Cd33 antibodies and methods of using the same to treat cancer

Abstract

To provide immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) that can bind to the CDS 3 protein.SOLUTION: An antibody or antigen binding fragment thereof comprises a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) the VH comprises a VH-CDR1 sequence of SEQ ID NO: 154, a VH CDR2 sequence of SEQ ID NO: 155, and a VH-CDR3 sequence of SEQ ID NO: 156; and (b) the VL comprises a VL-CDR1 sequence of SEQ ID NO: 157, a VL-CDR2 sequence of SEQ ID NO: 158, and a VL-CDR3 sequence of SEQ ID NO: 159.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 809,091, filed February 22, 2019, the entire disclosure of which is incorporated herein by reference. The present technology generally relates to the preparation and use of immunoglobulin-related constructs (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to the CD33 protein. In particular, the present technology relates to the preparation of CD33-binding antibodies and their use in the detection and treatment of CD33-associated cancers and Alzheimer's disease. [Background technology]

[0002] The following description of the background of the present technology is provided merely as an aid in the understanding of the present technology and is not admitted to describe or constitute prior art to the present technology. Acute myeloid leukemia (AML) accounts for 25% of childhood leukemia cases but more than half of childhood leukemia deaths. In contrast to acute lymphoblastic leukemia, which is curable in more than 80% of children, childhood AML has the poorest 5-year survival rate of any childhood cancer. In adults diagnosed before age 60, AML is curable in 35-40% of cases, but in patients who develop the disease after age 60, only 5-15% are curable (Dohner et al., N Engl J Med 373:1136-52 (2015)). Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure generally provides immunoglobulin-related constructs (eg, antibodies or antigen-binding fragments thereof) capable of specifically binding to a CD33 polypeptide. [Means for solving the problem]

[0004] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof comprising: H is the V of GYSFTDYN (SEQ ID NO: 154) H - CDR1 sequence, V of IDPYKGGT (SEQ ID NO: 155) H -CDR2 sequence and V of AREMITAYYFDY (SEQ ID NO: 156) H -CDR3 sequence, and (b) V L is the V of QDINKY (sequence number 157) L -CDR1 sequence, V of YAS (SEQ ID NO: 158) L -CDR2 sequence and V of LQYDNLLT (SEQ ID NO: 159) L - an antibody or antigen-binding fragment thereof comprising the CDR3 sequence. In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof comprising: H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133; and / or (b) V L provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0005] In any of the above embodiments, the antibody may further comprise an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen-binding fragment is a Fab, F(ab') 2 , Fab', scF v and F vIn some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In certain embodiments, the antibody or antigen-binding fragment binds to the IgC2 domain of CD33. In another aspect, the disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:141, or a variant thereof with one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:140, or a variant thereof with one or more conservative amino acid substitutions. In certain embodiments, the antibodies are selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:14 (chHIM34xCD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34xCD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34xCD3 BsAb; SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5xmC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5xhC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6xmC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6xhC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6xmC825); SEQ ID NO:22 and SEQ ID NO:29 (VL4VH6xhC825), SEQ ID NO:139 and SEQ ID NO:138 (mouse VL-mouse VHxmC825); and SEQ ID NO:141 and SEQ ID NO:140 (mouse VL-mouse VHxhC825).

[0006] In one aspect, the disclosure provides an antibody comprising: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 11, 12 or 13; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7 or 133. In another aspect, the disclosure provides an antibody comprising: (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to an LC sequence present in any one of SEQ ID NOs: 14, 18, 24, 26, 27, 28, 29, 134, 138 or 140; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to an HC sequence present in any one of SEQ ID NOs: 16, 20, 22, 136, 139 or 141.

[0007] In any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. In certain embodiments, the antibody of the present technology comprises an IgG4 constant region comprising an S228P mutation. In any of the above embodiments, the antibody binds to the IgC2 domain of CD33. Additionally or alternatively, in some embodiments, the antibody of the present technology lacks α-1,6-fucose modification. In one aspect, the disclosure provides a bispecific antibody or antigen-binding fragment comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 30-113 or 142-153. In certain embodiments, the bispecific antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 30-113 or 142-153.

[0008] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminus to C-terminus, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) an amino acid sequence (GGGGS), 6 (iii) a light chain variable domain of a first immunoglobulin; and (iv) an amino acid sequence (GGGGS). 4 (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) the amino acid sequence (GGGGS). 6 (vii) a light chain variable domain of a second immunoglobulin, (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT, and (ix) a self assembly disassembly (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0009] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminus to C-terminus, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) an amino acid sequence (GGGGS), 6(iii) a heavy chain variable domain of a first immunoglobulin; and (iv) an amino acid sequence (GGGGS). 4 (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) the amino acid sequence (GGGGS). 6 (vii) a flexible peptide linker comprising the amino acid sequence TPLGDTTHT; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly-disassembly (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises a tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs: 30-113 or 142-153.

[0010] In one aspect, the disclosure provides a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and (a) each of the first and fourth polypeptide chains comprises, from N-terminus to C-terminus, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, (iii) a light chain constant domain of the first immunoglobulin, and (iv) an amino acid sequence of (GGGGS). 3and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light and heavy chain variable domains of the second immunoglobulin are capable of specifically binding to a second epitope, and have the amino acid sequence (GGGGS) 6 and (b) each of the second and third polypeptide chains comprises, from N-terminus to C-terminus, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or a small molecule DOTA hapten.

[0011] In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments described herein. In some embodiments, the recombinant nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 135 and 137. In another aspect, the disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0012] In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharma- ceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0013] In some embodiments of the bispecific antibody or antigen-binding fragment of the present technology, the bispecific antibody binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibody or antigen-binding fragment binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or a small molecule DOTA hapten. The small molecule DOTA hapten is DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH 2 , Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH 2 , DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH 2 ;DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH 2 , Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH 2 , Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2、Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH 2 、Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 、DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 、(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH 2 、Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 、(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 、Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH 2 、Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 、Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH 2 、およびAc-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH 2 The lights were turned off.

[0014] In another aspect, the disclosure provides a method of treating a CD33-associated cancer or Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the antibodies are selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 14 (chHIM34xCD3 BsAb); SEQ ID NO: 20 and SEQ ID NO: 18 (BC249-hHIM34xCD3 BsAb); SEQ ID NO: 136 and SEQ ID NO: 134 (BC275-hHIM34xCD3 BsAb); SEQ ID NO: 22 and SEQ ID NO: 18 (BC267-hHIM34xCD3 BsAb); SEQ ID NO: 22 and SEQ ID NO: 24 (BC268-hHIM34xCD3 BsAb; SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5xmC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5xhC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6xmC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6xhC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6xmC825); SEQ ID NO:22 and SEQ ID NO:29 (VL4VH6xhC825), SEQ ID NO:139 and SEQ ID NO:138 (mouse VL-mouse VHxmC825); and SEQ ID NO:141 and SEQ ID NO:140 (mouse VL-mouse VHxhC825), and the antibody specifically binds to CD33. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs:30-113 or 142-153.

