein. In some embodiments, the antigen-binding domain of the antibody binds to CD3 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, where VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are the amino acid sequences SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, ... , SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49; SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57; SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65; or SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73. In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively. In some embodiments, the antigen-binding domain of the antibody comprises VH and VL amino acid sequences that are at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:18 and SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:46, SEQ ID NO:50 and SEQ ID NO:54, SEQ ID NO:58 and SEQ ID NO:62, or SEQ ID NO:66 and SEQ ID NO:70, respectively.In some embodiments, the antigen-binding domain of the antibody comprises the VH and VL amino acid sequences SEQ ID NO:18 and SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:46, SEQ ID NO:50 and SEQ ID NO:54, SEQ ID NO:58 and SEQ ID NO:62, or SEQ ID NO:66 and SEQ ID NO:70. In some embodiments, the antigen-binding domain of the antibody comprises the VH and VL amino acid sequences SEQ ID NO:26 and SEQ ID NO:30, respectively.

[0025] In some embodiments, the antibody or fragment or derivative thereof further comprises a secretory component or a fragment or variant thereof.

[0026] In some embodiments, the antibody or fragment or derivative thereof is multispecific, hi some embodiments, the antibody or fragment or derivative thereof is bispecific.

[0027] In some embodiments, the antibody or fragment or derivative thereof may specifically bind to human CD 123. In some embodiments, the antibody or fragment or derivative thereof specifically binds to human CD 123 with an affinity characterized by a dissociation constant KD of less than or equal to 500 nM, 100 nM, 50.0 nM, 40.0 nM, 30.0 nM, 20.0 nM, 10.0 nM, 9.0 nM, 8.0 nM, 7.0 nM, 6.0 nM, 5.0 nM, 4.0 nM, 3.0 nM, 2.0 nM, 1.0 nM, 0.50 nM, 0.10 nM, 0.050 nM, 0.01 nM, 0.005 nM, or 0.001 nM.

[0028] Further provided herein is a composition comprising the antibody or a fragment or derivative thereof disclosed herein. Further provided herein is a polynucleotide comprising a nucleic acid sequence encoding the antibody or a fragment or derivative thereof disclosed herein, or a subunit thereof. Further provided herein is a vector comprising the polynucleotide disclosed herein. Further provided herein is a host cell comprising the vector disclosed herein.

[0029] Further provided herein is a method of producing an antibody, or a fragment or derivative thereof, disclosed herein, comprising culturing a host cell disclosed herein and recovering the antibody, or a fragment or derivative thereof.

[0030] Further provided herein is a method of treating cancer, comprising administering to a subject in need of treatment an effective amount of an antibody or a fragment or derivative thereof disclosed herein. In some embodiments, the subject is a human. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is acute myeloid leukemia (AML). [Brief description of the drawings]

[0031] [Figure 1] Figures 1A-1D show a comparison of parental and humanized anti-CD123 VH (Figures 1A, 1C) and VL (Figures 1B, 1D) sequences based on the 4D9Q (Figures 1A, 1B) or 4NWT (Figures 1C, 1D) frameworks: h32716-VH: SEQ ID NO: 74, h32716-VL: SEQ ID NO: 75, h32716-VH1: SEQ ID NO: 76, h32716-VH2: SEQ ID NO: 77, h32716-VH3: SEQ ID NO: 78, h32716-VL1: SEQ ID NO: 79, h32716-VH4: SEQ ID NO: 80, h32716-VH5: SEQ ID NO: 81, h32716-VH6: SEQ ID NO: 82, and h32716-VL2: SEQ ID NO: 83. [Figure 2A] Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Figure 2B]Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Figure 2C] Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Figure 2D] Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Figure 2E] Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Figure 2F]Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Figure 2G] Figures 2A-2G show Octet measurements of the association and dissociation of 32716 IgG (Figure 2A), h32716-1-1 IgG (Figure 2B), h32716-2-1 IgG (Figure 2C), h32716-3-1 IgG (Figure 2D), h32716-4-2 IgG (Figure 2E), h32716-5-2 IgG (Figure 2F), and h32716-6-2 IgG (Figure 2G). The vertical line represents the start of the dissociation phase of the procedure. [Diagram 3] 1 shows the binding of anti-CD123 IgG antibodies at different concentrations to human CD123 on MV411 cells as measured by flow cytometry. [Figure 4A] 4A-4C show Octet measurements of the association and dissociation of 32716 IgM (FIG. 4A), h32716-1-1 IgM (FIG. 4B), and h32716-4-2 IgM (FIG. 4C). The vertical line represents the start of the dissociation phase of the procedure. [Figure 4B] 4A-4C show Octet measurements of the association and dissociation of 32716 IgM (FIG. 4A), h32716-1-1 IgM (FIG. 4B), and h32716-4-2 IgM (FIG. 4C). The vertical line represents the start of the dissociation phase of the procedure. [Figure 4C] 4A-4C show Octet measurements of the association and dissociation of 32716 IgM (FIG. 4A), h32716-1-1 IgM (FIG. 4B), and h32716-4-2 IgM (FIG. 4C). The vertical line represents the start of the dissociation phase of the procedure. [Diagram 5] 5A-5B show the binding of anti-CD123xCD3 IgM antibodies at different concentrations to human CD123 (FIG. 5A) or human CD3ε (FIG. 5B) as measured by ELISA. [Figure 6] Binding of anti-CD123xCD3 IgM antibodies at different concentrations to human CD123 on MV411 cells as measured by flow cytometry. [Figure 7] 7A-7B show tumor viability 72 hours after treatment with anti-CD123×CD3 IgM antibodies in a pan T-cell dependent cytotoxicity (TDCC) assay against KG1a (FIG. 7A) and MV411 cells (FIG. 7B). [Figure 8] 1 shows the percentage of high molecular weight aggregates (%HMW) in solutions of h32716-1-1 IgM, h32716-4-2 IgM, and 32716 IgM antibodies under various conditions. [Figure 9] Figure 9A shows the average tumor volume over time over 75 days for mice treated with vehicle, anti-CD123xCD3 IgG#1 treatment, h32716-1-1 IgM antibody at 5 mg / kg, and h32716-1-1 IgM antibody at 15 mg / kg. Figure 9B shows the individual tumor volumes at day 75 for mice treated with vehicle, anti-CD123xCD3 IgG#1 treatment, h32716-1-1 IgM antibody at 5 mg / kg, and h32716-1-1 IgM antibody at 15 mg / kg. [Figure 10] Figures 10A-10D show individual tumor volumes over time over 75 days for vehicle (Figure 10A), anti-CD123xCD3 IgG#1 (Figure 10B), 5 mg / kg h32716-1-1 IgM antibody (Figure 10C), and 15 mg / kg h32716-1-1 IgM antibody (Figure 10D). [Figure 11] Figure 1 shows in vitro colony formation of AML cells from four different donors following treatment with h32716-1-1 IgM antibody. Calculated values ​​are normalized to the solvent control. [Figure 12] 1 shows serum concentrations of antibodies over time following a single dose of 5 mg / kg h32716, h32716-4-2, or h32716-1-1 in a mouse model. [Figure 13]Figure 13A shows the number of viable tumor cells 48 hours after TDCC using either CD123xCD3 IgG#1 or h32716-1-1. Figures 13B-13D show the amount of IFNγ (Figure 13B), IL-6 (Figure 13C), and IL-10 (Figure 13D) detected in the medium 48 hours after TDCC using either CD123xCD3 IgG#1 or h32716-1-1. [Figure 14] Figure 14A shows the number of viable tumor cells 72 hours after TDCC using either CD123xCD3 IgG#1 or h32716-1-1. Figures 14B-14D show the amount of IFNγ (Figure 14B), IL-6 (Figure 14C), and IL-10 (Figure 14D) detected in the medium 72 hours after TDCC using either CD123xCD3 IgG#1 or h32716-1-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "a binding molecule" is understood to refer to one or more binding molecules. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0033] Furthermore, "and / or", when used herein, is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or", when used herein in a phrase such as "A and / or B", is intended to encompass A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or", when used herein in a phrase such as "A, B, and / or C", is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0034] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary for many of the terms used in this disclosure.

[0035] Units, prefixes, and symbols are expressed in the form accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various embodiments or embodiments of the present disclosure, which may be incorporated by reference to this specification in its entirety. Thus, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0036] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or other terms used to refer to chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, and derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. A polypeptide may be generated in any manner, including by chemical synthesis.

[0037] Polypeptides, as disclosed herein, can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Polypeptides can have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides that do not retain a defined three-dimensional structure, but rather can adopt a number of different conformations, are referred to as unfolded. As used herein, the term glycoprotein refers to a protein bound to at least one carbohydrate moiety that is attached to the protein via an oxygen- or nitrogen-containing side chain of an amino acid, such as serine or asparagine.

[0038] By "isolated" polypeptide, or a fragment, variant, or derivative thereof, is intended a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, and are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0039] As used herein, the term "non-naturally occurring polypeptide" or any grammatical variation thereof is a qualified definition that expressly excludes, but only excludes, forms that are or that can be determined or construed by a judicial or administrative agency or judicial body to be "naturally occurring" polypeptides.

[0040] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative," and "analog," as disclosed herein, encompass any polypeptide that retains at least some of the properties of the corresponding native antibody or polypeptide, such as specifically binding to an antigen. Fragments of polypeptides include, for example, proteolytic fragments, and deletion fragments, as well as specific antibody fragments discussed elsewhere herein. For example, variants of polypeptides include the fragments described above, and polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants may not be naturally occurring. Non-naturally occurring variants may be produced using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. Variant polypeptides may be referred to herein as "polypeptide analogs." As used herein, the "derivative" of a polypeptide may also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Peptides containing one or more derivatives of the 20 standard amino acids are also included as "derivatives". For example, 4-hydroxyproline can be substituted for proline, 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine. Homoserine can be substituted for serine. Ornithine can be substituted for lysine.

[0041] "Conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid that has a similar side chain. Families of amino acids with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the replacement of tyrosine with phenylalanine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abolish the binding of a polypeptide or antibody containing the amino acid sequence to an antigen to which the polypeptide or antibody binds. Methods for identifying conservative substitutions of nucleotides and amino acids that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0042] The term "polynucleotide" is intended to encompass single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct (e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA)). Polynucleotides can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds such as those found in peptide nucleic acids (PNA)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid segments (e.g., DNA or RNA fragments) present in a polynucleotide.

[0043] An "isolated" nucleic acid or polynucleotide is intended to mean any form of nucleic acid or polynucleotide that is separated from its natural environment. For example, a gel-purified polynucleotide or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered "isolated". Also, a polynucleotide segment (e.g., a PCR product) that has been engineered to have a restriction site for cloning would be considered "isolated". Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in non-native solutions (such as buffers or salt solutions). Isolated RNA molecules include in vivo or in vitro RNA transcripts of a polynucleotide, where the transcript is not found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid may be or may include regulatory elements (such as a promoter, a ribosome binding site, or a transcription terminator).

[0044] As used herein, the term "non-naturally occurring polynucleotide" or any grammatical variation thereof is a qualified definition that expressly excludes, but only excludes, forms that are or that can be determined or construed by a judicial or administrative agency or judicial body to be "naturally occurring" nucleic acids or polynucleotides.

[0045] As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into amino acids but can be considered part of a coding region. However, any adjacent sequences (e.g., promoters, ribosome binding sites, transcription terminators, introns, and the like) are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector can contain a single coding region or can include two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can include a heterologous coding region, either fused or unfused to another coding region. Heterologous coding regions include, but are not limited to, those that encode specialized elements or motifs, such as secretory signal peptides or heterologous functional domains.

[0046] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may usually include a promoter and / or other transcriptional or translational control elements operably associated with one or more coding regions. Operable association is when a coding region for a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in such a manner that the gene product is expressed under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of an mRNA encoding a desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA in a given cell. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

[0047] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter in conjunction with intron-A), Simian Virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes (such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin), and other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0048] Likewise, a variety of translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

[0049] In other embodiments, the polynucleotide may be in the form of RNA, such as messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0050] The coding regions of the polynucleotides and nucleic acids may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those skilled in the art are aware that polypeptides secreted by vertebrate cells may have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the completed peptide or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide (e.g., immunoglobulin heavy or light chain signal peptide) or a functional derivative thereof that retains the ability to direct the secretion of a polypeptide operably associated with the sequence is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0051] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to a binding target (e.g., an epitope or antigenic determinant). As further described herein, a binding molecule may contain one or more "antigen-binding domains" as described herein. Non-limiting examples of binding molecules are antibodies or antibody-like molecules that retain antigen-specific binding, or antibody-like molecules or fragments thereof as described in detail herein that retain antigen-specific binding. In certain embodiments, a "binding molecule" includes an antibody or antibody-like molecule as described in detail herein.

[0052] As used herein, the term "binding domain" or "antigen-binding domain" (which may be used interchangeably) refers to a region or fragment of a binding molecule (e.g., an antibody or antibody-like molecule) necessary and sufficient to specifically bind to a binding target (e.g., an epitope). For example, an "Fv" (e.g., an antibody heavy chain variable region and a light chain variable region, either as two separate polypeptide subunits or as a single chain) is considered to be a "binding domain". Other antigen-binding domains include, but are not limited to, the heavy chain variable region (VHH) of an antibody from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a scaffold (e.g., a fibronectin scaffold). A "binding molecule", e.g., an "antibody" or antibody-like molecule described herein, may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or in some cases more "antigen-binding domains". As used herein, a "binding unit-associated antigen-binding domain" refers to an antigen-binding domain that is a portion of an antibody heavy chain and / or an antibody light chain. The term "J chain-associated antigen-binding domain" refers to an antigen-binding domain associated with a modified J chain described herein (e.g., an scFv fused to a wild-type human J chain, or a functional fragment or variant thereof).

[0053] The terms "antibody" and "immunoglobulin" may be used interchangeably herein. An antibody provided in this disclosure must specifically bind to an antigen, i.e., it contains at least a heavy chain variable domain (for camelid species) or at least heavy and light chain variable domains. Basic immunoglobulin structure in vertebrate systems is relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise specified, the term "antibody" encompasses everything from small antigen-binding fragments of antibodies to full size antibodies (e.g., an IgG antibody comprising two IgG heavy chains or fragments thereof and two complete light chains, an IgA antibody comprising two, four or eight heavy chains or multimerized fragments thereof and two, four or eight light chains, optionally comprising a J chain and / or secretory component, or an IgM antibody comprising ten or twelve complete heavy chains and ten or twelve complete light chains, optionally comprising a J chain or a functional fragment or variant thereof).

[0054] The term "immunoglobulin" includes various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will recognize that heavy chains are classified, for example, as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within those (e.g., γ1-γ4 or α1-α2). The nature of the chain determines the "isotype" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the present disclosure, and thus are within the scope of the present disclosure.

[0055] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a κ or λ light chain. Generally, light and heavy chains are covalently linked to each other by disulfide bonds, and when the immunoglobulin is expressed, for example, by a hybridoma, a B cell, or a genetically engineered host cell, the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent linkages. In the heavy chains, the amino acid sequences run from the N-terminus at the bifurcated ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of a certain antibody (e.g., an IgG antibody) comprises two heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a "Y" structure, also referred to herein as the "H2L2" structure or "binding unit."