[0015] In some embodiments, the CD33-associated cancer is AML, biphenotypic leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloblastic crisis of chronic myelogenous leukemia, or acute lymphoblastic leukemia. Additionally or alternatively, in some embodiments of the methods, the antibody or antigen-binding fragment is administered to the subject separately, sequentially or simultaneously with an additional therapeutic agent, such as one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen agent, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, antimetabolite, an endocrine / hormonal agent, or a bisphosphonate therapy.

[0016] In another aspect, the present disclosure provides a method of detecting a tumor in a subject in vivo, comprising: (a) administering to the subject an effective amount of an antibody or antigen-binding fragment of the present technology, the antibody or antigen-binding fragment being configured to localize to tumors expressing CD33 and labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody or antigen-binding fragment that is higher than a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. The radioactivity level emitted by the antibody or antigen-binding fragment can be detected using positron emission tomography or single-photon emission computed tomography.

[0017] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody or antigen-binding fragment of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle radioisotope, a beta particle radioisotope, an Auger emitter, or any combination thereof. Examples of beta particle radioisotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., by the N297A mutation in the Fc region resulting in aglycosylation).

[0018] Also disclosed herein is a kit for detecting and / or treating CD33-associated cancer or Alzheimer's disease, comprising at least one immunoglobulin-related construct (e.g., any antibody or antigen-binding fragment described herein) or a functional variant (e.g., substitution variant) of the present technology and instructions for use. In certain embodiments, the immunoglobulin-related construct is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels include a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-CD33 immunoglobulin-related construct described herein, hi some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

[0019] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that binds to the radiolabeled DOTA hapten and the CD33 antigen, wherein the conjugate is configured to localize to a tumor that expresses the CD33 antigen recognized by the bispecific antibody or antigen-binding fragment of the conjugate; (b) detecting the level of radioactivity emitted by the conjugate; and (c) selecting the subject for pretargeted radioimmunotherapy if the level of radioactivity emitted by the conjugate is higher than a reference value.

[0020] In one aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD33-associated cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to a CD33 antigen target, wherein the conjugate is configured to localize to a tumor that expresses the CD33 antigen target recognized by the bispecific antibody or antigen-binding fragment of the conjugate. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to a CD33 antigen target, wherein the conjugate is configured to localize to a tumor that expresses the CD33 antigen target recognized by the bispecific antibody or antigen-binding fragment of the conjugate.

[0021] In any of the above embodiments of the methods disclosed herein, the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments of the methods disclosed herein, the subject is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re,177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th, or 64 It includes Cu and may include alpha particle radioisotopes, beta particle radioisotopes or Auger emitters.

[0022] In one aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD33-associated cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present technology, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to localize to a tumor expressing a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present technology, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to localize to a tumor expressing a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In some embodiments, the method of the present technology further comprises administering to the subject an effective amount of a clearance agent prior to administration of the radiolabeled DOTA hapten.

[0023] Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th, or 64 Cu and may include an alpha particle radioisotope, a beta particle radioisotope or an Auger emitter. In any of the above embodiments of the methods disclosed herein, the subject is a human. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 shows the schematic structure of CD33 isoforms, with the full-length CD33 protein on the left and shorter isoforms (splice variants lacking the outer IgV domain) on the right. [Diagram 2]FIG. 2 shows a list of existing anti-CD33 antibodies and their domain specificity as demonstrated by their ability to bind to full-length CD33 protein and / or short isoforms of CD33. [Diagram 3] FIG. 3 shows a schematic diagram of the modular format of tetravalent IgG-scFvs, consisting of an IgG molecule with two binding sites and covalently linked two scFvs with two additional binding domains. [Figure 4] FIG. 4 shows FACS data demonstrating that chimeric HIM34×CD3 bispecific antibody (chHIM34 BsAb) binds to the CD33(+) AML cell lines U937, THP1, SET2, C1498-CD33, and M-07e, but has no effect on the CD33(−) leukemia cell line CMLT1. [Figure 5A] FIG. 5(A) shows a schematic diagram of the full-length CD33 protein and the shorter isoforms (splice variants of CD33). [Figure 5B] FIG. 5(B) shows FACS data demonstrating that the chHIM34 BsAb binds both full-length and short isoforms of CD33, whereas the humanized M195-based BsAb (BC133) does not bind to the short isoform. [Figure 6A] 6(A) and 6(B) show T cell dependent cytotoxicity assays of chHIM34 BsAb against the CD33(+) AML cell lines C1498-CD33 and THP1, respectively. [Figure 6B] 6(A) and 6(B) show T cell dependent cytotoxicity assays of chHIM34 BsAb against the CD33(+) AML cell lines C1498-CD33 and THP1, respectively. [Figure 7A] FIG. 7(A) shows bioluminescence images showing the growth of MOLM13 AML tumors in different groups. [Figure 7B] FIG. 7(B) shows the quantification of the signals in the different groups of mice from FIG. 7(A). [Figure 8]8 shows the amino acid sequences of mouse and humanized HIM34 heavy chain variable domains (SEQ ID NOs: 1-7 and 133). HIM34_VH-1, HIM34_VH-2, HIM_34_VH-3, HIM34_VH-4, HIM34_VH-5, HIM34-VH-6, HIM-34-VH-7 were the seven versions of the humanized HIM34 heavy chain variable domain. The sequences of VH-CDR1, VH-CDR2, and VH-CDR3 are shown in bold. [Figure 9] 9 shows the amino acid sequences of mouse and humanized HIM34 light chain variable domains (SEQ ID NOs: 8-13). HIM34_VL-1, HIM34_VL-2, HIM_34_VL-3, HIM34_VL-4, and HIM34_VL-5 were five versions of the humanized HIM34 light chain variable domain. The sequences of VL-CDR1, VL-CDR2, and VL-CDR3 are shown in bold. [Figure 10A] Figures 10(A) and 10(B) show the amino acid and nucleotide sequences of the light and heavy chains, respectively, of chHIM34 BsAb (SEQ ID NOs: 14-17). The signal peptide is underlined, the variable domains of the chimeric anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. Also disclosed are the amino acid sequences of the VH and VL domains of chHIM34 BsAb (SEQ ID NOs: 114-115). [Figure 10B] Figures 10(A) and 10(B) show the amino acid and nucleotide sequences of the light and heavy chains, respectively, of chHIM34 BsAb (SEQ ID NOs: 14-17). The signal peptide is underlined, the variable domains of the chimeric anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. Also disclosed are the amino acid sequences of the VH and VL domains of chHIM34 BsAb (SEQ ID NOs: 114-115). [Figure 11A]Figures 11(A) and 11(B) show the amino acid and nucleotide sequences of the light and heavy chains of humanized HIM34xCD3 BsAb, BC249 (SEQ ID NOs: 18-21), respectively. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 11B] Figures 11(A) and 11(B) show the amino acid and nucleotide sequences of the light and heavy chains of humanized HIM34xCD3 BsAb, BC249 (SEQ ID NOs: 18-21), respectively. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 11C] Figures 11(C) and 11(D) show the amino acid and nucleotide sequences of the light and heavy chains of humanized HIM34xCD3 BsAb, BC275 (SEQ ID NOs: 134-137), respectively. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 11D] Figures 11(C) and 11(D) show the amino acid and nucleotide sequences of the light and heavy chains of humanized HIM34xCD3 BsAb, BC275 (SEQ ID NOs: 134-137), respectively. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 12A] Figures 12(A) and 12(B) show the amino acid and nucleotide sequences of the light and heavy chains, respectively, of the humanized HIM34xCD3 BsAb, BC267 (SEQ ID NOs: 18, 19, 22 and 23). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 12B]Figures 12(A) and 12(B) show the amino acid and nucleotide sequences of the light and heavy chains, respectively, of the humanized HIM34xCD3 BsAb, BC267 (SEQ ID NOs: 18, 19, 22 and 23). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 13A] Figures 13(A) and 13(B) show the amino acid and nucleotide sequences of the light and heavy chains, respectively, of the humanized HIM34xCD3 BsAb, BC268 (SEQ ID NOs: 24, 25, 22 and 23). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 13B] Figures 13(A) and 13(B) show the amino acid and nucleotide sequences of the light and heavy chains, respectively, of the humanized HIM34xCD3 BsAb, BC268 (SEQ ID NOs: 24, 25, 22 and 23). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14A] Figures 14(A)-14(F) show the amino acid sequences of the light and heavy chains of humanized HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequences of the light chain are shown in SEQ ID NOs: 26-29 and the heavy chain are shown in SEQ ID NOs: 20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14B] Figures 14(A)-14(F) show the amino acid sequences of the light and heavy chains of humanized HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequences of the light chain are shown in SEQ ID NOs: 26-29 and the heavy chain are shown in SEQ ID NOs: 20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14C]Figures 14(A)-14(F) show the amino acid sequences of the light and heavy chains of humanized HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequences of the light chain are shown in SEQ ID NOs: 26-29 and the heavy chain are shown in SEQ ID NOs: 20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14D] Figures 14(A)-14(F) show the amino acid sequences of the light and heavy chains of humanized HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequences of the light chain are shown in SEQ ID NOs: 26-29 and the heavy chain are shown in SEQ ID NOs: 20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14E] Figures 14(A)-14(F) show the amino acid sequences of the light and heavy chains of humanized HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequences of the light chain are shown in SEQ ID NOs: 26-29 and the heavy chain are shown in SEQ ID NOs: 20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14F] Figures 14(A)-14(F) show the amino acid sequences of the light and heavy chains of humanized HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequences of the light chain are shown in SEQ ID NOs: 26-29 and the heavy chain are shown in SEQ ID NOs: 20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14G]Figures 14(G)-14(H) show the amino acid sequences of the light and heavy chains of the murine HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequence of the light chain is shown in SEQ ID NOs: 138 and 140, and the amino acid sequence of the heavy chain is shown in SEQ ID NOs: 139 and 141. The signal peptide is underlined, the variable domains of the murine anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 14H] Figures 14(G)-14(H) show the amino acid sequences of the light and heavy chains of the murine HIM34xC825 (anti-DOTA) BsAb in IgG-scFv format. The amino acid sequence of the light chain is shown in SEQ ID NOs: 138 and 140, and the amino acid sequence of the heavy chain is shown in SEQ ID NOs: 139 and 141. The signal peptide is underlined, the variable domains of the murine anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15A] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15B] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15C] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15D]Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15E] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15F] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15G] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15H] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15I]Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15J] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15K] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15L] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15M] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15N]Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15O] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15P] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15Q] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15R] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15S]Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15T] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15U] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15V] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15W] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Fig. 15X]Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15Y] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15Z] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15AA] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Diagram 15BB] Figures 15(A)-15(BB) show the amino acid sequences (SEQ ID NOs: 30-113) of humanized HIM34xC825 (anti-DOTA) BsAb in single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15CC]Figures 15(CC)-15(FF) show the amino acid sequence (SEQ ID NOs: 142-153) of the murine HIM34xC825 (anti-DOTA) BsAb in a single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the murine-based anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Fig. 15DD] Figures 15(CC)-15(FF) show the amino acid sequence (SEQ ID NOs: 142-153) of the murine HIM34xC825 (anti-DOTA) BsAb in a single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the murine-based anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 15EE] Figures 15(CC)-15(FF) show the amino acid sequence (SEQ ID NOs: 142-153) of the murine HIM34xC825 (anti-DOTA) BsAb in a single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the murine-based anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Diagram 15FF] Figures 15(CC)-15(FF) show the amino acid sequence (SEQ ID NOs: 142-153) of the murine HIM34xC825 (anti-DOTA) BsAb in a single chain bispecific tandem fragment variable (scBsTaFv) format. The signal peptide is underlined, the variable domains of the murine-based anti-CD33 antibody are shown in bold, and the linker sequence is italicized and underlined. [Figure 16A] Figures 16(A) and 16(B) provide an overview of the characteristics, including binding kinetics, of chimeric and humanized HIM34 BsAbs. [Figure 16B] Figures 16(A) and 16(B) provide an overview of the characteristics, including binding kinetics, of chimeric and humanized HIM34 BsAbs. [Figure 17] FIG. 17 shows FACS analysis of MOLM13 CD33(+) cells contacted with different concentrations of humanized CD33 BsAb of the present technology. [Figure 18A] 18(A) to 18(B) show the cytotoxicity of the humanized BsAb of the present technology against MOLM13 CD33(+) cells in a T cell-dependent cytotoxicity assay (TDCC). [Figure 18B] 18(A) to 18(B) show the cytotoxicity of the humanized BsAb of the present technology against MOLM13 CD33(+) cells in a T cell-dependent cytotoxicity assay (TDCC). [Figure 19] FIG. 19 shows the stability of humanized BsAb of the present technology at 40° C. [Figure 20A] FIG. 20(A) shows T cell engaging bispecific antibodies (BsAbs) that recruit T cells to AML cells. [Figure 20B] FIG. 20(B) illustrates that the BC275 BsAb disclosed herein binds to the membrane proximal domain of the extracellular region of CD33 (IgC), whereas BC133 (lintuzumab) based on the M195 clone and BC269 (gemtuzumab) based on My96 bind to the membrane distal domain of the extracellular region of CD33. [Figure 20C] Figure 20(C) shows the CD33 binding characteristics of BC275, BC133, and BC269 BsAbs to various CD33(+) and CD33(-) human cancer cell lines. [Figure 21A] Figures 21(A)-21(C) show the T cell-dependent cell-mediated cytotoxicity effects of BC275, BC133, and BC269 BsAbs against CD33(+) and CD33(-) human cancer cell lines. [Figure 21B] Figures 21(A)-21(C) show the T cell-dependent cell-mediated cytotoxicity effects of BC275, BC133, and BC269 BsAbs against CD33(+) and CD33(-) human cancer cell lines. [Figure 21C] Figures 21(A)-21(C) show the T cell-dependent cell-mediated cytotoxicity effects of BC275, BC133, and BC269 BsAbs against CD33(+) and CD33(-) human cancer cell lines. [Figure 21D] Figure 21(D) plots the correlation between the expression level of CD33 on cancer cells in the TDCC assay and the potency of the BsAbs tested. Pooled data for all three CD3xCD33 BsAbs is shown. [Figure 22A] Figures 22(A)-22(B) show tumor size in immunodeficient NSG mice inoculated intravenously with CD33(+)MOLM13-luciferase (genotype CC at rs12459419 SNP) human AML xenografts (106 cells) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected once with 5x106 activated T cells and 0.025μg of BsAb (10μg / Kg / dose). Timing of T cell and antibody injection is shown in Figure 22(B). [Figure 22B] Figures 22(A)-22(B) show tumor size in immunodeficient NSG mice inoculated intravenously with CD33(+)MOLM13-luciferase (genotype CC at rs12459419 SNP) human AML xenografts (106 cells) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected once with 5x106 activated T cells and 0.025μg of BsAb (10μg / Kg / dose). Timing of T cell and antibody injection is shown in Figure 22(B). [Figure 23A] Figures 23(A)-23(B) show tumor size in immunodeficient NSG mice inoculated intravenously with human CD33-transduced NALM6-luciferase leukemia xenografts (0.5x106 cells) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected twice with activated T cells (2.7x106 and 5x106 cells, with a 1 week interval between doses) supplemented with 10ng BsAb / 106 T cells. The timing of T cell and BsAb injections is shown in Figure 23(B). [Figure 23B] Figures 23(A)-23(B) show tumor size in immunodeficient NSG mice inoculated intravenously with human CD33-transduced NALM6-luciferase leukemia xenografts (0.5x106 cells) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or the control BC119 BsAb (CD3xGD2 BsAb). Mice were injected twice with activated T cells (2.7x106 and 5x106 cells, with a 1 week interval between doses) supplemented with 10ng BsAb / 106 T cells. The timing of T cell and BsAb injections is shown in Figure 23(B). [Figure 23C] Figure 23(C) shows survival curves of animals treated with three CD3xCD33 BsAbs. The potency of CD3xCD33 BsAbs was ranked as follows: BC275>BC269=BC133. [Figure 24A] Figures 24(A)-24(D) show tumor size in immunodeficient DKO mice inoculated intravenously with THP1 human leukemia xenografts (genotype CC at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected twice (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 24(A). The potency of CD3xCD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 24B]Figures 24(A)-24(D) show tumor size in immunodeficient DKO mice inoculated intravenously with THP1 human leukemia xenografts (genotype CC at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected twice (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 24(A). The potency of CD3xCD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 24C] Figures 24(A)-24(D) show tumor size in immunodeficient DKO mice inoculated intravenously with THP1 human leukemia xenografts (genotype CC at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected twice (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 24(A). The potency of CD3xCD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 24D]Figures 24(A)-24(D) show tumor size in immunodeficient DKO mice inoculated intravenously with THP1 human leukemia xenografts (genotype CC at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3xCD33 BsAbs) or control BC119 BsAb (CD3xGD2 BsAb). Mice were injected twice (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 24(A). The potency of CD3xCD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 25A] Figures 25(A)-25(D) show tumor size in immunodeficient DKO mice inoculated intravenously with K562 human leukemia xenografts (genotype TT at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). Mice were injected three times (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 25(A). The potency of CD3×CD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 25B]Figures 25(A)-25(D) show tumor size in immunodeficient DKO mice inoculated intravenously with K562 human leukemia xenografts (genotype TT at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). Mice were injected three times (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 25(A). The potency of CD3×CD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 25C] Figures 25(A)-25(D) show tumor size in immunodeficient DKO mice inoculated intravenously with K562 human leukemia xenografts (genotype TT at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). Mice were injected three times (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 25(A). The potency of CD3×CD33 BsAbs was ranked as follows: BC275>BC269>BC133. [Figure 25D]Figures 25(A)-25(D) show tumor size in immunodeficient DKO mice inoculated intravenously with K562 human leukemia xenografts (genotype TT at rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). Mice were injected three times (107 cells, 1 week between doses) with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). The timing of T cell and antibody injections is shown in Figure 25(A). The potency of CD3×CD33 BsAbs was ranked as follows: BC275>BC269>BC133. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] It will be appreciated that certain aspects, modes, embodiments, variations and features of the methods are described below at varying levels of detail in order to provide a substantial understanding of the present technology. The present disclosure generally provides immunoglobulin-related constructs (e.g., antibodies or antigen-binding fragments thereof) that can specifically bind to CD33 polypeptides. The immunoglobulin-related constructs of the present technology are useful in methods for detecting or treating CD33-related cancer or Alzheimer's disease in subjects in need thereof. Thus, various aspects of the present method relate to the preparation, characterization and manipulation of anti-CD33 antibodies. The immunoglobulin-related constructs of the present technology are useful alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related constructs are humanized antibodies, chimeric antibodies or bispecific antibodies.