[0056] The term "binding unit" is used herein to refer to a portion of a binding molecule (e.g., an antibody or antibody-like molecule) that corresponds to a standard "H2L2" immunoglobulin structure (e.g., two heavy chains or fragments thereof and two light chains or fragments thereof). In certain embodiments, a binding unit may correspond to two heavy chains, e.g., in a camelid antibody. In certain embodiments, the terms "binding molecule" and "binding unit" are equivalent, e.g., when the binding molecule is a bivalent IgG antibody or antibody-like molecule. In other embodiments, the binding molecule comprises more than one "binding unit", e.g., when the binding molecule is a multimer (e.g., a dimeric or tetrameric IgA antibody or IgA-like antibody, a pentameric IgM antibody or IgM-like antibody, or a hexameric IgM antibody or IgM-like antibody). Two out of four in the case of an IgA dimer or tetramer, and five or six in the case of an IgM pentamer or hexamer, respectively. A binding unit need not comprise a heavy and light chain of a full-length antibody, but will typically be bivalent, i.e., comprise two "antigen-binding domains" as defined above. As used herein, certain binding molecules provided in the present disclosure are "dimers" or "tetramers" and comprise two or four bivalent binding units comprising an IgA heavy chain constant region or a multimerized fragment thereof. Certain binding molecules provided in the present disclosure are "pentamers" or "hexamers" and comprise five or six bivalent binding units comprising an IgM heavy chain constant region or a multimerized fragment thereof. A binding molecule (e.g., an antibody or antibody-like molecule) that comprises two or more (e.g., two, four, five, or six) binding units is referred to herein as "multimeric."

[0057] The term "J chain", as used herein, refers to a J chain associated with a pentameric IgM or dimeric or tetrameric IgA antibody of any animal species, any functional fragment thereof, derivative thereof, and / or variant thereof, including the mature human J chain whose amino acid sequence is presented as SEQ ID NO: 7. A variety of J chain variants and modified J chain derivatives are disclosed herein. One of skill in the art will recognize that a "functional fragment" or "functional variant" encompasses fragments and variants thereof that can associate with an IgM heavy chain constant region to form a pentameric IgM antibody (or alternatively, can associate with an IgA heavy chain constant region to form a dimeric or tetrameric IgA antibody).

[0058] The term "modified J chain" is used herein to refer to a derivative of a native sequence J chain polypeptide that includes a heterologous moiety (e.g., a heterologous polypeptide, e.g., an exogenous binding domain introduced into the native sequence). Introduction can be accomplished by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety, or attachment via a peptide or chemical linker. The term "modified human J chain" encompasses, without limitation, a human J chain comprising the amino acid sequence of SEQ ID NO:7, or a functional fragment or functional variant thereof, that has been modified by the introduction of a heterologous moiety (e.g., a heterologous polypeptide, e.g., an exogenous binding domain). In certain embodiments, the heterologous moiety does not interfere with efficient polymerization of IgM into pentamers, or IgA into dimers or tetramers, and binding of such polymers to targets. Exemplary modified J chains can be found, for example, in U.S. Pat. Nos. 9,951,134, 10,975,147, 10,400,038, and 10,618,978, and U.S. Patent Application Publication No. US-2019-0185570, each of which is incorporated by reference in its entirety.

[0059] As used herein, the terms "IgM-derived binding molecule", "IgM-like antibody", "IgM-like binding unit" or "IgM-like heavy chain constant region" refer to a binding molecule, antibody, binding unit, or heavy chain constant region derived from a variant antibody that still retains the structural portion of the IgM heavy chain necessary to confer the ability to bind to antigen and form multimers (i.e., hexamers) or associate with a J chain to form a pentamer. An IgM-like antibody or IgM-derived binding molecule typically contains at least the Cμ4 and μ tailpiece (μtp) domains of the IgM constant region and its antigen-binding domain or subunit, but may contain heavy chain constant region domains from other antibody isotypes (e.g., IgG) from the same or different species. An IgM-like antibody or IgM-derived binding molecule may also be an antibody fragment in which one or more constant regions have been deleted, so long as the IgM-like antibody is capable of binding to antigen and forming hexamers and / or pentamers. Thus, an IgM-like antibody or an IgM-derived binding molecule may be, for example, a hybrid IgM / IgG antibody, or may be a "multimerized fragment" of an IgM antibody.

[0060] As used herein, the terms "IgA-derived binding molecule", "IgA-like antibody", "IgA-like binding unit" or "IgA-like heavy chain constant region" refer to a binding molecule, antibody, binding unit, or heavy chain constant region derived from a variant antibody that still retains the structural portion of the IgA heavy chain necessary to confer the ability to associate with the J chain to bind antigen and form multimers (i.e., dimers or tetramers). An IgA-like antibody or IgA-derived binding molecule typically contains the IgA constant region and at least the Cα3 and α tailpiece (αtp) domains of its antigen-binding domain or subunit, but may contain heavy chain constant region domains from other antibody isotypes (e.g., IgG) from the same or different species. An IgA-like antibody or IgA-derived binding molecule may also be an antibody fragment in which one or more constant regions are deleted, so long as the IgA-like antibody is capable of associating with the J chain to bind antigen and form dimers. Thus, an IgA-like antibody or an IgA-derived binding molecule may be, for example, a hybrid IgA / IgG antibody, or may be a "multimerized fragment" of an IgA antibody.

[0061] The terms "valency", "monovalent", "bivalent", "multivalent" and grammatical equivalents refer to the number of antigen-binding domains in a given binding molecule (e.g., an antibody or antibody-like molecule) or a given binding unit. Thus, the terms "bivalent", "tetravalent", and "hexavalent" with reference to a given binding molecule (e.g., an IgM antibody, an IgM-like antibody, or a multimerized fragment thereof) refer to the presence of two antigen-binding domains, four antigen-binding domains, and six antigen-binding domains, respectively. A typical IgM antibody, or an IgM-like antibody or IgM-derived binding molecule, each of which is bivalent, may have a valency of 10 or 12. A bivalent or multivalent binding molecule (e.g., an antibody or antibody-like molecule) may be monospecific (i.e., all of the antigen-binding domains are the same) or bispecific or multispecific (e.g., where two or more antigen-binding domains are different, e.g., binding to different epitopes on the same antigen or binding to completely different antigens).

[0062] The term "epitope" includes any molecular determinant capable of specific binding to the antigen-binding domain of an antibody or antibody-like molecule. In certain embodiments, an epitope can include chemically active surface groups of a molecule (such as amino acids, sugar side chains, phosphoryl, or sulfonyl) and, in certain embodiments, can have three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the area of ​​a target that is bound by the antigen-binding domain of an antibody.

[0063] The term "target" is used in the broadest sense to include any substance that can be bound by a binding molecule (e.g., an antibody or antibody-like molecule). A target can be, for example, a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule. Furthermore, a "target" can be, for example, a cell, an organ, or an organism that contains an epitope that can be bound by a binding molecule (e.g., an antibody or antibody-like molecule).

[0064] Both the light and heavy chains of an antibody or antibody-like molecule are classified into regions of structural and functional homology. The terms "constant" and "variable" are used functionally, but refer to the specific structure of the molecule. The variable regions of both the light (VL) and heavy (VH) chains determine antigen recognition and specificity. Conversely, the constant domains (e.g. CH1, CH2, CH3, or CH4) of the light (CL) and heavy chains confer biological properties (ability to multimerize, secretion, transplacental mobility, Fc receptor binding, complement binding, etc.). By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding region or amino-terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 (or CH4 for IgM) and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0065] A "full-length IgM antibody heavy chain" is a polypeptide that comprises, from N-terminal to C-terminal direction, an antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CM1 or Cμ1), antibody heavy chain constant domain 2 (CM2 or Cμ2), antibody heavy chain constant domain 3 (CM3 or Cμ3), and antibody heavy chain constant domain 4 (CM4 or Cμ4), which may include the tailpiece.

[0066] As mentioned above, the variable region(s) form the antigen-binding domain of an antibody or antibody-like molecule, allowing it to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and VH domain, or an antigen-binding subset of complementarity determining regions (CDRs) of a binding molecule (e.g., an antibody or antibody-like molecule) combine to form the antigen-binding domain. More specifically, the antigen-binding domain can be defined by three CDRs on each of the VH and VL chains. Certain antibodies or antibody-like molecules form larger structures. For example, an IgA heavy chain can form a molecule that includes two or four H2L2 binding units and a J chain covalently linked by disulfide bonds, which can further associate with secretory components, and an IgM heavy chain can form a pentameric or hexameric molecule that includes five or six H2L2 binding units and optionally a J chain covalently linked by disulfide bonds.

[0067] The six "complementarity determining regions" or "CDRs" present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain are called "framework" regions and show less inter-molecular variation. The framework regions adopt a predominantly β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that provides for the positioning of the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to the epitope on the target antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids which constitute the CDRs and framework regions, respectively, have been defined in a variety of different ways and can be readily identified by one of skill in the art for any given heavy or light chain variable region (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated by reference in their entireties).

[0068] In cases where there is more than one definition of a term used and / or accepted within the art, the definition of the term used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described, for example, by Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of amino acids when compared to each other. Nevertheless, application of either definition (or other definitions known to those of skill in the art) to refer to the CDRs of an antibody or variants thereof is intended to be within the scope of the term defined and used herein, unless otherwise indicated. The appropriate amino acids that cover the CDRs defined by each of the above cited references are set forth below in Table 1 for comparison. The exact amino acid numbers that cover a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which amino acids constitute a particular CDR, given the variable region amino acid sequence of an antibody.

[0069] [Table 1]

[0070] Antibody variable domains can also be analyzed, for example, using the IMGT information system (imgt_dot_cines_dot_fr / ) (IMGT® / V-Quest) to identify variable region segments containing the CDRs (see, for example, Brochet et al., Nucl. Acids Res., 36:W503-508, 2008).

[0071] Kabat et al. also defined a numbering system for variable and constant region sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). However, for all amino acid sequences in this disclosure, sequential numbering is used unless the use of the Kabat numbering system is explicitly indicated.

[0072] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et al. "Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, β-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, α-2 Macroglobulins, and Other Related Proteins," USDept. of Health and Human Services (1991). The IgM constant region can be numbered sequentially (i.e., amino acid #1 starts with the first amino acid of the constant region) or by use of the Kabat numbering scheme. A comparison of the sequential numbering of the two alleles of the human IgM constant region (presented herein as SEQ ID NO:1 (allele IGHM*03) and SEQ ID NO:2 (allele IGHM*04)) and the numbering according to the Kabat system is given below. The underlined amino acid residues are not taken into account in the Kabat system (double underlined below). TIFF2024508709000003.tif6128 can be serine (S) (SEQ ID NO:1) or glycine (G) (SEQ ID NO:2). TIFF2024508709000004.tif89165

[0073] Binding molecules (e.g., antibodies, antibody-like molecules, antigen-binding fragments, variants or derivatives thereof, and / or multimerized fragments thereof) include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments produced by a Fab expression library). scFv molecules are described, for example, in U.S. Patent No. 5,892,019.

[0074] "Specifically bind" generally means that a binding molecule (e.g., an antibody or antibody-like molecule) binds to an epitope through its antigen-binding domain, and the binding causes some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule (e.g., an antibody or antibody-like molecule) is said to "specifically bind" to an epitope if it binds to the epitope through its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular binding molecule binds to a particular epitope. For example, binding molecule "A" may be considered to have a higher specificity for a given epitope than binding molecule "B", or binding molecule "A" may be said to bind epitope "C" with higher specificity than related epitope "D".

[0075] A binding molecule (e.g., an antibody or antibody-like molecule disclosed herein) may be administered at a concentration of 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , 10 -3 seconds -1 , 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds-1 , 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 , or 10 -7 seconds -1 Binding to a target antigen can be expressed as follows:

[0076] A binding molecule (e.g., an antibody or antibody-like molecule disclosed herein) may be administered at up to 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , 5×10 4 M -1 seconds -1 , 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , 5×10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 It can be expressed as binding to a target antigen with an on rate (k(on)) equal to or greater than this.

[0077] A binding molecule (e.g., an antibody or antibody-like molecule) is said to competitively inhibit the binding of a reference antibody or antibody-like molecule to a given epitope if it preferentially binds to that epitope to such an extent that it blocks the binding of a reference antibody or antigen-binding fragment to that epitope to some extent. Competitive inhibition can be determined by any method known in the art (e.g., competitive ELISA assay or OCTET assay). A binding molecule can be said to competitively inhibit the binding of a reference antibody or antibody-like molecule to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0078] As used herein, the term "affinity" refers to a measure of the strength of binding of one or more antigen-binding domains (e.g., of an antibody or antibody-like molecule) to an individual epitope. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., Harlow, pages 29-34. Avidity is related to both the affinity of an individual antigen-binding domain in a population to a specific epitope, as well as the valency of the immunoglobulin and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure (such as a polymer) would be one of high avidity. Similarly, the interaction of a multimeric antibody with a valency of 4, 8, 10 or 12 with a population of specific epitopes would be one of high avidity. The interaction between a bivalent monoclonal antibody and a receptor that is present at high density on a cell surface will also be of high avidity.

[0079] Binding molecules (e.g., antibodies disclosed herein, or fragments, variants, or derivatives thereof) can also be described or defined in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule (e.g., an antibody, or fragments, variants, or derivatives thereof) specific for one antigen to react with a second antigen, and is a measure of the correlation between two different antigenic substances. Thus, a binding molecule is cross-reactive if it binds to an epitope other than the epitope that induced the formation of the binding molecule. Cross-reactive epitopes generally contain many of the same complementary structural features as the induced epitope, and in some cases may actually fit better than the original one.

[0080] Binding molecules (e.g., antibodies, or fragments, variants, or derivatives thereof) may also be described or specified in terms of their binding affinity to an antigen. For example, a binding molecule may have a binding affinity of 5×10 -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15 It can bind to the antigen with a dissociation constant, or KD, equal to or less than M.

[0081] The "antigen-binding fragments of binding molecules or antibodies" (including single chain antibodies or other antigen-binding domains) provided herein may be present alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component. Also included are antigen-binding fragments that may include any combination of one or more of hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component, and sufficient variable region(s) to bind antigen. The binding molecules (e.g., antibodies or antibody-like molecules) may be from any animal origin, including birds and mammals. The antibodies may be, for example, human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable regions may be of condricthoid origin (e.g., from sharks). As used herein, a "human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, and includes antibodies isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins, which may or may not express endogenous immunoglobulins in some instances, as described below and in, for example, U.S. Patent No. 5,939,598 by Kucherlapati et al. In accordance with embodiments of the present disclosure, the IgM, or IgM-like antibodies or IgM-derived binding molecules provided herein may include antigen-binding fragments of antibodies (e.g., scFvs), so long as the IgM or IgM-like antibodies are capable of forming multimers (e.g., hexamers or pentamers).

[0082] As used herein, the term "heavy chain subunit" includes an amino acid sequence derived from an immunoglobulin heavy chain. A binding molecule (e.g., an antibody or antibody-like molecule) that includes a heavy chain subunit can include a VH domain and one or more of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, a μ tailpiece (μtp), or a variant or fragment thereof. For example, a binding molecule (e.g., an antibody, antibody-like molecule, or a fragment, variant, or derivative thereof) can include, in addition to a VH domain, any combination of a CH1 domain, a hinge, a CH2 domain, a CH3 domain, a CH4 domain, or a μ tailpiece (μtp) of one or more antibody isotypes and / or species, without limitation. In certain embodiments, a binding molecule (e.g., an antibody, antibody-like molecule, or a fragment, variant, or derivative thereof) may include, in addition to a VH domain, one or more of a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a μ tailpiece (μtp) domain, and a J chain (for IgM), or one or more of a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, an α tailpiece (αtp) domain, and a J chain (for IgA). Additionally, a binding molecule (e.g., an antibody or antibody-like molecule) provided in this disclosure may lack certain constant region portions (e.g., all or a portion of a CH1 domain, a hinge, a CH2 domain, or a CH3 domain). These domains (e.g., heavy chain subunits) may be modified such that they vary in amino acid sequence from the original immunoglobulin molecule. According to embodiments of the present disclosure, an IgM or IgM-like antibody provided herein comprises a sufficient portion of an IgM heavy chain constant region (e.g., an IgM heavy chain constant region comprises a "multimerization fragment" of an IgM heavy chain constant region) to enable the IgM or IgM-like antibody to form a multimer (e.g., a hexamer or pentamer).

[0083] As used herein, the term "light chain subunit" includes amino acid sequences derived from an immunoglobulin light chain. A light chain subunit contains at least a VL and may further contain a CL (e.g., a Cκ or Cλ) domain.

[0084] Binding molecules (e.g., antibodies, antibody-like molecules, antigen-binding fragments, variants or derivatives thereof, or multimerized fragments thereof) may be described or defined in terms of the epitope(s) or portion(s) of an antigen that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant." A target antigen may contain a single epitope or more than one epitope, and may contain any number of epitopes depending on the size, conformation, and type of antigen.

[0085] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain in IgG, IgA, and IgD heavy chains. This hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0086] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group.

[0087] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified) is obtained from a second species. In some embodiments, the target binding region or site is from a non-human source (e.g., mouse or primate) and the constant region is human.

[0088] The term "multispecific antibody" or "bispecific antibody" refers to an antibody or antibody-like molecule that has antigen-binding domains directed to two or more different epitopes within a single antibody molecule. In addition to the canonical antibody structure, other binding molecules can be constructed with two binding specificities.

[0089] As used herein, the term "engineered antibody" refers to an antibody in which the variable domains of either the heavy and light chains or both are altered by partial replacement of one or more amino acids at least in either the CDRs or framework regions. In certain embodiments, the entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody. The replacement CDRs can be from an antibody of the same class or possibly the same subclass as the antibody from which the framework region is derived, but the CDRs can also be from an antibody of a different class (e.g., from an antibody from a different species). An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are grafted into the framework regions of a human heavy or light chain is referred to herein as a "humanized antibody". In certain embodiments, not all CDRs are replaced by complete CDRs from the donor variable region, but the antigen-binding capacity of the donor can still be transferred to the variable domains of the recipient. Exemplary methods of humanization are described in U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.

[0090] As used herein, the term "engineered" includes the processing of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).

[0091] As used herein, the terms "linked", "fused" or "fusion" or other grammatical equivalents may be used interchangeably. These terms refer to the joining together of two or more elements or components by any means, including chemical conjugation or recombinant means. "In-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (these segments are not normally so joined in nature). Although the reading frame is thus made continuous through the fused segments, the segments may be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding the CDRs of immunoglobulin variable regions may be fused in-frame, but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a continuous polypeptide. The term "associated," and its grammatical equivalents, refers to the interaction of two or more elements that function together, may be linked or fused, yet may be in close proximity without being linked in any particular way, e.g., interacting in trans.

[0092] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide in the amino-terminal to carboxyl-terminal direction, where amino acids adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is "amino-terminal" or "N-terminal" to another portion of the polypeptide is the portion that occurs earlier in the contiguous polypeptide chain. Similarly, a portion of a polypeptide that is "carboxy-terminal" or "C-terminal" to another portion of the polypeptide is the portion that occurs later in the contiguous polypeptide chain. For example, in a typical antibody, the variable domain is "N-terminal" to the constant region, and the constant region is "C-terminal" to the variable domain.

[0093] The term "expression" as used herein refers to the process by which a gene produces a biochemical (e.g., a polypeptide). The process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockdown, and both transient and stable expression. The process includes, but is not limited to, transcription of a gene into RNA (e.g., messenger RNA (mRNA)) and translation of such mRNA into a polypeptide(s). If the final desired product is a biochemical, expression includes the production of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid (e.g., messenger RNA) produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids), association with other protein subunits, proteolytic cleavage, and the like.

[0094] Terms such as "treat" or "treatment" or "treating" or "alleviate" or "alleviating" refer to therapeutic treatments that cure, slow, alleviate symptoms, and / or halt the progression or slowing of an existing diagnosed disease, pathological condition, or disorder. Terms such as "prevent", "prevention", "avoid", "inhibit", and the like refer to prophylactic or preventative measures that prevent the onset of an undiagnosed targeted disease, pathological condition, or disorder. Thus, a "subject in need of treatment" may include a subject that already has a disease, condition, or disorder. The term "subject in need of prevention" refers to a subject that is prone to having a disease, condition, or disorder, and a subject in whom a disease, condition, or disorder is to be prevented.

[0095] As used herein, the term "serum half-life" or "plasma half-life" refers to the time (e.g., in minutes, hours or days) it takes for the serum or plasma concentration of a protein or drug (such as a binding molecule, antibody or antibody-like molecule described herein) to decrease by 50% after administration. Two half-lives may be described: alpha half-life, α half-life, or t 1 / 2 α is the rate of decline in plasma concentration due to the process of drug redistribution from the central compartment (e.g., blood in the case of intravenous delivery) to the peripheral compartment (e.g., tissue or organ), and is also known as the beta half-life, β half-life, or t 1 / 2 β is the rate of decline due to excretory or metabolic processes.

[0096] As used herein, the term "area under the plasma drug concentration-time curve" or "AUC" reflects the actual body exposure to a drug after administration of a dose of the drug and is expressed in mg*h / L. This area under the curve is measured from time 0 (t0) to infinity (∞) and depends on the rate of elimination of the drug from the body and the dose administered.

[0097] As used herein, the term "mean residence time" or "MRT" refers to the average length of time that a drug remains in the body.

[0098] "Subject" or "individual" or "animal" or "patient" or "mammal" means any mammalian subject for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, farm animals, livestock, and zoo animals, sport animals, or pets (dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.).

[0099] Anti-CD123 antigen-binding domain Provided herein is an antibody or antigen-binding fragment or derivative thereof comprising an antigen-binding domain that specifically binds to CD123, wherein the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL comprise the amino acid sequences SEQ ID NO:76 and SEQ ID NO:79, SEQ ID NO:77 and SEQ ID NO:79, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:83, SEQ ID NO:81 and SEQ ID NO:83, and SEQ ID NO:82 and SEQ ID NO:83, respectively.

[0100] In certain embodiments, the antigen-binding domain provided above is an Fv fragment, such as a single chain Fv fragment (scFv) or a disulfide-linked Fv fragment (sdFv). In certain embodiments, the antigen-binding domain provided above is an scFv.

[0101] In certain embodiments, the antigen-binding domains provided above are comprised in an antibody or antibody-like molecule, as described elsewhere herein. In some embodiments, the antigen-binding domains provided above are comprised in an antibody or antibody-like molecule, and the antibody or antibody-like molecule is multispecific, e.g., bispecific, trispecific, or tetraspecific. In some embodiments, the multispecific antibody or antibody-like molecule provided herein specifically binds to CD123 and to a target on an effector cell, e.g., CD16 or CD3.

[0102] In certain embodiments, the antibody or antibody-like molecule comprises a bivalent binding unit comprising two antigen-binding domains, at least one of which specifically binds to CD123. According to this embodiment, the binding unit comprises two heavy chains, each of which comprises a heavy chain constant region or a fragment or variant thereof, and at least one of the heavy chain constant regions or a fragment or variant thereof of the binding unit is associated, e.g., fused, with a copy of the VH provided of the antigen-binding domain. In certain embodiments, both of the heavy chain constant regions or a fragment or variant thereof of the binding unit are associated, e.g., fused, with a copy of the VH provided of the antigen-binding domain. In certain embodiments, the heavy chain comprises an IgG heavy chain constant region or a fragment or variant thereof. Various IgG heavy chain constant regions and fragments or variants thereof are known, including those described in Kang, et al., 2019, Experimental & Molecular Medicine, 51:1-9; Brezski, et al., 2016, Current Opinion in Immunology, 40:62-69; Okazaki, et al., 2004, Journal of Molecular Biology, 336(5):1239-1249; Kang, et al., 2019, Front. Immunol., 10(562):1-11; and Saxena, et al., 2016, Front. Immunol., 7(580):1-11. In certain embodiments, the bivalent binding unit further comprises two light chains, each of which comprises a light chain constant region or a fragment or variant thereof. In certain embodiments, at least one light chain constant region is fused to a copy of the provided VL of the antigen binding domain. In certain embodiments, the bivalent binding unit comprises a complete antibody, such as a complete IgG antibody or a F(ab')2 fragment. In certain embodiments, both light chain constant regions or fragments or variants thereof of the binding unit are fused to a copy of the provided VL of the antigen binding domain. In certain embodiments, the bivalent binding unit comprises a complete antibody, such as a complete IgG antibody or a F(ab')2 fragment.In certain embodiments, the bivalent binding unit comprises a complete antibody, such as a complete IgG heavy chain constant region, hi certain embodiments, the bivalent binding unit is a human IgG antibody, a fragment or derivative thereof.

[0103] In certain embodiments, the provided antigen-binding domains are comprised in a multimeric antibody or antibody-like molecule comprising 2, 5 or 6 bivalent binding units, and the antibody comprises 4, 8, 10 or 12 antigen-binding domains. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigen-binding domains specifically bind to CD123. As provided herein, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigen-binding domains comprise the VH and VL amino acid sequences provided herein. According to these embodiments, each binding unit comprises two heavy chains, each of the two heavy chains comprising an IgA or IgM constant region, or a multimerized fragment or variant thereof, and at least one of the heavy chain constant regions of the binding unit is associated, e.g., fused, with a copy of the provided VH of the provided antigen-binding domain. In certain embodiments, the multimeric antibody or antibody-like molecule is a human antibody.

[0104] In certain embodiments, the provided multimeric antibody or antibody-like molecule is a dimer or tetramer and comprises two bivalent IgA binding units and a J chain or a functional fragment or variant thereof, each binding unit comprising two IgA heavy chain constant regions, such as IgA1 or IgA2 heavy chain constant regions, or multimerized fragments or variants thereof. In certain embodiments, the dimeric or tetrameric antibody or antibody-like molecule may further comprise a secretory component or a fragment or variant thereof. In certain embodiments, each of the IgA heavy chain constant regions or multimerized fragments or variants thereof comprises a Cα3 domain and an α tailpiece (αtp) domain, and may further comprise a Cα1 domain, a Cα2 domain, an IgA hinge region, or any combination thereof.

[0105] In certain embodiments, the provided multimeric antibody or antibody-like molecule is a hexamer or pentamer and comprises 5 or 6 bivalent IgM binding units, each of which comprises two IgM heavy chain constant regions or multimerized fragments or variants thereof. In certain embodiments, each of the IgM heavy chain constant regions or multimerized fragments or variants thereof comprises a Cμ4 domain and a μ tailpiece (μtp) domain or a fragment or variant thereof, and may further comprise a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof. In certain embodiments, the multimeric antibody or antibody-like molecule is a pentamer and further comprises a J chain or a functional fragment or functional variant thereof. In certain embodiments, each binding unit further comprises two light chains, each of which comprises a light chain constant region or a fragment or variant thereof, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 light chain constant regions are fused to the provided VL copy of the antigen binding domain. In certain embodiments, the multimeric antibody or antibody-like molecule is a human antibody.

[0106] The antibodies or antibody-like molecules provided herein can be multispecific in certain embodiments.

[0107] In certain embodiments, the antigen-binding domains provided, or antibodies or fragments or derivatives or antibody-like molecules comprising the antigen-binding domain, can specifically bind to human CD 123. In certain embodiments, the antigen-binding domains provided, or antibodies or fragments or derivatives comprising the antigen-binding domain, bind to CD 123 with an affinity characterized by a dissociation constant KD of 500 nM, 100 nM, 50.0 nM, 40.0 nM, 30.0 nM, 20.0 nM, 10.0 nM, 9.0 nM, 8.0 nM, 7.0 nM, 6.0 nM, 5.0 nM, 4.0 nM, 3.0 nM, 2.0 nM, 1.0 nM, 0.50 nM, 0.10 nM, 0.050 nM, 0.01 nM, 0.005 nM or 0.001 nM or less, and CD 123 is human CD 123.

[0108] IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules IgM is the first immunoglobulin produced by B cells in response to stimulation by an antigen, and is naturally present in serum at approximately 1.5 mg / ml with a half-life of about 5 days. IgM is a pentameric or hexameric molecule, and therefore contains 5 or 6 binding units. An IgM binding unit typically contains two light chains and two heavy chains. The IgG heavy chain constant region contains three heavy chain constant domains (CH1, CH2, and CH3), while the heavy chain (μ) constant region of IgM contains an additional fourth constant domain (CH4), including a C-terminal μ "tailpiece" (μtp). Although multiple human alleles exist, the human IgM constant region typically contains the amino acid sequence SEQ ID NO: 1 (identical to the IMGT allele IGHM*03, e.g., GenBank accession number pir||S37768) or SEQ ID NO: 2 (identical to the IMGT allele IGHM*04, e.g., GenBank accession number sp|P01871.4). The human Cμ1 region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO:1 or SEQ ID NO:2, the human Cμ2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO:1 or SEQ ID NO:2, the human Cμ3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO:1 or SEQ ID NO:2, the Cμ4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO:1 or SEQ ID NO:2, and the tailpiece ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO:1 or SEQ ID NO:2.

[0109] Other forms of the human IgM constant region with minor sequence variations exist, including, but not limited to, GenBank Accession Nos. CAB37838.1 and pir||MHHU. Amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO:1 or SEQ ID NO:2 can be incorporated into alternative human IgM sequences described and claimed elsewhere in this disclosure, and into IgM constant region amino acid sequences of other species as well.

[0110] Each IgM heavy chain constant region associates with an antigen-binding domain (eg, an scFv) or a subunit of an antigen-binding domain (eg, a VH region).

[0111] The five IgM binding units may be complexed with an additional small polypeptide chain (J chain), or a functional fragment, variant, or derivative thereof, to form a pentameric IgM or IgM-like antibody. The precursor form of the human J chain is shown as SEQ ID NO:1. The signal peptide (underlined) extends from amino acid 1 to about amino acid 22 of SEQ ID NO:1, and the mature human J chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO:1. The mature human J chain has the amino acid sequence SEQ ID NO:2.

[0112] Exemplary variants and modified J chains are provided elsewhere herein. In the absence of a J chain, an IgM or IgM-like antibody typically assembles into a hexamer that includes six binding units and an antigen-binding domain associated with up to 12 binding units. In the presence of a J chain, an IgM or IgM-like antibody typically assembles into a pentamer that includes five binding units and an antigen-binding domain associated with up to 10 binding units or more, if the J chain is a modified J chain that includes one or more heterologous polypeptides that may be, for example, additional J chain-associated antigen-binding domain(s). The assembly of five or six IgM binding units into a pentameric or hexameric IgM or IgM-like antibody is believed to involve interactions between the Cμ4 and μ tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Thus, the pentameric or hexameric IgM antibody constant region or antibody-like molecule provided in the present disclosure typically comprises at least the Cμ4 and / or μ tailpiece domains. A "multimerized fragment" of an IgM heavy chain constant region thus comprises at least the Cμ4 domain and the μtp domain. The IgM heavy chain constant region may additionally comprise a Cμ3 domain or fragment thereof, a Cμ2 domain or fragment thereof, and / or a Cμ1 domain or fragment thereof. In certain embodiments, a binding molecule (e.g., an IgM antibody or IgM-like antibody provided herein) may comprise the complete IgM heavy (μ) chain constant domain (e.g., SEQ ID NO: 1, SEQ ID NO: 2), or a multimerized variant, derivative, or analog thereof, e.g., as provided herein.