[0026] The practice of the subject methods employs many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0027] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this technology belongs. As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly indicates otherwise. For example, reference to a "cell" includes a combination of two or more cells, and the like. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are well known and commonly used in the art.

[0028] As used herein, the term "about" in connection with numbers is generally interpreted as including numbers that fall within 1%, 5% or 10% in either direction (more or less) of the numerical value, unless otherwise stated or apparent from the context (except where such number is less than 0% or more than 100% of the possible value). As used herein, "administration" of an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be performed by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and administration by another.

[0029] "Adjuvant" refers to one or more substances that cause the stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants can be administered to a subject before, in combination with, or after the administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and costimulatory factors.

[0030] As used herein, the term "antibody" collectively refers to immunoglobulin or immunoglobulin-like molecules including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as in non-mammalian species, such as shark immunoglobulins. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragments" specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to substantially exclude binding to other molecules (e.g., with a binding constant at least 10 times higher than the binding constant for other molecules in a biological sample). 3 M -1 Greater than, at least 10 4 M -1 Greater than or at least 10 5 M -1 (Antibodies and antibody fragments having a larger binding constant for a molecule of interest). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.);Kuby, J., Immunology,3 rd Ed., W. H. Freeman & Co., New York, 1997. See also

[0031] More specifically, an antibody refers to a polypeptide ligand that contains at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region and specifically recognizes and binds to an epitope of an antigen. An antibody is composed of heavy and light chains, each of which has a heavy chain variable domain (V H) region and the light chain variable (V L ) regions. H Area and V L The regions are involved in binding to the antigen recognized by the antibody. Usually, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of various light or heavy chains are relatively conserved within a species. The framework regions of antibodies, which are the combined framework regions of the component light and heavy chains, mainly adopt a β-sheet conformation, and the CDRs form loops that connect with, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that allows the CDRs to be positioned in the correct orientation by interchain, non-covalent interactions.

[0032] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus, and are usually identified by the chain in which the individual CDR is located. Thus, V HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, V L CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the CD33 protein have a particular V H Area and V L Each CDR has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although the CDRs are different for each antibody, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. These positions in the CDRs are called specificity determining residues (SDRs). "Immunoglobulin-related constructs" as used herein refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or an antigen-binding fragment thereof specifically binds to an antigen.

[0033] As used herein, the term "antibody-related polypeptide" refers to a variable region alone or in combination with the following polypeptide elements: hinge region, CH, 1 , C.H. 2 and C.H. 3 The term "antibody fragment" refers to an antigen-binding antibody fragment, including a single chain antibody, which may comprise a combination of all or a portion of the variable region, as well as the hinge region, CH domain, or the like. 1 , C.H. 2 and C.H. 3 Any combination of domains is encompassed by the present technology. Antibody-related molecules useful in the present methods include, but are not limited to, Fab, Fab' and F(ab') 2 , Fd, single chain Fv (scFv), single chain antibody, disulfide-linked Fv (sdFv) and V L or V H A fragment that contains any of the domains, such as (i) a Fab fragment, a V L , V H , C L and C.H. 1 (ii) a monovalent fragment comprising the F(ab') 2(iii) a V fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; H and C.H. 1 (iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V H (vi) dAb fragments consisting of domains (Ward et al., Nature 341: 544-546, 1989) and (vi) isolated complementarity determining regions (CDRs). As such, an "antibody fragment" or "antigen-binding fragment" may include a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab') and F(ab') fragments. 2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0034] "Bispecific antibody" or "BsAb" as used herein refers to an antibody that can simultaneously bind two targets having distinct structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in a bispecific antibody is a V H and / or V L In some such embodiments, the V H and / or V L The V domains are those found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each derived from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody comprises two antigen-binding moieties, one of which comprises a V domain containing the CDRs from a first monoclonal antibody. H and / or V LThe other antigen-binding portion comprises an immunoglobulin molecule having a V domain containing CDRs derived from a second monoclonal antibody. H and / or V L Antibody fragments having regions (e.g., Fab, F(ab'), F(ab') 2 , Fd, Fv, dAB, scFv, etc.

[0035] As used herein, a "clearance agent" is a substance that binds to excess bispecific antibodies present in the subject's blood compartment to facilitate rapid clearance by the kidney. The use of a clearance agent prior to hapten administration (e.g., DOTA) promotes better tumor-to-background ratios in pretargeted radioimmunotherapy (PRIT) systems. Examples of clearance agents include 500kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365-1372 (2012)), 500kD aminodextran-DOTA conjugates, antibodies against pretargeting antibodies, and the like.

[0036] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art.Suitable types of association include chemical bonds and physical bonds.Chemical bonds include, for example, covalent bonds and coordinate bonds.Physical bonds include, for example, hydrogen bonds, dipolar interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0037] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment containing a light chain variable domain (V L ) connected to a heavy chain variable domain (V H )Include(V H V L(By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0038] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to an Fv fragment, V L and V H A single chain antibody molecule may comprise a polymer having several individual molecules, such as a dimer, trimer or other polymer. v Fragment, V L and V H The two domains are encoded by separate genes, but they are L and V H The domains pair to form a monovalent molecule (single-chain F v (scF v The antibodies may be joined using recombinant methods by synthetic linkers which allow them to be made as a single protein chain forming a single chain (known as a single chain antibody) (Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0039] Any of the above antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies. As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., a CD33 polypeptide). The antigen can be administered to an animal to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide that is involved in binding to the antigen. Examples of antigen-binding fragments useful in the present technology include scFv, (scFv) 2 , scFvFc, Fab, Fab' and F(ab') 2 These include, but are not limited to:

[0040] "Binding affinity" refers to the strength of the overall non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, whereas high affinity complexes generally contain antibodies that tend to remain bound to the antigen for extended periods of time. As used herein, the term "biological sample" refers to sample material derived from living cells.Biological samples can include tissues, cells, protein or membrane extracts of cells and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from subjects, as well as tissues, cells and fluids present within subjects.Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological samples can also be obtained from biopsies of internal organs or from cancer. Biological samples can be obtained from subjects for diagnosis or research, or from unaffected individuals as controls or for basic research by standard methods including venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0041] As used herein, the term "CDR-grafted antibody" refers to an antibody in which at least one CDR of an "acceptor" antibody has been replaced by a CDR "graft" from a "donor" antibody with the desired antigen specificity.

[0042] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody derived from one species (e.g., a murine Fc constant region) has been replaced, using recombinant DNA techniques, with the Fc constant region derived from an antibody of another species (e.g., a human Fc constant region). In general, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO86 / 01533; Cabilly et al. al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al. See Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988. As used herein, the term "consensus FR" refers to a framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0043] As used herein, "control" refers to a substitute sample used in an experiment for comparison purposes. Controls can be "positive" or "negative". For example, when the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are usually used. As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will vary depending on the composition, the extent, type and severity of the disease, and individual characteristics, e.g., general health, age, sex, weight and tolerance to drugs. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount may be given in one or more administrations.

[0044] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and retain specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes that express FcαR are involved in the specific killing of target cells and in presenting or binding to cells that present antigens to other components of the immune system.

[0045] As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost. In some embodiments, the "epitope" of the CD33 protein is the region of the protein to which the anti-CD33 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. To screen for anti-CD33 antibodies that bind to the epitope, a conventional cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), may be performed. This assay can be used to determine whether an anti-CD33 antibody binds to the same site or epitope as the anti-CD33 antibody of the present technology.Alternatively, or in addition, epitope mapping can be performed by methods known in the art.For example, the antibody sequence can be mutated, such as by alanine scanning, to identify contact residues.In a different method, peptides corresponding to different regions of the CD33 protein can be used in a competitive assay with a test antibody, or with a test antibody and an antibody with a characterized or known epitope.

[0046] As used herein, "expression" includes one or more of the following, as necessary for proper expression and function: transcription of a gene into precursor mRNA, splicing and other processing of the precursor mRNA to produce a mature mRNA, mRNA stability; translation of the mature mRNA into a protein (including codon usage and tRNA availability), and glycosylation and / or other modifications of the translation product.

[0047] As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns and other untranslated regions that control expression.

[0048] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region), when aligned, has a certain percentage of "sequence identity" to another sequence (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%), meaning that the percentage of bases (or amino acids) are identical in the comparison of the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs include BLASTN and BLASTP, using the following default parameters: genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, matrix=BLOSUM62, description=50 sequences, sorted=HIGH SCORE, database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percentage of homology and encode polypeptides that have the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity or less than 25% identity with each other.

[0049] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance, such as binding affinity. In general, humanized antibodies contain at least one, and usually two, variable domains (e.g., Fab, Fab', F(ab') 2 The humanized antibody generally comprises substantially all of the Fc region (Fv) of an immunoglobulin, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR region being of a human immunoglobulin consensus FR sequence, but the FR region may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is usually not more than six in the H chain and not more than three in the L chain. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297(2014).

[0050] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. A hypervariable region generally consists of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V LApproximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in H Approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the H1 sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from the "hypervariable loops" (e.g., L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in H Among these are 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0051] As used herein, the term "identical" or percent "identity", when used in relation to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison window or specified region as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then said to be "substantially identical". The term may also refer to or apply to the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0052] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein HCVR or V H The heavy chain constant region is made up of three domains, CH 1 , C.H. 2 and C.H. 3 Each light chain is composed of a light chain variable region (referred to herein as LCVR or V L The light chain constant region is composed of one domain, C L It consists of: VH and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are separated by more conserved regions called framework regions (FRs). H and V L The order is as follows: FR 1 , CDR 1 , F.R. 2 , CDR 2 , F.R. 3 , CDR 3 , F.R. 4 The variable regions of the heavy and light chains are composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0053] As used herein, the term "individual," "patient," or "subject" can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0054] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody may be an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody construct exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies may be prepared using a variety of techniques known in the art, including, for example, but not limited to, hybridoma, recombinant, and phage display techniques. For example, the monoclonal antibodies to be used according to the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0055] As used herein, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulation are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa. As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two different antibody-producing cell lines. Use of this term includes at least two antibody preparations that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0056] As used herein, the term "polynucleotide" or "nucleic acid" refers to any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotide includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules that contain DNA and RNA that may be single-stranded or, more usually, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, polynucleotide refers to triple-stranded regions that contain RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases and DNA or RNA that has a backbone that is modified for stability or for other reasons.