[0113] In certain embodiments, the present disclosure provides a pentameric IgM or IgM-like antibody comprising five bivalent binding units, each of which comprises two IgM heavy chain constant regions, or multimerized fragments or variants thereof, each of which is associated with an antigen-binding domain or subunit of an antigen-binding domain. In certain embodiments, the two IgM heavy chain constant regions are human heavy chain constant regions.

[0114] When the IgM or IgM-like antibody provided herein is a pentamer, the IgM or IgM-like antibody typically further comprises a J chain, or a functional fragment or variant thereof. In some embodiments, the J chain is a modified J chain that comprises a heterologous moiety, such as a J chain-associated antigen-binding domain. In certain embodiments, the J chain-associated antigen-binding domain specifically binds to an immune effector cell (e.g., a CD8+ cytotoxic T cell or a NK cell). In certain embodiments, the modified J chain comprises one or more heterologous moieties (e.g., an immunostimulatory agent) added thereto. In certain embodiments, the J chain can be mutated to affect (e.g., improve) the serum half-life of the IgM or IgM-like antibody provided herein, as discussed elsewhere in this disclosure. In certain embodiments, the J chain can be mutated to affect glycosylation, as discussed elsewhere in this disclosure.

[0115] In some embodiments, the IgM or IgM-like antibodies provided herein are hexamers and comprise six bivalent binding units, in some embodiments, each binding unit comprises two IgM heavy chain constant regions, or multimerized fragments or variants thereof.

[0116] An IgM heavy chain constant region may comprise one or more of a Cμ1 domain or a fragment or variant thereof, a Cμ2 domain or a fragment or variant thereof, a Cμ3 domain or a fragment or variant thereof, a Cμ4 domain or a fragment or variant thereof, and / or a μ tailpiece (μtp) or a fragment or variant thereof, provided that the constant region can, for example, associate with a second IgM constant region to form a binding unit with one, two or more antigen binding domain(s) and / or associate with other binding units (and in the case of pentamers, a J chain) to form a hexamer or pentamer to provide a desired function in an IgM or IgM-like antibody. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a Cμ4 domain or a fragment or variant thereof, a μ tailpiece (μtp) or a fragment or variant thereof, or a combination of a Cμ4 domain and a μtp, or a fragment or variant thereof. In certain embodiments, the two IgM heavy chain constant regions, or fragments or variants thereof, in an individual binding unit each further comprises a Cμ3 domain, or fragment or variant thereof, a Cμ2 domain, or fragment or variant thereof, a Cμ1 domain, or fragment or variant thereof, or any combination thereof.

[0117] In some embodiments, the binding unit of an IgM or IgM-like antibody comprises two light chains. In some embodiments, the binding unit of an IgM or IgM-like antibody comprises fragments of two light chains. In some embodiments, the light chain is a κ light chain. In some embodiments, the light chain is a λ light chain. In some embodiments, the light chain is a hybrid κ and λ light chain. In some embodiments, each binding unit comprises two immunoglobulin light chains, each comprising a VL positioned amino terminal to an immunoglobulin light chain constant region.

[0118] IgM ANTIBODIES, IgM-LIKE ANTIBODIES, AND IgM-DERIVED BINDING MOLECULES WITH IMPROVED SERUM HALF-LIFE Certain IgM-derived multimeric binding molecules, such as antibodies or antibody-like molecules, provided herein can be modified to have improved serum half-life. Exemplary IgM heavy chain constant region mutations that can improve the serum half-life of IgM-derived binding molecules are disclosed in U.S. Patent No. 10,899,835, which is incorporated herein by reference in its entirety. For example, the variant IgM heavy chain constant region of the IgM-derived binding molecules provided herein can include amino acid substitutions at amino acid positions corresponding to amino acids S401, E402, E403, R344, and / or E345 of wild-type human IgM constant region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). "Amino acids corresponding to amino acids S401, E402, E403, R344, and / or E345 of the wild-type human IgM constant region" means amino acids in the sequence of an IgM constant region of any species that are homologous to S401, E402, E403, R344, and / or E345 in the human IgM constant region. In certain embodiments, amino acids corresponding to S401, E402, E403, R344, and / or E345 of SEQ ID NO:1 or SEQ ID NO:2 can be substituted with any amino acid (e.g., alanine).

[0119] IgM ANTIBODIES, IgM-LIKE ANTIBODIES, AND IgM-DERIVED BINDING MOLECULES WITH REDUCED CDC ACTIVITY Certain IgM-derived multimeric binding molecules, e.g., antibodies or antibody-like molecules, provided herein can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity on cells in the presence of complement, compared to a reference IgM antibody or IgM-like antibody with a corresponding reference human IgM constant region that is identical except for mutations that confer reduced CDC activity. These CDC mutations can also be combined with any of the mutations provided herein that block N-linked glycosylation and / or confer increased serum half-life. A "corresponding reference human IgM constant region" refers to a human IgM constant region or portion thereof (e.g., Cμ3 domain) that is identical to the variant IgM constant region, except for the modification(s) in the constant region that affect CDC activity. In certain embodiments, the variant human IgM constant region comprises one or more amino acid substitutions, e.g., in the Cμ3 domain, relative to a wild-type human IgM constant region, e.g., as described in U.S. Patent Application Publication No. US2021-0147567, which is incorporated by reference in its entirety. Assays for measuring CDC are well known to those of skill in the art, and exemplary assays are described, e.g., in U.S. Patent Application Publication No. 2021-0147567, which is incorporated by reference in its entirety.

[0120] In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution corresponding to the wild-type human IgM constant region at positions L310, P311, P313, and / or K315 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the variant IgM constant region provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the variant IgM constant region provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and / or P313 of SEQ ID NO: 1 or SEQ ID NO: 2. These proline residues may be independently substituted by any amino acid (e.g., by alanine or serine or glycine). In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO:1 or SEQ ID NO:2. The lysine residues may be independently substituted by any amino acid (e.g., by alanine, serine, glycine, or aspartic acid). In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution corresponding to the wild-type human IgM constant region at position L310 of SEQ ID NO:1 or SEQ ID NO:2. The lysine residues may be independently substituted by any amino acid (e.g., by alanine, serine, glycine, or aspartic acid).In certain embodiments, the variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution with aspartic acid corresponding to the wild-type human IgM constant region at position L310 of SEQ ID NO:1 or SEQ ID NO:2.

[0121] Glycosylated IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules Human and certain non-human primate IgM constant regions typically contain five naturally occurring asparagine (N)-linked glycosylation motifs or sites. As used herein, an "N-linked glycosylation motif" comprises or consists of the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. The N-linked glycosylation motifs occur in the human IgM heavy chain constant region of SEQ ID NO:1 or SEQ ID NO:2, beginning at positions 46 ("N1"), 209 ("N2"), 272 ("N3"), 279 ("N4"), and 440 ("N5"). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in mouse IgM heavy chain constant regions. Thus, in some embodiments, the IgM heavy chain constant region of the multimeric binding molecules provided herein comprises five N-linked glycosylation motifs: N1, N2, N3, N4, and N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., N1, N2, and N3) on each IgM heavy chain constant region are populated by complex glycans.

[0122] In certain embodiments, at least one, at least two, at least three, or at least four of the N-X1-S / T motifs may contain an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif. In certain embodiments, the IgM-derived multimeric binding molecule may contain an amino acid insertion, deletion, or substitution at motif N1, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs N1, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif. In some embodiments, the IgM constant region contains one or more substitutions compared to the wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO:1 (human IgM constant region allele IGHM*03) or SEQ ID NO:2 (human IgM constant region allele IGHM*04). See, for example, PCT Publication No. WO2021 / 041250, which is incorporated by reference in its entirety.

[0123] IgA antibodies, IgA-like antibodies, and IgA-derived binding molecules IgA plays a key role in mucosal immunity and constitutes approximately 15% of the total immunoglobulins produced. IgA can be monomeric or polymeric, forming primarily dimeric molecules, but also as trimers, tetramers, and / or pentamers. See, e.g., de Sousa-Pereira, P., and JM Woof, Antibodies 8:57 (2019).

[0124] In some embodiments, the multimeric binding molecule is a dimer and comprises two bivalent binding units, or variants or fragments thereof. In some embodiments, the multimeric binding molecule is a dimer or a tetramer, comprising two or four bivalent binding units, or variants or fragments thereof, respectively, and further comprising a J chain, or a functional fragment or variant thereof, as described herein. In some embodiments, the multimeric binding molecule is a dimer and comprises two bivalent binding units, or variants or fragments thereof, and further comprising a J chain, or a functional fragment or variant thereof, as described herein, each binding unit comprising two IgA heavy chain constant regions, or a multimerization fragment or variant thereof.

[0125] In some embodiments, the multimeric binding molecule is a tetramer and comprises four bivalent binding units, or variants or fragments thereof. In some embodiments, the multimeric binding molecule is a tetramer and comprises four bivalent binding units, or variants or fragments thereof, and further comprises a J chain, or functional fragments or variants thereof, as described herein. In some embodiments, the multimeric binding molecule is a tetramer and comprises four bivalent binding units, or variants or fragments thereof, and further comprises a J chain, or functional fragments or variants thereof, as described herein, and each binding unit comprises two IgA heavy chain constant regions, or multimerized fragments or variants thereof.

[0126] In certain embodiments, the multimeric binding molecule provided by the present disclosure is a dimeric binding molecule comprising four IgA heavy chain constant regions or multimerized fragments thereof, each associated with an antigen binding domain for a total of four antigen binding domains. As provided herein, the dimeric IgA antibody, IgA-derived binding molecule, or IgA-like antibody comprises two binding units and one J chain (e.g., a modified J chain comprising an scFv antibody fragment that binds to CD3, or an IL-15 and / or IL-15 receptor-α sushi domain fused to a modified J chain comprising an scFv antibody fragment, as described elsewhere herein). Each binding unit provided comprises two IgA heavy chain constant regions, or multimerized fragments or variants thereof. In certain embodiments, at least three or all four antigen binding domains of the multimeric binding molecule bind to the same target antigen. In certain embodiments, at least three or all four binding polypeptides of the multimeric binding molecule are identical.

[0127] A bivalent IgA-derived binding unit comprises two IgA heavy chain constant regions, and a dimeric IgA-derived binding molecule comprises two binding units. IgA contains the following heavy chain constant domains: Cα1 (or alternatively CA1 or CH1), hinge region, Cα2 (or alternatively CA2 or CH2), and Cα3 (or alternatively CA3 or CH3), and a C-terminal "tailpiece". Human IgA has two subtypes: IgA1 and IgA2. The human IgA1 constant region typically comprises the amino acid sequence SEQ ID NO:3. The human Cα1 domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO:3, the human IgA1 hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO:3, the human Cα2 domain extends from about amino acid 125 to about amino acid 219 of SEQ ID NO:3, the human Cα3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO:3, and the tailpiece extends from about amino acid 331 to about amino acid 352 of SEQ ID NO:3. The human IgA2 constant region typically comprises the amino acid sequence SEQ ID NO:4. The human Cα1 domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO:4, the human IgA2 hinge region extends from about amino acid 102 to about amino acid 111 of SEQ ID NO:4, the human Cα2 domain extends from about amino acid 113 to about amino acid 206 of SEQ ID NO:4, the human Cα3 domain extends from about amino acid 215 to about amino acid 317 of SEQ ID NO:4, and the tailpiece extends from about amino acid 318 to about amino acid 340 of SEQ ID NO:4.

[0128] Two IgA binding units may be complexed with two additional polypeptide chains (J chain (e.g., SEQ ID NO: 7) and secretory component (precursor SEQ ID NO: 5, mature from about amino acid 19 to about amino acid 764 of SEQ ID NO: 5) to form a bivalent secretory IgA (sIgA)-derived binding molecule provided herein. Assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the Cα3 and tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Thus, a multimerized dimeric IgA-derived binding molecule provided in this disclosure typically includes an IgA constant region that includes at least the Cα3 and α tailpiece domains. Four IgA binding units may similarly form a tetrameric complex with a J chain. sIgA antibodies may also form higher order multimers (e.g., tetramers).

[0129] The IgA heavy chain constant region may additionally comprise a Cα2 domain or a fragment thereof, an IgA hinge region or a fragment thereof, a Cα1 domain or a fragment thereof, and / or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, for example, an IgG hinge region. In certain embodiments, a binding molecule provided herein may comprise a complete IgA heavy (α) chain constant domain (e.g., SEQ ID NO: 3 or SEQ ID NO: 4), or a variant, derivative, or analog thereof. In some embodiments, the IgA heavy chain constant region or multimerization fragment thereof is a human IgA constant region.

[0130] In certain embodiments, each binding unit of the multimeric binding molecules provided herein comprises two IgA heavy chain constant regions, or multimerized fragments or variants thereof, each comprising at least an IgA Cα3 domain and an IgA tailpiece domain. In certain embodiments, the IgA heavy chain constant regions may each further comprise an IgA Cα2 domain located N-terminal to the IgA Cα3 and IgA tailpiece domains. For example, the IgA heavy chain constant region may comprise amino acids 125-353 of SEQ ID NO: 3 or amino acids 113-340 of SEQ ID NO: 4. In certain embodiments, the IgA heavy chain constant region may each further comprise an IgA or IgG hinge region located N-terminal to the IgA Cα2 domain. For example, the IgA heavy chain constant region may comprise amino acids 102-353 of SEQ ID NO: 3 or amino acids 102-340 of SEQ ID NO: 4. In certain embodiments, the IgA heavy chain constant regions may each further comprise an IgA Cα1 domain positioned N-terminal to the IgA hinge region.

[0131] In some embodiments, each binding unit of an IgA antibody, an IgA-like antibody, or other IgA-derived binding molecule comprises two light chains. In some embodiments, each binding unit of an IgA antibody, an IgA-like antibody, or other IgA-derived binding molecule comprises fragments of two light chains. In some embodiments, the light chain is a κ light chain. In some embodiments, the light chain is a λ light chain. In some embodiments, the light chain is a chimeric κ-λ light chain. In some embodiments, each binding unit comprises two immunoglobulin light chains, each comprising a VL positioned amino-terminal to an immunoglobulin light chain constant region.

[0132] Modified and / or variant J chains In certain embodiments, the multimeric binding molecules provided herein, such as antibodies or antibody-like molecules, comprise a J chain, or a functional fragment or variant thereof. In certain embodiments, the multimeric binding molecules provided herein are pentameric IgM antibodies or IgM antibody-like molecules and comprise a J chain, or a functional fragment or variant thereof. In certain embodiments, the multimeric binding molecules provided herein are dimeric IgA antibodies or IgA antibody-like molecules and comprise a J chain, or a functional fragment or variant thereof. In some embodiments, the multimeric binding molecules may comprise a naturally occurring J chain, such as a mature human J chain sequence (e.g., SEQ ID NO: 7). In some embodiments, the multimeric binding molecules may comprise a functional fragment or variant of a naturally occurring J chain.