[0057] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic textbooks and more detailed monographs, as well as in a voluminous research literature.

[0058] As used herein, "PRIT" or "pretargeted radioimmunotherapy" refers to a multi-step process that resolves the slow blood clearance of tumor-targeting antibodies, which contributes to undesirable toxicity to normal tissues such as bone marrow. In pretargeting, a radionuclide or other diagnostic or therapeutic agent is attached to a small hapten. A pretargeting bispecific antibody with a binding site for the hapten as well as the target antigen is administered first. Unbound antibodies are then allowed to clear from the circulation, followed by administration of the hapten. As used herein, the term "recombinant," for example, when used in reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0059] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration route is the same or different. More specifically, sequential use refers to the total administration of one of the active ingredients before the start of administration of another one or more. Thus, it is possible to administer one of the active ingredients over a period of minutes, hours or days, and then administer the other active ingredient or ingredients. In this case, there is no simultaneous treatment.

[0060] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind other molecules. As used herein, the term "specific binding," "specifically binds to" or "is specific for" a particular molecule (e.g., a polypeptide or epitope on a polypeptide) refers to, for example, a binding affinity of about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K for the molecule to which M binds D The term "specifically binds" can also refer to binding by a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., a CD33 polypeptide) or an epitope on a particular polypeptide without substantially binding to any other polypeptides or epitopes on the polypeptide. As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route at the same time, or substantially the same time.

[0061] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, provides a desired therapeutic effect in a subject in need thereof. "Treating" or "treatment," as used herein, refers to the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., arresting its development, (ii) relieving the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission.

[0062] It should also be understood that the various modes of treatment of disorders as described herein include not only complete treatment, but also less than complete treatment, and are intended to mean "substantial" in which some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic disease or a single or a few doses for acute condition treatment.

[0063] Amino acid sequence modification(s) of the anti-CD33 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-CD33 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions may be made to obtain the antibody of interest, so long as the resulting antibody has the desired properties. Modifications also include changes in the glycosylation pattern of the protein. The sites of greatest interest for substitutional mutagenesis include hypervariable regions, although FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0064] [Table 1]

[0065] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants may be subjected to screening as described herein to select antibodies with similar or superior properties in one or more relevant assays for further development.

[0066] CD33 CD33 (also known as Siglec-3, SIGLEC3, gp67, or p67) is a 67 kDa cell membrane protein that binds sialic acid and is a member of the sialic acid-binding Ig-related lectin (SIGLEC) family of proteins. Siglec proteins are thought to be involved in diverse biological processes, including hematopoiesis, neuronal development, and immunity (Vinson et al., J. Biol.Chem.271 :9267-9272 (1996)). Studies have also suggested that Siglec proteins mediate cell adhesion / cell signaling by recognizing sialylated cell surface glycans (Kelm et al., Glycoconj. J. 13:913-926 (1996); Kelm et al., Eur. J. Biochem. 255:663- 672 (1998); Vinson et al., J. Biol. Chem. 271 :9267-9272 (1996)). The extracellular portion of CD33 contains two immunoglobulin domains, one IgV domain and one IgC2 domain (Figure 1). The intracellular portion of CD33 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). In immune responses, CD33 may function as an inhibitory receptor by recruiting cytoplasmic phosphatases upon ligand-induced tyrosine phosphorylation, inhibiting signaling by dephosphorylating signaling molecules.

[0067] CD33 is known to be expressed on myeloid cells. Expression of CD33 has also been reported on many malignant cells. Anti-CD33 agents are generally classified into four groups: naked antibodies, antibody-toxin conjugates, radionuclide conjugates, and bispecific antibodies. CD33 has been targeted in the treatment of cancers such as acute myeloid leukemia, but currently, no effective CD33-targeting therapeutic drugs are commercially available. Existing anti-CD33 agents have problems, particularly weak binding to tumor antigens and short half-lives in vivo. Furthermore, none of these antibodies can target the short isoforms of CD33 (splice variants of CD33) (Figure 2). CD33 is expressed on the majority of leukemic cells, but nearly 50% of leukemic cells lack the outer IgV domain due to the rs12459419 polymorphism (J Clin Oncol.35(23):2674-2682 (2017)). These splice variants, which represent nearly 50% of AML patients, do not benefit from current anti-CD33-IgV domain antibody-based therapies (e.g., gemtuzumab ozogamicin, lintuzumab, AMV564).

[0068] Immunoglobulin-related constructs of the present technology The present technology describes methods and compositions for making and using anti-CD33 immunoglobulin-related constructs (e.g., anti-CD33 antibodies or antigen-binding fragments thereof). The anti-CD33 immunoglobulin-related constructs of the present disclosure may be useful in the diagnosis or treatment of CD33-related cancers or Alzheimer's disease. Anti-CD33 immunoglobulin-related constructs within the scope of the present technology include, for example, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies and diabodies that specifically bind to target polypeptides, homologs, derivatives or fragments thereof. The present disclosure also provides antigen-binding fragments of any of the anti-CD33 antibodies disclosed herein, the antigen-binding fragments being Fab, F(ab)'2, Fab', scF, F(ab)'2 ... v and F v The present technology discloses an anti-CD33 bispecific antibody format that resolves the existing problems of weak binding to tumor antigens and short half-life in vivo, in addition to binding to both full-length CD33 protein and short isoforms of CD33. Importantly, the short isoform of CD33 is expressed in half of AML patients. In one aspect, the present technology provides chimeric and humanized variants of HIM34, including multispecific immunoglobulin-related constructs (e.g., bispecific antibody agents).

[0069] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof comprising:H is the V of GYSFTDYN (SEQ ID NO: 154) H - CDR1 sequence, V of IDPYKGGT (SEQ ID NO: 155) H - CDR2 sequence, and V of AREMITAYYFDY (SEQ ID NO: 156) H -CDR3 sequence, and (b) V L is the V of QDINKY (sequence number 157) L -CDR1 sequence, V of YAS (SEQ ID NO: 158) L -CDR2 sequence, and V of LQYDNLLT (SEQ ID NO: 159) L - an antibody or antigen-binding fragment thereof comprising the CDR3 sequence.