[0133] In certain embodiments, the J chain of a pentameric IgM or IgM-like antibody or a dimeric IgA or IgA-like antibody provided herein can be modified, for example, by the introduction of a heterologous moiety or two or more heterologous moieties (e.g., polypeptides) without interfering with the ability of the IgM or IgM-like antibody or IgA or IgA-like antibody to assemble and bind to its binding target(s). See U.S. Patent Nos. 9,951,134, 10,975,147, 10,400,038 and 10,618,978, and U.S. Patent Application Publication No. US-2019-0185570, each of which is incorporated herein by reference in its entirety. Thus, the IgM or IgM-like antibodies or IgA or IgA-like antibodies provided herein (including bispecific or multispecific IgM or IgM-like antibodies or IgA or IgA-like antibodies described elsewhere herein) comprise a modified J chain, or a functional fragment or variant thereof, which may further comprise a heterologous moiety (e.g., a heterologous polypeptide) introduced therein. In certain embodiments, the heterologous moiety may be a peptide or polypeptide that is fused in frame or chemically conjugated to the J chain, or a fragment or variant thereof. For example, a heterologous polypeptide may be fused to the J chain, or a functional fragment or variant thereof. In certain embodiments, the heterologous polypeptide is fused to the J chain, or a functional fragment or variant thereof, by a linker (e.g., a peptide linker consisting of at least 5 amino acids, but typically no more than 25 amino acids). In certain embodiments, the peptide linker consists of GGGGS (SEQ ID NO:9), GGGGSGGGGS (SEQ ID NO:10), GGGSGGGGSGGGGGS (SEQ ID NO:11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:12), or GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:13). In certain embodiments, the heterologous moiety can be a chemical moiety that is conjugated to the J chain.Heterologous moieties added to the J chain can include, but are not limited to, a binding moiety (e.g., an antibody or antigen-binding fragment thereof, such as a single chain Fv (scFv) molecule), a stabilizing peptide that can increase the half-life of an IgM or IgM-like antibody, or a chemical moiety (such as a polymer or cytotoxin). In some embodiments, the heterologous moiety comprises a stabilizing peptide that can increase the half-life of a binding molecule (e.g., human serum albumin (HSA) or an HSA binding molecule).

[0134] In some embodiments, the modified J chain may comprise a J chain-associated antigen-binding domain (e.g., a polypeptide capable of specifically binding to a target antigen). In certain embodiments, the J chain-associated antigen-binding domain may be an antibody or antigen-binding fragment thereof described elsewhere herein. In certain embodiments, the J chain-associated antigen-binding domain may be a single-chain Fv (scFv) antigen-binding domain or a single-chain antigen-binding domain derived from, for example, a camelid or cartilaginous fish antibody. The J chain-associated antigen-binding domain may be introduced into the J chain at any position that allows the J chain-associated antigen-binding domain to bind to its binding target without interfering with J chain function or the function of the associated IgM or IgA antibody. Insertion positions include, but are not limited to, at or near the C-terminus, at or near the N-terminus, or at an internal location accessible based on the three-dimensional structure of the J chain. In certain embodiments, the J chain-associated antigen-binding domain may be introduced into the mature human J chain of SEQ ID NO:7 between cysteine ​​residues 92 and 101 of SEQ ID NO:7. In a further embodiment, a J chain-associated antigen binding domain may be introduced into or near a glycosylation site in the human J chain of SEQ ID NO: 7. In a further embodiment, a J chain-associated antigen binding domain may be introduced into the human J chain of SEQ ID NO: 7 within about 10 amino acid residues from the C-terminus or within about 10 amino acids from the N-terminus. As described elsewhere herein, the present disclosure provides multimeric bispecific binding molecules comprising a modified J chain, where the modified J chain comprises a J chain-associated antigen binding domain that specifically binds to an immune effector cell (e.g., a T cell (such as a CD4+ T cell or a CD8+ cytotoxic T cell) or a NK cell).

[0135] In some embodiments, the modified J chain further comprises an immunostimulatory agent (ISA), such as a cytokine (e.g., interleukin-2 (IL-2) or interleukin-15 (IL-15)), or a receptor-binding fragment or variant thereof, which in certain embodiments may be associated with a portion of its receptor (e.g., the sushi domain of the IL-15 receptor-α) by either binding or covalent attachment. Such ISAs are described in detail in co-pending PCT Publication No. WO2021 / 030688, which is incorporated herein by reference in its entirety.

[0136] In certain embodiments, the J chain of an IgM antibody, an IgM-like antibody, an IgA antibody, an IgA-like antibody, an IgM-derived binding molecule, or an IgA-derived binding molecule provided herein is a variant J chain that contains one or more amino acid substitutions that can, for example, alter the serum half-life of an IgM antibody, an IgM-like antibody, an IgA antibody, an IgA-like antibody, an IgM-derived binding molecule, or an IgA-derived binding molecule provided herein. For example, a certain amino acid substitution, deletion, or insertion can result in an IgM-derived binding molecule that exhibits an increased serum half-life upon administration to a subject animal compared to a reference IgM-derived binding molecule that is identical except for the substitution, deletion, or insertion of one or more single amino acids in the variant J chain and is administered to the same animal species using the same method. In certain embodiments, a variant J chain can contain one, two, three, or four single amino acid substitutions, deletions, or insertions compared to the reference J chain.

[0137] In some embodiments, the multimeric binding molecule may comprise a variant J chain sequence (such as a variant sequence described herein with reduced glycosylation or with reduced binding to one or more polymeric Ig receptors (e.g., pIgR, Fc alpha-mu receptor (FcαμR), or Fc mu receptor (FcμR)). See, e.g., U.S. Pat. No. 10,899,835, which is incorporated herein by reference in its entirety). In certain embodiments, the variant J chain may comprise an amino acid substitution at an amino acid position corresponding to amino acid Y102 of mature wild-type human J chain (SEQ ID NO: 7). An "amino acid corresponding to amino acid Y102 of mature wild-type human J chain" means an amino acid in the sequence of the J chain of any species that is homologous to Y102 in the human J chain. See, U.S. Pat. No. 10,899,835, which is incorporated herein by reference in its entirety. The position corresponding to Y102 in SEQ ID NO: 7 is conserved in the J chain amino acid sequences of at least 43 other species. See Figure 4 of U.S. Patent No. 9,951,134, which is incorporated herein by reference. Certain mutations at the position corresponding to Y102 in SEQ ID NO:7 can inhibit certain immunoglobulin receptors (e.g., human or mouse Fcαμ receptors, mouse Fcμ receptors, and / or human or mouse polymeric Ig receptors (pIg receptors)) from binding to IgM pentamers containing mutant J chains. IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules containing a mutation at the amino acid corresponding to Y102 in SEQ ID NO:7 have improved serum half-lives when administered to animals than corresponding antibodies, antibody-like molecules, or binding molecules that are identical except for the substitution and are administered in the same manner to the same species. In certain embodiments, the amino acid corresponding to Y102 in SEQ ID NO:7 can be substituted with any amino acid. In certain embodiments, the amino acid corresponding to Y102 in SEQ ID NO:7 can be substituted with alanine (A), serine (S), or arginine (R). In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO:7 may be substituted with alanine.In certain embodiments, the J chain, or functional fragment or variant thereof, is a variant human J chain and comprises the amino acid sequence SEQ ID NO:8 (a J chain referred to herein as "J*").

[0138] A wild-type J chain typically contains one N-linked glycosylation site. In certain embodiments, a variant J chain or functional fragment thereof of a multimeric binding molecule provided herein contains a mutation within an asparagine (N)-linked glycosylation motif N-X1-S / T (e.g., starting at an amino acid position corresponding to amino acid 49 (motif N6) of mature human J chain (SEQ ID NO: 7) or J* (SEQ ID NO: 8), where N is asparagine, X1 is any amino acid other than proline, and S / T is serine or threonine, and the mutation prevents glycosylation at that motif. As demonstrated in U.S. Pat. No. 10,899,835, a mutation that prevents glycosylation at this site can result in a multimeric binding molecule provided herein exhibiting an increased serum half-life upon administration to a subject animal compared to a reference multimeric binding molecule that is identical except for the mutation(s) that prevent glycosylation in the variant J chain and that is administered in the same manner to the same animal species.

[0139] For example, in certain embodiments, a variant J chain or functional fragment thereof of a binding molecule comprising a J chain provided herein can include an amino acid substitution at an amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO:7 or SEQ ID NO:8, with the proviso that the amino acid corresponding to S51 is not substituted with threonine (T), or the variant J chain includes an amino acid substitution at an amino acid position corresponding to both amino acids N49 and S51 of SEQ ID NO:7 or SEQ ID NO:8. In certain embodiments, the position corresponding to N49 of SEQ ID NO:7 or SEQ ID NO:8 is substituted with any amino acid (e.g., alanine (A), glycine (G), threonine (T), serine (S), aspartic acid (D)). In certain embodiments, the position corresponding to N49 of SEQ ID NO:7 or SEQ ID NO:8 can be substituted with alanine (A). In another embodiment, the position corresponding to N49 of SEQ ID NO:7 or SEQ ID NO:8 can be substituted with aspartic acid (D). In some embodiments, the position corresponding to S51 of SEQ ID NO:7 or SEQ ID NO:8 is substituted with alanine (A) or glycine (G). In some embodiments, the position corresponding to S51 of SEQ ID NO:7 or SEQ ID NO:8 is substituted with alanine (A).

[0140] Multimeric bispecific or multispecific anti-CD123 binding molecules with modified J chains that bind to immune effector cells The present disclosure provides multimeric bispecific or multispecific binding molecules for use in the treatment of cancer (e.g., hematological cancers, e.g., acute myeloid leukemia (AML)), which are bispecific and target CD123 (IL-3Rα) on cancer cells with high avidity while also targeting immune effector cells (e.g., CD4+ or CD8+ T cells or NK cells) via one antigen-binding domain, thereby facilitating effector cell-mediated killing of cancer cells while simultaneously minimizing excessive release of cytokines. In certain embodiments, the multimeric bispecific anti-CD123 binding molecule is an anti-CD123×anti-CD3 binding molecule.

[0141] Thus, the present disclosure provides a multimeric bispecific or multispecific binding molecule comprising two IgA or IgA-like or five IgM or IgM-like bivalent binding units and one modified J chain, the modified J chain comprising at least a wild-type J chain, or a functional fragment or variant thereof, and a J chain-associated antigen-binding domain that specifically binds to an immune effector cell. Each binding unit comprises two antibody heavy chains, each of which comprises an IgA, IgA-like, IgM, or IgM-like heavy chain constant region, or a multimerization fragment thereof (as described elsewhere herein), and at least a heavy chain variable region (VH) portion of the binding unit-associated antigen-binding domain. At least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 of the binding unit-associated antigen-binding domains specifically bind to CD123. The binding molecules provided herein can induce immune effector cell-dependent killing of cells expressing CD123 (e.g., cancer cells).

[0142] In certain embodiments, the modified J chain of the binding molecules provided herein comprises a variant of a wild-type J chain, or a fragment thereof, where the variant comprises one or more single amino acid substitutions, deletions, or insertions that may affect the serum half-life of the binding molecule relative to the wild-type J chain, and the binding molecule exhibits increased serum half-life upon administration to an animal relative to a reference binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the J chain and is administered in the same manner to the same animal species. For example, in certain embodiments, the J chain is a variant human J chain ("J*") comprising the amino acid sequence SEQ ID NO:8.

[0143] In certain embodiments, the J chain-associated antigen-binding domain of the provided binding molecule comprises an antibody or a fragment thereof. In certain embodiments, the antibody fragment is a single chain Fv (scFv). The scFv can be fused or chemically conjugated to a J chain or a fragment or variant (e.g., J*). In certain embodiments, the scFv is fused to the J chain by a peptide linker (e.g., SEQ ID NOs: 9-13). As noted elsewhere in this disclosure, the scFv can be fused to the J chain, or a fragment or variant thereof, in any manner so long as the function of the J chain (i.e., assembly with IgM, IgM-like, IgA, or IgA-like binding units to form dimers or pentamers) is not affected. For example, the scFv can be fused to the N-terminus of the J chain, or a fragment or variant thereof, the C-terminus of the J chain, or a fragment or variant thereof, or to both the N-terminus and C-terminus of the J chain, or a fragment or variant thereof.

[0144] The immune effector cell to which the antigen-binding domain of the modified J chain binds can be any immune effector cell that confers a beneficial effect (e.g., mediates cell-based killing of CD123+ cancer cells) when associated with cancer cells targeted by CD123. In certain embodiments, the immune effector cell can be, without limitation, a T cell (e.g., a CD4+ T cell, a CD8+ T cell, a NKT cell, or a γδ T cell), a B cell, a plasma cell, a macrophage, a dendritic cell, or a natural killer (NK) cell. In certain embodiments, the immune effector cell is a T cell (e.g., a CD4+ or CD8+ T cell). In certain embodiments, the immune effector cell is a CD8+ cytotoxic T cell. In certain embodiments, the immune effector cell is a NK cell.

[0145] When the immune effector cell is a T cell (e.g., a CD8+ T cell), the J chain associated antibody or fragment thereof, e.g., scFv, can specifically bind to the T cell surface antigen CD3 (e.g., CD3ε). In certain embodiments, the anti-CD3 scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, where VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are the amino acid sequences SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. No. 33; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41; SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49; SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57; SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65; or SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73 with 0, 1 or 2 amino acid substitutions. In some embodiments, the scFv comprises the VH and VL amino acid sequences SEQ ID NO:18 and SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:46, SEQ ID NO:50 and SEQ ID NO:54, SEQ ID NO:58 and SEQ ID NO:62, or SEQ ID NO:66 and SEQ ID NO:70, respectively.

[0146] In certain other embodiments, the immune effector cell is a NK cell and the J chain associated antibody or fragment thereof, e.g., scFv, is capable of specifically binding to CD16 or CD56. Many CD16 and CD56 scFvs are known, including those disclosed in U.S. Patent Nos. 9,035,026, 9,701,750, 10,730,941, 11,001,633, and McCall et al., 1999. Mol Immunol. 7:433-445.

[0147] The modified J chain of a multimeric bispecific anti-CD123 binding molecule (e.g., an anti-CD123×anti-CD3 binding molecule provided herein) can be further modified to include an additional heterologous moiety added to the J chain. Exemplary moieties are described, for example, in U.S. Pat. No. 9,951,134, U.S. Patent Application Publication No. US2019-0185570, U.S. Pat. No. 10,618,978, and PCT Publication No. WO2021 / 030688, all of which are incorporated herein by reference in their entirety. In certain embodiments, the modified J chain of a multimeric bispecific anti-CD123 binding molecule (e.g., an anti-CD123×anti-CD3 binding molecule provided herein) can further include an immune stimulatory agent ("ISA") fused or chemically conjugated to the J chain, or a fragment or variant thereof. For example, the ISA can include a cytokine, or a receptor-binding fragment or variant thereof. In certain embodiments, the J chain-associated ISA comprises (a) an interleukin-15 (IL-15) protein, or a receptor-binding fragment or variant thereof ("I"), and (b) an interleukin-15 receptor-alpha (IL-15Rα) fragment ("R") that includes a sushi domain or a variant thereof that can associate with I, wherein the J chain, or fragment or variant thereof, and at least one of I and R, or both I and R, associate as a fusion protein, and I and R can associate to function as an ISA. In certain embodiments, the ISA may be fused to the J chain via a peptide linker.

[0148] Polynucleotides, Vectors, and Host Cells The present disclosure further provides polynucleotides (e.g., isolated polynucleotides, recombinant polynucleotides, and / or non-naturally occurring polynucleotides) that comprise a nucleic acid sequence encoding an antigen-binding domain provided herein, or a polypeptide subunit of an antibody or antibody-like molecule provided herein (e.g., a dimeric, hexameric, or pentameric antibody or antibody-like molecule). By "polypeptide subunit" is meant a portion, binding unit, or antigen-binding domain of an antibody or antibody-like molecule that can be translated independently. Examples include, but are not limited to, an antibody variable domain (e.g., VH or VL), a J chain (including modified J chains provided herein), a secretory component, a single chain Fv, an antibody heavy chain, an antibody light chain, an antibody heavy chain constant region, an antibody light chain constant region, and / or any fragment, variant, or derivative thereof.

[0149] In certain embodiments, a polynucleotide comprises a nucleic acid sequence encoding a polypeptide subunit of a binding molecule described herein. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a heavy chain constant region of the binding molecule and at least an antibody VH portion of the binding domain. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a heavy chain of the binding molecule. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a VH comprising HCDR1, HCDR2 and HCDR3 regions, where HCDR1, HCDR2 and HCDR3 comprise the amino acid sequence of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID NO:82, respectively.

[0150] In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a light chain constant region of the binding molecule and at least an antibody VL portion of the binding domain. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a light chain of the binding molecule. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a VL comprising LCDR1, LCDR2 and LCDR3 regions, where LCDR1, LCDR2 and LCDR3 each comprise the amino acid sequence of SEQ ID NO:79 or SEQ ID NO:83.

[0151] In certain embodiments, the polypeptide subunits may include an IgM or IgM-like heavy chain constant region, or a multimerized fragment thereof, or an IgA or IgA-like heavy chain constant region, or a multimerized fragment thereof, all fused to an antigen binding domain or a subunit thereof (e.g., to the VH portion of the antigen binding domain, or to the VL portion of the antigen binding domain), all as provided herein. In certain embodiments, the polynucleotide encodes a polypeptide subunit comprising a human IgM heavy chain constant region, a human IgM-like heavy chain constant region, a human IgA heavy chain constant region, a human IgA-like heavy chain constant region, or a multimerized fragment thereof (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), any of which is associated with, e.g., fused to, an antigen binding domain or a subunit thereof (e.g., to the C-terminus of the VH).

[0152] To form the antigen-binding domain or variable region of an antibody that specifically binds to CD123, the provided polynucleotides can be inserted into an expression vector template, for example, for a monomeric antibody (e.g., an IgG antibody) or for an IgG and / or IgA construct, thereby generating a monomeric antibody containing a single binding unit, or a multimeric antibody, or a multimerized fragment or derivative thereof having at least two bivalent binding units. Briefly, nucleic acid sequences encoding heavy and light chain variable domain sequences can be synthesized or amplified from an existing molecule and inserted into a vector in the appropriate orientation and in frame such that upon expression, the vector will generate a full-length heavy or light chain. Vectors useful for these purposes are known in the art. Such vectors can also include enhancers and other sequences required to achieve expression of the desired chain. Multiple vectors or a single vector can be used. These vectors are transfected into a host cell, after which the chains are expressed and purified. Upon expression, the chains form a fully functional multimeric binding molecule, as reported in the literature. The fully assembled multimeric binding molecule can then be purified by standard methods. The expression and purification process can be carried out on a commercial scale, if desired.

[0153] The present disclosure further provides compositions comprising two or more polynucleotides that can collectively encode the antigen binding domains or antibodies or antibody-like molecules (e.g., monomeric, dimeric, hexameric or pentameric antibodies) described herein. In certain embodiments, the compositions can comprise polynucleotides encoding the above-mentioned IgG, IgM and / or IgA heavy chains or fragments thereof (e.g., human IgG, IgM or IgA heavy chains), where the IgG, IgM and / or IgA heavy chains comprise at least the provided VH of the CD123 antigen binding domains provided herein, and can further comprise polynucleotides encoding light chains or fragments thereof (e.g., human κ or λ light chains) that comprise at least the provided VL of the CD123 antigen binding domains provided herein. The provided polynucleotide compositions can further comprise polynucleotides encoding a J chain, e.g., a human J chain, or a fragment, variant or derivative thereof. In certain embodiments, the polynucleotides constituting the compositions provided herein can be located on two, three or more separate vectors, e.g., expression vectors. Such vectors are provided by the present disclosure.In certain embodiments, two or more of the polynucleotides that constitute the compositions provided herein can be located on a single vector, such as an expression vector.Such vectors are provided by the present disclosure.

[0154] In certain embodiments, the present disclosure provides a composition comprising two, three, or more polynucleotides provided herein, which together can encode an anti-CD123 binding molecule, such as a multimeric bispecific anti-CD123 binding molecule (e.g., an anti-CD123 x anti-CD3 binding molecule provided herein). In certain embodiments, the polynucleotides can be located on separate vectors. In certain embodiments, two or more polynucleotides can be located on the same vector. Such vectors are similarly provided by the present disclosure.

[0155] The present disclosure further provides a host cell (e.g., a prokaryotic or eukaryotic host cell) comprising a polynucleotide or two or more polynucleotides encoding an anti-CD123 binding molecule, e.g., a multimeric, bispecific anti-CD123 binding molecule (e.g., an anti-CD123 x anti-CD3 binding molecule provided herein, or any subunit thereof), a polynucleotide composition provided herein, or a vector that collectively encodes a binding molecule provided herein, or any subunit thereof, or two, three, or more vectors.

[0156] In a related embodiment, the present disclosure provides a method for producing a multimeric binding molecule provided by the present disclosure, the method comprising culturing a host cell provided herein and recovering the multimeric binding molecule.

[0157] How to use The present disclosure further provides a method of treating a disease or disorder (e.g., cancer or other malignancy, e.g., a hematological cancer or hematological malignancy) in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of an anti-CD123 antibody or antigen-binding fragment or derivative thereof (e.g., an anti-CD123 x anti-CD3 antibody provided herein). By "therapeutically effective dose or amount" or "effective amount" is intended the amount of binding molecule that, when administered, results in a positive response (e.g., killing of tumor cells in the subject).

[0158] In certain embodiments, the cancer to be treated can be any cancer whose malignant cell expresses or overexpresses CD123.For example, the cancer can be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-cell ALL), classical Hodgkin's lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), systemic mastocytosis, or plasmacytoid dendritic cell leukemia).

[0159] The effective dose of a composition for the treatment of cancer can vary depending on many different factors, including the means of administration, the target site, the physiological condition of the subject, whether the subject is human or animal, other pharmaceutical agents being administered, and whether the treatment is prophylactic or therapeutic. Typically, the subject is a human, although non-human mammals, including transgenic mammals, can also be treated.

[0160] The subject to be treated can be any mammal in need of treatment, and in certain embodiments, the subject is a human subject.

[0161] In its simplest form, the preparation administered to a subject is an anti-CD123 antibody, or an antigen-binding fragment of a derivative thereof (e.g., an anti-CD123 x anti-CD3 antibody provided herein) administered in a conventional dosage form, which may be combined with a pharmaceutical excipient, carrier, or diluent substance described elsewhere herein.

[0162] Compositions of the present disclosure may be administered by any suitable method, such as parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0163] Pharmaceutical Compositions and Methods of Administration Methods for preparing and administering an anti-CD123 antibody or antigen-binding fragment or derivative thereof (e.g., an anti-CD123 x anti-CD3 antibody provided herein) to a subject in need thereof are well known to those of skill in the art or can be readily determined by those of skill in the art in light of the present disclosure. The route of administration can be, for example, intratumoral, oral, parenteral, by inhalation, or topical. The term parenteral, as used herein, includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Although these forms of administration are contemplated as suitable forms, examples of other forms of administration would be solutions for injection, intratumoral, intravenous, or intraarterial injection or infusion. Suitable pharmaceutical compositions can include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin), and the like.

[0164] As discussed herein, anti-CD123 antibodies, or antigen-binding fragments or derivatives thereof (e.g., anti-CD123×anti-CD3 antibodies provided herein) can be administered in a pharmacologic effective amount for the treatment of a subject in need thereof. In this regard, it will be understood that the disclosed antibodies, or antigen-binding fragments or derivatives thereof, can be formulated to facilitate administration and promote stability of the active agent. The pharmaceutical composition can thus include a pharmacologic acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like. A pharmacologic effective amount of an antibody, or antigen-binding fragment or derivative thereof, including an ISA, provided herein means an amount sufficient to achieve effective binding to a target and achieve a therapeutic benefit. Suitable formulations are described in Remington's Pharmaceutical Sciences, e.g., 21st Edition (Lippincott Williams & Wilkins) (2005).

[0165] Certain pharmaceutical compositions provided herein may be administered orally in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions, or solutions. Certain pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions may be prepared as a solution in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers that improve bioavailability, and / or other conventional solubilizing or dispersing agents.

[0166] The amount of anti-CD123 antibody or antibody-like molecule (e.g., anti-CD123 x anti-CD3 antibody) disclosed herein that can be combined with a carrier material to produce a single dosage form will vary depending, for example, on the subject being treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses, or over an established period of time in an infusion. Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0167] In accordance with the scope of the present disclosure, anti-CD123 antibodies or antibody-like molecules (e.g., anti-CD123xanti-CD3 antibodies provided herein) may be administered to a subject in need of therapy in an amount sufficient to produce a therapeutic effect. Anti-CD123 antibodies or antibody-like molecules (e.g., anti-CD123xanti-CD3 antibodies provided herein) may be administered to a subject in conventional dosage forms prepared by combining an antibody or antibody-like molecule of the present disclosure with a conventional pharma- ceutically acceptable carrier or diluent substance according to known techniques. The form and nature of the pharma-ceutically acceptable carrier or diluent substance may be determined by the amount of active ingredient with which it is combined, the route of administration, and other well-known variables.

[0168] The present disclosure also provides for the use of an anti-CD123 antibody or antibody-like molecule (e.g., an anti-CD123x anti-CD3 antibody provided herein) in the manufacture of a medicament for treating, preventing, or managing cancer or other malignancies. The present disclosure also provides for an anti-CD123 antibody or antibody-like molecule (e.g., an anti-CD123x anti-CD3 binding molecule provided herein) for use in treating, preventing, or managing cancer.

[0169] The present disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art and are fully explained in the literature. For example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press), Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY), DNGlover and BD Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes. 1-4, Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press), Mullis et al. U.S. Patent No. 4,683,195, Hames and Higgins, eds. Press), Freshney(2016)Culture Of Animal Cells,7th Edition (Wiley-Blackwell), Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985), Perbal (1988) A Practical Guide To Molecular Cloning;2d Edition (Wiley-Interscience), Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory), SCSee Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science), Methods in Enzymology, Vols. 151-155 (Academic Press, Inc., N.Y.), Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), Weir and Blackwell, eds., and Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0170] General principles of antibody engineering are described, for example, in Strohl, WR, and LM Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). General principles of protein engineering are described, for example, in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). General principles of immunology are described, for example, in Abbas and Lichtman (2017) Cellular and Molecular Immunology 9th Edition (Elsevier). Additionally, standard methods in immunology known in the art may be followed, e.g., those in Current Protocols in Immunology (Wiley Online Library), Wild, D. (2013), The Immunoassay Handbook 4th Edition (Elsevier Science), Greenfield, ed. (2013), Antibodies, a Laboratory Manual, 2d Edition (Cold Spring Harbor Press), and Ossipow and Fischer, eds., (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press).

[0171] All of the above references, as well as all references cited herein, are hereby incorporated by reference in their entirety.

[0172] The following examples are offered by way of illustration and not by way of limitation.

[0173] Exemplary embodiments Among the embodiments provided are the following:

[0174] EMBODIMENT 1 An antibody or antigen-binding fragment or derivative thereof comprising an antigen-binding domain that specifically binds to CD123, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); The VH and the VL comprise the amino acid sequences SEQ ID NO:76 and SEQ ID NO:79, SEQ ID NO:77 and SEQ ID NO:79, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:83, SEQ ID NO:81 and SEQ ID NO:83, and SEQ ID NO:82 and SEQ ID NO:83, respectively; The antibody or an antigen-binding fragment or derivative thereof.

[0175] EMBODIMENT 2 The antibody or fragment or derivative thereof of embodiment 1, wherein the VH and the VL comprise the amino acid sequences SEQ ID NO: 76 and SEQ ID NO: 79, respectively.

[0176] EMBODIMENT 3 3. The antibody or fragment or derivative thereof of embodiment 1 or embodiment 2, which is a multimeric antibody comprising 5, 6 or 2 bivalent binding units and 10, 12 or 4 antigen binding domains, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 antigen binding domains specifically bind to CD123, each binding unit comprising two heavy chains, each of said two heavy chains comprising an IgM or IgA constant region, or a multimerized fragment or variant thereof, and wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of said heavy chain constant regions of said multimeric antibody are associated with a copy of said VH.

[0177] EMBODIMENT 4 comprising a single bivalent binding unit comprising two antigen-binding domains, at least one antigen-binding domain specifically binds to CD123, said binding unit comprising two heavy chains, each of said two heavy chains comprising a heavy chain constant region or a fragment or variant thereof, said heavy chain constant region or a fragment or variant thereof of at least one of said binding units being associated with a copy of said VH; The antibody or fragment or derivative thereof of embodiment 1 or embodiment 2.

[0178] EMBODIMENT 5 The antibody or fragment or derivative thereof of embodiment 4, wherein the heavy chain comprises an IgG heavy chain constant region, or a fragment or variant thereof.

[0179] EMBODIMENT 6 The antibody or fragment or derivative thereof of embodiment 1 or embodiment 2, which is an Fv fragment, a single chain Fv fragment (scFv) or a disulfide-linked Fv fragment (sdFv).

[0180] EMBODIMENT 7 7. The antibody or fragment or derivative thereof of any one of embodiments 1 to 6, which is multispecific.

[0181] EMBODIMENT 8 8. The antibody or fragment or derivative thereof of embodiment 7, which is bispecific.

[0182] EMBODIMENT 9 The antibody or fragment or derivative thereof of embodiment 7 or embodiment 8, which is capable of binding to CD3.

[0183] EMBODIMENT 10 10. The antibody or fragment or derivative thereof of any one of embodiments 1 to 9, capable of specifically binding to human CD123.

[0184] EMBODIMENT 11 11. The antibody or fragment or derivative thereof of embodiment 10, which specifically binds to human CD123 with an affinity characterized by a dissociation constant KD of less than or equal to 500nM, 100nM, 50.0nM, 40.0nM, 30.0nM, 20.0nM, 10.0nM, 9.0nM, 8.0nM, 7.0nM, 6.0nM, 5.0nM, 4.0nM, 3.0nM, 2.0nM, 1.0nM, 0.50nM, 0.10nM, 0.050nM, 0.01nM, 0.005nM or 0.001nM.

[0185] EMBODIMENT 12 A multimeric antibody comprising 5, 6 or 2 bivalent binding units and 10, 12 or 4 antigen-binding domains, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 antigen-binding domains specifically bind to CD123; the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); the VH and the VL respectively comprise the amino acid sequences SEQ ID NO:76 and SEQ ID NO:79, SEQ ID NO:77 and SEQ ID NO:79, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:83, SEQ ID NO:81 and SEQ ID NO:83, and SEQ ID NO:82 and SEQ ID NO:83; each binding unit comprises two heavy chains, each of the two heavy chains comprising an IgM or IgA constant region, or a multimerized fragment or variant thereof; At least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 heavy chain constant regions of said multimeric antibody are associated with said VH copies; The multimeric antibody.

[0186] EMBODIMENT 13 The multimeric antibody of embodiment 12, wherein the VH and the VL comprise the amino acid sequences SEQ ID NO: 76 and SEQ ID NO: 79, respectively.

[0187] EMBODIMENT 14 is pentameric or hexameric and contains 5 or 6 bivalent IgM binding units; Each binding unit comprises a Cμ4 domain and a μ tailpiece (μtp) domain, or a multimerized fragment or variant thereof; The multimeric antibody of embodiment 12 or embodiment 13.

[0188] EMBODIMENT 15 The multimeric antibody of embodiment 14, wherein each of the IgM heavy chain constant regions or multimerized fragments or variants thereof further comprises a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof.

[0189] EMBODIMENT 16 16. The multimeric antibody of any one of embodiments 12 to 15, wherein each IgM heavy chain constant region is a human IgM constant region or a multimerized variant or fragment thereof comprising the amino acid sequence SEQ ID NO:1, SEQ ID NO:2, or a multimerized variant or fragment thereof.

[0190] EMBODIMENT 17 A multimeric antibody comprising a variant human IgM constant region and having reduced CDC activity compared to a multimeric antibody comprising an IgM heavy chain constant region comprising the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2. The multimeric antibody of embodiment 15 or embodiment 16.