[0070] In one aspect, the present technology provides a method for the production of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof comprising: H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133; and / or (b) V L provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0071] In any of the above embodiments, the antibody may be of any isotype, such as, but not limited to, IgG (including IgG1, IgG2, IgG3 and IgG4), IgA (IgA 1 and IgA 2 and further comprising the Fc domains of IgD, IgE or IgM and IgY. Non-limiting examples of constant region sequences include: Human IgD constant region, Uniprot:P01880 (SEQ ID NO:116) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0072] Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO:117) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0073] Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO:118) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0074] Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO:119) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0075] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 120) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0076] Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO:121) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0077] Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO:122) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0078] Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO:123) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 124) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0079] In some embodiments, the immunoglobulin-related constructs of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 116-123. Additionally or alternatively, in some embodiments, the immunoglobulin-related constructs of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 124. In some embodiments, the immunoglobulin-related constructs of the present technology bind to the IgC2 domain of CD33. In some embodiments, the epitope is a conformational epitope. In another aspect, the disclosure provides an isolated immunoglobulin related construct (e.g., an antibody or antigen-binding fragment thereof) comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:141, or a variant thereof having one or more conservative amino acid substitutions.

[0080] Additionally or alternatively, in some embodiments, the immunoglobulin related constructs of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:140, or a variant thereof having one or more conservative amino acid substitutions. In some embodiments, the immunoglobulin-related constructs of the present technology are, respectively, SEQ ID NO:16 and SEQ ID NO:14 (chHIM34xCD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34xCD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34xCD3 BsAb; SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5×mC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5×hc825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6×mC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6×hc825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6×mC825); SEQ ID NO:22 and SEQ ID NO:29 (VL4VH6×hc825); SEQ ID NO:139 and SEQ ID NO:138 (mouse VL-mouse VH×mC825); and SEQ ID NO:141 and SEQ ID NO:140 (mouse VL-mouse VH×hC825).

[0081] In any of the above embodiments of the immunoglobulin related construct, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to the IgC2 domain of CD33. In some embodiments, the epitope is a conformational epitope. In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0082] In some embodiments, the immunoglobulin-related constructs of the present technology specifically bind to at least one CD33 polypeptide. In some embodiments, the immunoglobulin-related constructs of the present technology specifically bind to at least one CD33 polypeptide. -3 M, 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Dissociation constant of M (K D ) binds to at least one CD33 polypeptide. In certain embodiments, the immunoglobulin-related construct is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0083] In certain embodiments, the immunoglobulin-related construct comprises one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 11, 12, or 13, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, or 133. In another aspect, one or more amino acid residues in the immunoglobulin-related constructs provided herein are replaced with another amino acid. The substitution may be a "conservative substitution" as defined herein.

[0084] In some embodiments, the immunoglobulin related construct comprises (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to an LC sequence present in any one of SEQ ID NOs: 14, 18, 24, 26, 27, 28, 29, 134, 138 or 140, and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to an HC sequence present in any one of SEQ ID NOs: 16, 20, 22, 136, 139 or 141. In one aspect, the disclosure provides an immunoglobulin-related construct comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 30-113 or 142-153. In certain embodiments, the immunoglobulin-related construct of the disclosure comprises an amino acid sequence selected from SEQ ID NOs: 30-113 or 142-153.

[0085] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminus to C-terminus, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) an amino acid sequence (GGGGS), 6 (iii) a light chain variable domain of a first immunoglobulin; and (iv) an amino acid sequence (GGGGS). 4 (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) the amino acid sequence (GGGGS). 6 (vii) a light chain variable domain of a second immunoglobulin, (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT, and (ix) a self assembly disassembly (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0086] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminus to C-terminus, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) an amino acid sequence (GGGGS), 6(iii) a heavy chain variable domain of a first immunoglobulin; and (iv) an amino acid sequence (GGGGS). 4 (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) the amino acid sequence (GGGGS). 6 (vii) a light chain variable domain of a second immunoglobulin, (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT, and (ix) a self assembly disassembly (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0087] In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises a tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs: 30-113 or 142-153.

[0088] In one aspect, the disclosure provides a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and (a) each of the first and fourth polypeptide chains comprises, from N-terminus to C-terminus, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, (iii) a light chain constant domain of the first immunoglobulin, and (iv) an amino acid sequence of (GGGGS). 3 and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light and heavy chain variable domains of the second immunoglobulin are capable of specifically binding to a second epitope, and have the amino acid sequence (GGGGS) 6 and (b) each of the second and third polypeptide chains comprises, from N-terminus to C-terminus, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or a small molecule DOTA hapten.

[0089] In certain embodiments, the immunoglobulin-related construct contains an IgG1 constant region that includes one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related construct contains an IgG4 constant region that includes a S228P mutation.

[0090] In some embodiments, the anti-CD33 immunoglobulin-related constructs described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some embodiments, the anti-CD33 immunoglobulin-related constructs (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some embodiments, Fab fragments are used to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, F(ab)' is used to promote rapid binding and cellular uptake and / or sustained release. 2 Fragments are used. In one aspect, the present technology provides a nucleic acid sequence that encodes any of the immunoglobulin-related constructs described herein.Also disclosed herein is a recombinant nucleic acid sequence that encodes any of the antibodies described herein.In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 15, 17, 19, 21, 23, 25, 135 and 137.

[0091] In another aspect, the technology provides host cells that express any nucleic acid sequence encoding any of the immunoglobulin related constructs described herein. The immunoglobulin-related constructs of the present technology (e.g., anti-CD33 antibodies) can be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more CD33 polypeptides, or can be specific for both a CD33 polypeptide and a heterologous construct, such as a heterologous polypeptide or solid support material. See, for example, WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., J. Immunol. 147: 60-69 (1991), U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, and 6,106,835, and Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related construct is chimeric. In certain embodiments, the immunoglobulin-related construct is humanized.

[0092] The immunoglobulin-related construct of the present technology may further be recombinantly fused to heterologous polypeptides at N-terminus or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation).For example, the immunoglobulin-related construct of the present technology may be recombinantly fused to or conjugated to molecules that are useful as labels in detection assays and effector molecules, such as heterologous polypeptides, drugs or toxins.See, for example, WO92 / 08495, WO91 / 14438, WO89 / 12624, U.S. Patent No. 5,314,995 and EP0396387.

[0093] In any of the above embodiments of the immunoglobulin-related construct of the present technology, the antibody or antigen-binding fragment may be conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. For chemical or physical binding, a functional group on the immunoglobulin-related construct is usually associated with a functional group on the agent. Alternatively, a functional group on the agent is associated with a functional group on the immunoglobulin-related construct.

[0094] The functional groups on the agent and the immunoglobulin-related construct may be directly associated. For example, a functional group (e.g., a sulfhydryl group) on the agent may be associated with a functional group (e.g., a sulfhydryl group) on the immunoglobulin-related construct to form a disulfide. Alternatively, the functional groups may be associated via a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent may be attached to either the agent or the immunoglobulin-related construct. The number of agents or immunoglobulin-related constructs in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related construct. Alternatively, the maximum number of immunoglobulin-related constructs associated with the agent depends on the number of functional groups present on the agent.

[0095] In yet another embodiment, the conjugate comprises one immunoglobulin-related construct associated with one agent. In one embodiment, the conjugate comprises at least one agent chemically bound (e.g., conjugated) to at least one immunoglobulin-related construct. The agent may be chemically bound to the immunoglobulin-related construct by any method known to those skilled in the art. For example, a functional group on the agent may be directly attached to a functional group on the immunoglobulin-related construct. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl. Agents can also be chemically linked to immunoglobulin-related constructs by cross-linking agents such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. website can be of assistance. Additional cross-linking agents include platinum cross-linkers described in U.S. Patents 5,580,990, 5,985,566, and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands.