[0191] EMBODIMENT 18 18. The multimeric antibody of embodiment 17, wherein each variant human IgM constant region comprises an amino acid substitution corresponding to position P311 of SEQ ID NO:1 or SEQ ID NO:2, an amino acid substitution corresponding to position P313 of SEQ ID NO:1 or SEQ ID NO:2, or an amino acid substitution corresponding to positions P311 and P313 of SEQ ID NO:1 or SEQ ID NO:2.

[0192] EMBODIMENT 19 19. The multimeric antibody of any one of embodiments 15-18, wherein each IgM heavy chain constant region or multimerized variant or fragment thereof is a variant human IgM constant region having one or more single amino acid substitutions, deletions or insertions compared to a reference IgM heavy chain constant region, said reference IgM heavy chain constant region being identical to said variant IgM heavy chain constant region except for said one or more single amino acid substitutions, deletions or insertions; and said multimeric antibody, upon administration to a subject animal, exhibits increased serum half-life compared to a multimeric antibody comprising a reference IgM heavy chain constant region administered in the same manner to the same animal species.

[0193] EMBODIMENT 20 20. The multimeric antibody of embodiment 19, wherein said variant IgM heavy chain constant region comprises an amino acid substitution at one or more amino acid positions corresponding to amino acids E345, S401, E402 or E403 of SEQ ID NO:1 or SEQ ID NO:2.

[0194] EMBODIMENT 21 21. The multimeric antibody of any one of embodiments 15-20, wherein each of said IgM heavy chain constant regions or multimerized variants or fragments thereof comprises one or more amino acid substitutions corresponding to amino acid positions N46, N209, N272 or N440 of SEQ ID NO:1 or SEQ ID NO:2, wherein the one or more single amino acid substitutions prevent asparagine (N)-linked glycosylation.

[0195] EMBODIMENT 22 The multimeric antibody of any one of embodiments 12 to 21, wherein each heavy chain constant region or a multimerized fragment or variant thereof is associated with a copy of said VH.

[0196] EMBODIMENT 23 each binding unit further comprises two light chains, each of the two light chains comprising a light chain constant region or a fragment or variant thereof; at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 light chain constant regions or fragments or variants thereof associated with said VL copy; The multimeric antibody of any one of embodiments 12 to 22.

[0197] EMBODIMENT 24 The multimeric antibody of embodiment 23, wherein each light chain constant region, or a fragment or variant thereof, is associated with a copy of said VL.

[0198] EMBODIMENT 25 21. The multimeric antibody of any one of embodiments 14 to 20, which is a pentamer and further comprises a J chain or a fragment or variant thereof.

[0199] EMBODIMENT 26 is a dimer and comprises two bivalent IgA binding units and a J chain or a fragment or variant thereof; Each binding unit comprises a Cα3 domain and an α tailpiece (αtp) domain; The multimeric antibody of embodiment 12 or embodiment 13.

[0200] EMBODIMENT 27 27. The multimeric antibody of embodiment 26, wherein the J chain or fragment or variant thereof is a mature human J chain comprising the amino acid sequence SEQ ID NO:7, or a fragment or variant thereof.

[0201] EMBODIMENT 28 The multimeric antibody of embodiment 26 or embodiment 27, wherein each of said IgA heavy chain constant regions or multimerized fragments or variants thereof further comprises a Cα1 domain, a Cα2 domain, an IgA hinge region, or any combination thereof.

[0202] EMBODIMENT 29 29. The multimeric antibody of embodiment 28, wherein said IgA heavy chain constant region, or a multimerized fragment or variant thereof, is an IgA1 heavy chain constant region, or a multimerized fragment or variant thereof.

[0203] EMBODIMENT 30 The multimeric antibody of embodiment 29, wherein said IgA heavy chain constant region comprises SEQ ID NO:3.

[0204] EMBODIMENT 31 29. The multimeric antibody of embodiment 28, wherein said IgA heavy chain constant region, or a multimerized fragment or variant thereof, is an IgA2 heavy chain, or a multimerized fragment or variant thereof.

[0205] EMBODIMENT 32 The multimeric antibody of embodiment 31, wherein the IgA heavy chain constant region comprises SEQ ID NO:4.

[0206] EMBODIMENT 33 The multimeric antibody of any one of embodiments 25-32, further comprising a secretory component or a fragment or variant thereof.

[0207] EMBODIMENT 34 The multimeric antibody of any one of embodiments 12 to 33, which is multispecific.

[0208] EMBODIMENT 35 The multimeric antibody of embodiment 34, which is bispecific.

[0209] EMBODIMENT 36 The multimeric antibody of embodiment 34 or embodiment 35, which is capable of binding to CD3.

[0210] EMBODIMENT 37 26. The multimeric antibody of embodiment 25, wherein the J chain or fragment or variant thereof is a mature human J chain comprising the amino acid sequence SEQ ID NO:7, or a fragment or variant thereof.

[0211] EMBODIMENT 38 38. The multimeric antibody of embodiment 37, wherein the J chain or fragment thereof is a variant J chain comprising an amino acid substitution at an amino acid position corresponding to amino acid Y102 of SEQ ID NO: 7, and an IgM antibody comprising said variant J chain exhibits increased serum half-life upon administration to an animal compared to a reference IgM antibody that is identical except for said amino acid substitution in the J chain and administered in the same manner to the same animal species.

[0212] EMBODIMENT 39 The multimeric antibody of embodiment 38, wherein the amino acid corresponding to Y102 of SEQ ID NO: 7 is replaced with alanine (A).

[0213] EMBODIMENT 40 The multimeric antibody of embodiment 39, wherein the variant J chain comprises the amino acid sequence SEQ ID NO:8.

[0214] EMBODIMENT 41 The multimeric antibody of any one of embodiments 25 to 40, wherein the J chain, or a fragment or variant thereof, is a modified J chain further comprising a heterologous moiety, wherein the heterologous moiety is fused or conjugated to the J chain, or a fragment or variant thereof.

[0215] EMBODIMENT 42 The multimeric antibody of embodiment 41, wherein the heterologous moiety is a heterologous polypeptide fused to the J chain, or a fragment or variant thereof.

[0216] EMBODIMENT 43 The multimeric antibody of embodiment 42, wherein the heterologous polypeptide is fused to the J chain, or a fragment or variant thereof, via a peptide linker comprising at least 5 amino acids and not more than 25 amino acids.

[0217] EMBODIMENT 44 The multimeric antibody of embodiment 42 or embodiment 43, wherein the heterologous polypeptide is fused to the N-terminus of the J chain or fragment or variant thereof, to the C-terminus of the J chain or fragment or variant thereof, or to both the N-terminus and C-terminus of the J chain or fragment or variant thereof, and the heterologous polypeptides fused to both the N-terminus and C-terminus may be the same or different.

[0218] EMBODIMENT 45 The multimeric antibody of any one of embodiments 42 to 44, wherein said heterologous polypeptide is an antigen-binding domain of an antibody or a subunit thereof.

[0219] EMBODIMENT 46 The multimeric antibody of embodiment 45, wherein the antigen-binding domain of the antibody comprises an scFv fragment.

[0220] EMBODIMENT 47 The multimeric antibody of any one of embodiments 44 to 46, wherein said heterologous polypeptide binds to CD3.

[0221] EMBODIMENT 48 The antigen-binding domain of the antibody binds to CD3 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, and wherein the VHCDR1, the VHCDR2, the VHCDR3, the VLCDR1, the VLCDR2, and the VLCDR3 are the amino acid sequences SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, The multimeric antibody of embodiment 46 or embodiment 47, comprising SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41; SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49; SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57; SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65; or SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73.

[0222] EMBODIMENT 49 The multimeric antibody of embodiment 48, wherein the VHCDR1, the VHCDR2, the VHCDR3, the VLCDR1, the VLCDR2, and the VLCDR3 each comprise the amino acid sequences SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively.

[0223] EMBODIMENT 50 49. The multimeric antibody of embodiment 48, wherein the antigen-binding domain of the antibody comprises VH and VL amino acid sequences, respectively, that are at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO:18 and SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:46, SEQ ID NO:50 and SEQ ID NO:54, SEQ ID NO:58 and SEQ ID NO:62, or SEQ ID NO:66 and SEQ ID NO:70.

[0224] EMBODIMENT 51 The multimeric antibody of embodiment 50, wherein the antigen-binding domains of the antibody comprise the VH and VL amino acid sequences SEQ ID NO:18 and SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:46, SEQ ID NO:50 and SEQ ID NO:54, SEQ ID NO:58 and SEQ ID NO:62, or SEQ ID NO:66 and SEQ ID NO:70, respectively.

[0225] EMBODIMENT 52 The multimeric antibody of embodiment 51, wherein the antigen-binding domain of the antibody comprises the VH and VL amino acid sequences SEQ ID NO:26 and SEQ ID NO:30, respectively.

[0226] EMBODIMENT 53 The multimeric antibody of any one of embodiments 12 to 52, capable of specifically binding to human CD123.

[0227] EMBODIMENT 54 54. The multimeric antibody of embodiment 53, which specifically binds to human CD123 with an affinity characterized by a dissociation constant KD of less than or equal to 500 nM, 100 nM, 50.0 nM, 40.0 nM, 30.0 nM, 20.0 nM, 10.0 nM, 9.0 nM, 8.0 nM, 7.0 nM, 6.0 nM, 5.0 nM, 4.0 nM, 3.0 nM, 2.0 nM, 1.0 nM, 0.50 nM, 0.10 nM, 0.050 nM, 0.01 nM, 0.005 nM or 0.001 nM.

[0228] EMBODIMENT 55 A composition comprising the antibody or fragment or derivative thereof of any one of embodiments 1 to 11, or the multimeric antibody of any one of embodiments 12 to 54.

[0229] EMBODIMENT 56 A polynucleotide comprising a nucleic acid sequence encoding the antibody or fragment or derivative thereof of any one of embodiments 1 to 11, or the multimeric antibody or subunit thereof of any one of embodiments 12 to 54.

[0230] EMBODIMENT 57 A vector comprising the polynucleotide of embodiment 56.

[0231] EMBODIMENT 58 A host cell comprising the vector of embodiment 57.

[0232] EMBODIMENT 59 A method for producing an antibody or a fragment or derivative thereof according to any one of embodiments 1 to 11, or a multimeric antibody according to any one of embodiments 12 to 54, comprising: Culturing the host cell of embodiment 58; and recovering said antibody or a fragment or derivative thereof, or said multimeric antibody. The method comprising:

[0233] EMBODIMENT 60 A method for treating cancer, comprising administering to a subject in need of treatment an effective amount of the antibody or fragment or derivative thereof of any one of embodiments 1-11, or the multimeric antibody of any one of embodiments 12-54.

[0234] EMBODIMENT 61 The method of embodiment 60, wherein the subject is a human.

[0235] EMBODIMENT 62 The method of embodiment 60 or embodiment 61, wherein the cancer is a hematological cancer.

[0236] EMBODIMENT 63 The method of embodiment 62, wherein said hematological cancer is acute myeloid leukemia (AML). EXAMPLES

[0237] Example 1: Humanization of anti-CD123 antibodies The sequence of anti-CD123 antibody 32716 is described in Du (2007) J Immunother 30:607-13. A 32716 homolog model was generated in BioLuminate based on the crystal structure of mouse Ab 2H4 (PDB 5YWF). The homology of 32716 to Ab 2H4 was determined using sequence alignment to be the following: VH identity 81%, VL identity 71%, VH framework identity 91%, VL framework identity 80%, and furthermore, the CDR lengths were identical in 5 / 6 CDRs (excluding light chain CDR1).

[0238] Two human antibodies were selected as framework acceptors: 4D9Q, with 75% VH framework identity (Figure 1A) and 70% VK framework identity (Figure 1B), and 4NWT, with 65% VH framework identity (Figure 1C) and 80% VK framework identity (Figure 1D). Several humanized sequences were designed based on each framework in Figure 1A-D. Six combinations of parental 32716 VH and VL, and humanized 32716 VH and VL sequences were cloned into human IgG format according to standard cloning protocols, as described in Table 2. Human IgG constructs were synthesized, expressed and purified by commercial suppliers (ATUM and Celltheon).

[0239] [Table 2]

[0240] Example 2: Biolayer Interferometry Affinity Measurement Assay of Humanized 32716 IgG Antibody The binding affinity of parental 32716 IgG and humanized variants to recombinant human CD123 protein (CD123)-His Acrobio#ILA-H52H6) was determined by BLI using an anti-pentaHis biosensor (Sartorius) on Octet-384 (Sartorius / Fortebio, NY, USA). PBST (1x PBS + 1% BSA + 0.05% Tween-20) buffer was used as antibody / CD123 dilution and sensor hydration buffer. The experiment was performed following a 5-step sequential assay at 24°C. First, the biosensor was hydrated for 10 min. Samples and buffer were applied to a 384-well plate. After an initial baseline of 60 s, the sensor was loaded with 7 nM hu IL-3R alpha (CD123)-His Acrobio#ILA-H52H6. The biosensor was immersed in PBST for 20 seconds to reach baseline, then incubated with 2-fold serially diluted anti-CD123 antibodies starting at 10 nM for association for 420 seconds, followed by incubation in PBST for 900 seconds for dissociation. Results were analyzed by ForteBio Data Analysis Software 9.0 using a 1:1 global fit model.

[0241] The binding properties of the parent 32716 IgG and the humanized variants are shown in Figures 2A-G, respectively. D , k on and k dis are shown in Table 3. The humanized IgG antibodies retained affinity for CD123 within 1.5-fold of the original mouse antibody. h32716-3-1 IgG showed slightly better affinity compared to the original mouse IgG antibody.

[0242] [Table 3]

[0243] Example 3: Binding to CD123-expressing cell lines A binding assay was performed to evaluate the binding ability of IgG antibodies against CD123 on CHO cells expressing human CD123. CHO cells expressing human CD123 were detached from flasks using trypsin. Cells (1×10 5 ) were pipetted into wells in a round-bottom 96-well plate, washed with FACS staining buffer (BD Pharmigen catalog #554656), pre-incubated with Fc Block (BD, #564220) for 10 minutes at room temperature, and then incubated with serial dilutions of 32716 or h32716 IgG antibodies for 30 minutes at 4°C. Cells were washed twice and stained with mouse anti-human kappa antibody conjugated with Alexa647 (Southern Biotech, clone SB81a). Cells were analyzed by flow cytometry. Results are shown in Figure 3. Mean fluorescence intensity (MFI) values ​​were analyzed by GraphPad Prism using a four-parameter logistic model. Data are shown in Table 4. All 32716 anti-CD123 humanized IgG antibodies bound with similar affinity and potency.

[0244] [Table 4]

[0245] Example 4: Conversion of humanized 32716 anti-CD123 to IgM and binding kinetics to CD123 To generate the IgM humanized construct, the two VH and VL regions of the humanized anti-CD123 sequence were assembled into an IgM with a modified J chain containing a CD3-binding scFv (SEQ ID NO: 84) according to standard cloning protocols to form a bispecific IgM antibody. The IgM antibody construct was expressed in Expi293 or CHO cells. The IgM antibody was purified according to the method described in Keyt, B., et al. Antibodies: 9: 53, doi: 10.3390 / antib9040053 (2020). The IgM antibody was assembled as a pentamer by the J chain.

[0246] The binding affinity of the CD123xCD3 IgM antibodies 32716IgM, h32716-1-1 IgM, and h32716-4-2 IgM to recombinant human CD123 protein (CD123, Fc-Fusion (IgG1) Avi-Tag, Biotin-Labeled, BPS Bioscience Cat. 100068-2) was determined by BLI with an Octet-384 (Sartorius / Fortebio, NY, USA) using an anti-human Fc (AFC) biosensor (Sartorius) as described in Example 2.

[0247] The binding of parental 32716 IgM, h32716-1-1 IgM, and h32716-4-2 IgM to CD123 is shown in Figures 4A-C, respectively. D , k on and k dis are shown in Table 5. The humanized IgM antibodies retained affinity for CD123 within 1.5-fold of the original murine antibody.

[0248] [Table 5]

[0249] Example 5: IgM antibody specificity measured by ELISA The specificity of anti-CD123xCD3 IgM antibodies 32716IgM, h32716-1-1 IgM, and h32716-4-2 IgM to human CD123 and CD3ε was measured in an ELISA assay as follows: 96-well white polystyrene ELISA plates (Pierce 15042) were coated with 100 μL per well of 0.2 μg / mL recombinant human CD123 protein (Sino Biological 10518-H08H-50) or 0.5 μg / ml recombinant human CD3ε protein (Acro Biosystems, CDE-H5256-100) overnight at 4° C. The plates were then washed five times with 0.05% PBS-Tween and blocked with 2% BSA-PBS. After blocking, 100 μL of serial dilutions of CD123×CD3 IgM antibodies, standards, and controls were added to the wells and incubated for 2 hours at room temperature. Plates were then washed 10 times and incubated with HRP-conjugated mouse anti-human kappa (Southern Biotech, 9230-05, diluted 1:6000 in 2% BSA-PBS) for 30 minutes. After 10 final washes with 0.05% PBS-Tween, plates were read using SuperSignal chemiluminescent substrate (ThermoFisher, 37070). Luminescence data was collected on an EnVision plate reader (Perkin-Elmer) and analyzed by GraphPad Prism using a 4-parameter logistic model. Binding of IgM bispecific antibodies to CD123 is shown in FIG. 5A and binding to CD3ε is shown in FIG. 5B.

[0250] Example 6: Binding of IgM antibodies to AML cell lines Binding assays were performed to assess the ability of anti-CD123xCD3 IgM antibodies 32716IgM, h32716-1-1 IgM, and h32716-4-2 IgM to bind to CD123 on AML cells expressing CD123 protein. MV411 cells were washed with FACS staining buffer (BD Pharmigen Cat#554656) and pre-incubated with Fc block (BD, 564220) for 10 min at room temperature. 5x10 4 The cells were stained with serial dilutions of IgM antibodies for 30 minutes at 4° C. The cells were washed twice and then stained with 5 μg / mL of anti-human IgM-PE-labeled secondary antibody (SB, clone SA-DA4) for 30 minutes at 4° C. The cells were washed twice, resuspended in FACS staining buffer, and captured by flow cytometry. The results are shown in FIG. 6.

[0251] Example 7: Humanized 32716 IgM bispecific antibody retains T cell directed AML cytotoxicity potency To demonstrate the ability of anti-CD123xCD3 IgM antibodies 32716IgM, h32716-1-1 IgM, and h32716-4-2 IgM to kill target cells in the presence of human T cells, we performed co-culture experiments. 3Tumor cells KG1a and MV4-11 were co-cultured with T cells at an effector to target (E:T) ratio of 7:1 in the presence of serial dilutions of anti-CD123xCD3 IgM antibodies in a total volume of 100 μL per well of AIM-V cell culture medium (GIBCO, #12055091) supplemented with 3% heat-inactivated fetal bovine serum (FBS, HyClone, #SH3007103HI). After 72 h of incubation at 37°C in a 5% CO2 incubator, 50 μl of supernatant was removed and frozen at -80°C for later cytokine release analysis. 50 μl of luciferase substrate (e.g. ONE-Glo EX Luciferase Assay System, Promega) was added to the wells. The plate was briefly shaken to mix the reagents and the luciferase luminescence signal was measured in an EnVision plate reader (Perkin-Elmer). The data was then analyzed by GraphPad Prism to obtain EC 50 Representative dose-response curves for KG1a and MV411 are shown in Figures 7A-7B, and the maximum percent killing and EC50 values ​​are shown in Table 6.

[0252] [Table 6]

[0253] Example 8: Effect of humanization on IgM stability and aggregation Equal concentrations of 32716 IgM, h32716-1-1 IgM, and h32716-4-2 IgM were formulated in the same buffer. The initial (t=0) percentage of high molecular weight aggregates, i.e., molecules with molecular weights greater than pentameric IgM with a J chain (%HMW), was determined by size-exclusion high performance liquid chromatography (HPLC). Aliquots of each antibody were subjected to 3-5 freeze-thaw cycles storing the aliquots at -80°C for 2-20 h and at 25°C for 2 h. Other aliquots were stored at 4°C or 40°C for 1 week. The %HMW of all aliquots was measured after processing. The results are shown in Figure 8.

[0254] h32716-1-1 IgM showed a better stability profile and lower aggregation tendency compared to 32716IgM and h32716-4-2 IgM.

[0255] Example 9: In Vivo Treatment with Humanized 32716 Eight-week-old female MHC- / -NSG mice were purchased from The Jackson Laboratory. 10 × 10 per mouse. 6 Mice were humanized by engraftment with 5 × 10 healthy human donor-derived peripheral blood mononuclear cells (PBMCs). Ten days after human PBMC engraftment, 5 × 10 6 MV4-11-gfp-luc tumor cells were mixed with Matrigel (1:1 with 1× phosphate buffered saline (PBS)) and implanted subcutaneously on the right flank of mice. One day after tumor implantation, mice were dosed intravenously (iv) every 3 days with vehicle for a total of 8 doses, iv with anti-CD123×CD3 IgG #1 (comprising CD123 VH and VL of SEQ ID NO:85 and SEQ ID NO:86, and CD3 scFv of SEQ ID NO:87) at 0.1 mg / kg every 3 days for a total of 5 doses, iv with h32716-1-1 IgM antibody at 5 mg / kg every 3 days for a total of 8 doses, or iv with h32716-1-1 IgM antibody at 15 mg / kg every 3 days for a total of 8 doses (n=10 animals / group).

[0256] The mean tumor volume over time over 75 days is shown in Figure 9A. Individual tumor volumes at day 75 are shown in Figure 9B. Individual tumor volumes over time over 75 days for vehicle, anti-CD123xCD3 IgG #1 treatment, 5 mg / kg h32716-1-1 IgM antibody, and 15 mg / kg h32716-1-1 IgM antibody are shown in Figures 10A-D, respectively.

[0257] Treatment with 5 mg / kg h32716-1-1 IgM antibody significantly reduced tumor volume compared to vehicle treatment: on day 75 of the study, 7 of 10 mice in the 5 mg / kg h32716-1-1 IgM antibody group were tumor-free, and 6 of 10 mice in the 15 mg / kg h32716-1-1 IgM antibody group were tumor-free.

[0258] Example 10: CD123×CD3 IgM Inhibits In Vitro Human AML Colony Formation To test the effect of CD123×CD3 IgM on the growth of multiple myeloma cells in vitro, a colony formation assay was used. Frozen bone marrow mononuclear cells from four separate acute myeloid leukemia (AML) donors were thawed and cultured at 0.5×10 in liquid medium containing cytokines (IL-3, GM-CSF, and SCF). 6 Cells were resuspended at 1000 cells / mL and seeded into wells of a 12-well plate. Test antibodies were added to each well at seven different concentrations (150, 50, 10, 2, 0.4, 0.08, 0.06 μg / mL). In addition, 50, 10 and 1 nM daunorubicin controls were evaluated. Wells containing only a solvent control were also included. The 12-well plates were incubated for 72 hours in a humidified incubator at 37° C. and 5% CO2. After incubation, the cells in each well were carefully dispersed by pipetting. 400 μL of cells (and media) were removed from each well and added to 4.0 mL of methylcellulose containing IL-3, GM-CSF and SCF. The tube of methylcellulose was vortexed to ensure that the cells were evenly distributed throughout the matrix. Triplicate 35 mm dishes of cultures were prepared per condition. Replicate dishes were placed at 37°C under 5% CO2 for a total of 14-16 days, after which the resulting colonies were assessed and counted based on morphology. The effector cell to T cell (E:T) ratios used for each donor are shown in Figure 11.

[0259] Figure 11 shows in vitro colony formation of multiple myeloma cells from four different donors after treatment with h32716-1-1. Treatment with h32716-1-1 reduced colony formation for all donors tested.

[0260] Example 11: Pharmacokinetic properties of CD123xCD3 IgM antibodies Pharmacokinetic parameters were measured for various CD123xCD3 IgM antibodies in an in vivo mouse model as follows: Balb / c mice were injected with 5 mg / kg h32716, h32716-4-2 or h32716-1-1 by intravenous bolus. Blood samples were collected at eight time points per antibody, with two mice per time point. Plasma concentrations of each antibody at each time point were measured using a sandwich ELISA assay. All ELISAs were validated for quality criteria and t 1 / 2 PK parameters, including clearance (CL), area under the curve (AUC) and maximum concentration (Cmax), were derived using non-linear curve fitting techniques (WinNonLin, Phoenix Software). A graph of concentration over time is shown in Figure 12, and the PK parameters are shown in Table 7.

[0261] [Table 7]

[0262] Example 12: CD123xCD3 IgM induces lower levels of cytokines from T cells TDCC assay method MV4-11 leukemia cells were labeled with the PKH26 red fluorescent tag using the PKH26 red fluorescent cell linker kit (Sigma-Aldrich, Cat#MINI26-1kt) according to the manufacturer's instructions. Briefly, 5 × 10 6MV4-11 cells were washed with serum-free RPMI-1640 medium (Gibco, REF22400-071). After centrifugation, 0.25 ml of Diluent C was transferred to the cell pellet and resuspended by gentle pipetting. Just before staining, 4 μl of ethanolic dye solution of PKH26 was added to 1.5 ml of Diluent C (2×) in a polypropylene centrifuge tube and mixed well to disperse, resulting in a 2× dye solution. 0.25 ml of 2× dye solution was quickly added to 0.25 ml of 2× cell suspension and the sample was mixed immediately. The sample was incubated for 5 min and staining was stopped by adding 0.5 ml of inactivated FBS. Cells were centrifuged and resuspended in culture medium to obtain 2.5×10 5 Human peripheral blood mononuclear cells (PBMCs) (IQ Biosciences, IQB-PBMC103) were washed and cultured in culture medium at a final concentration of 2 × 10 6 The cells were resuspended at 100 μl / ml. 80 μl of MV4-11 cells, 100 μl of PBMCs, and 20 μl of 5-fold serially diluted test antibody were added to each well at a highest final concentration of 5 nM. Plates were incubated for 48 and 72 hours. FACS analysis was performed to determine the number of viable tumor cells.

[0263] MSD Assay Methods Cytokine levels from supernatants collected from MV4-11 cell TDCC assays at 48 and 72 hours were measured by MSD Pro-Inflammatory Panel 1 (Human) Kit (V-PLEX, Cat#K15049D-2). Briefly, TDCC media was diluted 25-fold, 5-fold or 2-fold with Diluent 2. Detection antibody solution was prepared by adding 60 μL of SULFO-TAG anti-human IFN-γ, IL-6 or IL-10 to 2.4 ml of Diluent 3. Plates were washed 3 times with 200 μl / well of wash buffer and then incubated with 50 μL / well of diluted samples or standards for 2 hours at room temperature with shaking. After washing 3 times with 200 μl / well of wash buffer, plates were incubated with 25 μL of detection antibody solution for 2 hours at room temperature with shaking. Plates were then washed 3 times with 200 μl / well of wash buffer. 150 uL of 2x Read Buffer T was added to each well of the plate and the plate was analyzed on the MSD instrument.

[0264] The calibration curve used to calculate the analyte concentration was calculated by multiplying the signal from the standards by 1 / Y 2 The ECL coefficients were established by fitting a four-parameter logistic model with weighting of . Analyte concentrations were determined from the ECL signals by backfitting to a calibration curve. Calculations to establish the calibration curve and to determine concentrations were performed using MSD DISCOVERY WORKBENCH analysis software.

[0265] The number of viable cells, IFNγ, IL-6 and IL-10 levels after 48 hours of TDCC using either CD123×CD3 IgG #1 or h32716-1-1 are shown in Figures 13A-D, respectively, and after 72 hours of TDCC using either CD123×CD3 IgG #1 or h32716-1-1 are shown in Figures 14A-D, respectively. At concentrations that produced equivalent levels of cell killing, h32716-1-1 produced much lower levels of cytokine production compared to CD123×CD3 IgG #1.

[0266]

Table 8

[0267]

Table 9

Claims

1. A multimeric antibody comprising 5, 6 or 2 bivalent binding units and 10, 12 or 4 antigen-binding domains, each of which specifically binds to CD123, Each antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); the VH and the VL each comprise the amino acid sequence SEQ ID NO:76 and SEQ ID NO:79, SEQ ID NO:77 and SEQ ID NO:79, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:83, SEQ ID NO:81 and SEQ ID NO:83, or SEQ ID NO:82 and SEQ ID NO:83; and Each heavy chain constant region comprises two heavy chains, each of which comprises an IgM or IgA constant region associated with a copy of VH, and two light chains, each of which comprises a light chain constant region associated with a copy of VL; The multimeric antibody.

2. is a pentamer and contains five bivalent binding units, and further contains a J chain or a variant thereof; each binding unit is an IgM heavy chain constant region comprising a Cμ4 domain and a μ tailpiece (μtp) domain, and optionally further comprising a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof; The multimeric antibody of claim 1.

3. 3. The multimeric antibody of claim 2, wherein each IgM heavy chain constant region is a human IgM constant region comprising the amino acid sequence SEQ ID NO:1, SEQ ID NO:2, or a multimerized variant or fragment thereof.

4. The multimeric antibody described in claim 3, wherein each IgM heavy chain constant region is a variant human IgM heavy chain constant region comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, except for amino acid substitutions at one or more positions L310, P311, P313, and / or K315 of SEQ ID NO:1 or SEQ ID NO:

2.

5. The multimeric antibody of claim 2 , wherein the J chain is a mature human J chain comprising the amino acid sequence SEQ ID NO: 7, or a variant thereof.

6. the J chain is a variant J chain comprising an amino acid substitution at an amino acid position corresponding to amino acid Y102 of SEQ ID NO:7; said multimeric antibody exhibits an increased serum half-life upon administration to an animal compared to a reference multimeric antibody that is identical except for said amino acid substitutions in the J chain and that is administered in the same manner to the same animal species. The multimeric antibody of claim 5.

7. The multimeric antibody of claim 6, wherein the amino acid position corresponding to amino acid Y102 of SEQ ID NO:7 is substituted with alanine (A), and the variant J chain comprises the amino acid sequence SEQ ID NO:

8.

8. The multimeric antibody of claim 2, wherein the J chain, or a fragment or variant thereof, is a modified J chain further comprising a heterologous moiety fused or conjugated to the J chain, or a fragment or variant thereof.

9. The multimeric antibody of claim 8 , wherein the heterologous moiety is a heterologous polypeptide fused to the J chain or a fragment or variant thereof.

10. The multimeric antibody of claim 9 , wherein the heterologous polypeptide comprises an antigen-binding domain of an antibody that is a scfv fragment.

11. The multimeric antibody of claim 10, wherein the scFv fragment binds to CD3.

12. A composition comprising a multimeric antibody described in any one of claims 1 to 11.

13. A polynucleotide comprising a nucleic acid sequence encoding a multimeric antibody described in any one of claims 1 to 11.

14. A host cell comprising the polynucleotide of claim 13.

15. A pharmaceutical composition for use in the treatment of blood cancer, comprising a multimeric antibody described in any one of claims 1 to 11.

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