[0096] Alternatively, the functional groups on the agent and the immunoglobulin-related construct can be the same. Homo-bifunctional crosslinkers are usually used to crosslink the same functional groups. Examples of homo-bifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane) and BMH (i.e., bis-maleimidohexane). Such homo-bifunctional crosslinkers are also available from Pierce Biotechnology, Inc.

[0097] In other cases, it may be beneficial to cleave the agent from the immunoglobulin-related construct. The Pierce Biotechnology, Inc. website above may also assist those skilled in the art in selecting suitable crosslinkers that can be cleaved by enzymes in cells, for example. Thus, the agent can be separated from the immunoglobulin-related construct. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0098] In another embodiment, the conjugate comprises at least one active substance that is physically bound to at least one immunoglobulin-related construct. Any method known to those skilled in the art can be used to physically bind the active substance to the immunoglobulin-related construct. For example, the immunoglobulin-related construct and the active substance can be mixed together by any method known to those skilled in the art. The order of mixing is not important. For example, the active substance can be physically mixed with the immunoglobulin-related construct by any method known to those skilled in the art. For example, the immunoglobulin-related construct and the active substance can be placed in a container and agitated, for example by shaking the container, to mix the immunoglobulin-related construct and the active substance. The immunoglobulin related constructs can be modified by any method known to those of skill in the art, for example, the immunoglobulin related constructs can be modified with crosslinking agents or functional groups as described above.

[0099] A. Methods of Preparing the Anti-CD33 Antibodies of the Present Technology General overview. First, a target polypeptide is selected for which the antibody of the present technology can be generated. For example, the antibody can be generated against full-length CD33 protein, CD33 protein lacking the outer IgV domain, or a part of the extracellular domain of CD33 protein (e.g., IgC2 domain). Techniques for generating antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, display libraries, xenogeneic or human mice, hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from CD33 protein that includes an extracellular domain (e.g., IgC2 domain) that can induce an immune response.

[0100] It should be understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, directed against the CD33 protein and fragments thereof are suitable for use in accordance with the present disclosure. Anti-CD33 antibodies that can be subjected to the techniques described herein include monoclonal and polyclonal antibodies and antibody fragments, such as Fab, Fab', F(ab') 2 Antibody Fv-containing polypeptides include, for example, Fab' and F(ab') 2 Methods useful for the high yield production of antibody fragments have been described, see US Patent No. 5,648,237.

[0101] Generally, antibody is obtained from original species.More specifically, the nucleic acid or amino acid sequence of the variable part of light chain, heavy chain or both of the original species antibody with specificity to target polypeptide antigen is obtained.Original species can be any species that is useful for making the antibody or antibody library of the present technology, such as rat, mouse, rabbit, chicken, monkey, human, etc. Phage or phagemid display technology is a useful technique for deriving the antibody of the present technology. The techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art. The expression of the sequence encoding the antibody of the present technology can be carried out in E. coli.

[0102] Due to the degeneracy of nucleic acid encoding sequences, other sequences that code for substantially the same amino acid sequence as that of naturally occurring proteins may be used in the practice of the present technology. These include, but are not limited to, nucleic acid sequences that include all or part of the nucleic acid sequences encoding the above polypeptides, which are altered by substitution of different codons that code for functionally equivalent amino acid residues in the sequence, thus resulting in silent changes. It is understood that the nucleotide sequences of the immunoglobulins of the present technology allow for up to 25% sequence homology variation as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), so long as such variants form operative antibodies that recognize CD33 protein. For example, one or more amino acid residues in a polypeptide sequence may be replaced by another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent change. Substitutes for amino acids in a sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar natural amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage with antibody molecules or other cellular ligands, etc.Additionally, nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to create and / or destroy translation, initiation and / or termination sequences to create variations in the coding region and / or to form new restriction endonuclease sites or destroy existing ones, facilitating further in vitro modifications. Any technique for mutagenesis known in the art may be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), and the like.

[0103] Preparation of polyclonal antisera and immunogens. The method of producing the antibody or antibody fragment of the present technology usually involves immunotherapy of a subject (generally a non-human subject such as a mouse or rabbit) with purified CD33 protein or a fragment thereof, or with a cell expressing CD33 protein or a fragment thereof. Suitable immunogenic preparations can contain, for example, recombinantly expressed CD33 protein or chemically synthesized CD33 peptide. The extracellular domain of CD33 protein, or a part or fragment thereof (e.g., IgC2 domain), can be used as an immunogen to produce anti-CD33 antibodies that bind to CD33 protein, or a part or fragment thereof, using standard techniques for polyclonal and monoclonal antibody preparations.

[0104] Full-length CD33 protein or fragments thereof are useful as immunogens. In some embodiments, the CD33 fragment comprises the IgC2 domain of CD33, such that antibodies raised against the peptide form specific immune complexes with CD33 proteins that contain short isoforms of CD33 (CD33 splice variants lacking the IgV domain). The IgC2 domain of CD33 is 84 amino acids long. In some embodiments, the antigenic CD33 peptide comprises at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, or 80 amino acid residues. Depending on the application and according to methods well known to those skilled in the art, a long antigenic peptide may be more desirable than a short antigenic peptide. Multimers of a given epitope may be more effective than monomers.

[0105] If necessary, the immunogenicity of the CD33 protein (or fragments thereof) may be increased by fusion or conjugation with a hapten such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. To increase the subject's immune response to the polypeptide, the CD33 protein may be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), inorganic gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptide or oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacillus Calmette-Guerin and Corynebacterium parvum or similar immunostimulatory compounds. These techniques are standard in the art.

[0106] In the description of the present technology, immune responses can be described as "primary" or "secondary" immune responses. Primary immune responses are also described as "protective" immune responses and refer to immune responses generated in individuals as a result of some initial exposure to a particular antigen, e.g., CD33 protein (e.g., initial "immunotherapy"). In some embodiments, immunotherapy can result from vaccination of an individual with a vaccine containing an antigen. For example, the vaccine can be a CD33 vaccine that includes antigens from one or more CD33 proteins. Primary immune responses can become weaker or attenuated over time, and can even disappear, or at least become attenuated so that they cannot be detected. Thus, the present technology also relates to "secondary" immune responses, also described herein as "memory immune responses". The term secondary immune response refers to an immune response that is induced in an individual after a primary immune response has already been generated.

[0107] Thus, a secondary immune response can be elicited to boost a pre-existing immune response that has become weakened or attenuated, or to reconstitute a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells generated upon the first presentation of the antigen. A delayed type hypersensitivity (DTH) response is initiated by CD4 + It is a type of cellular secondary or memory immune response mediated by T cells. Initial exposure to an antigen primes the immune system, and further exposure results in DTH.

[0108] After appropriate immunotherapy, anti-CD33 antibodies can be prepared from the subject's serum. If desired, antibody molecules directed against the CD33 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain the IgG fraction.

[0109] Monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-CD33 monoclonal antibody. For example, in some embodiments, the anti-CD33 monoclonal antibody can be a human or mouse anti-CD33 monoclonal antibody. For the preparation of monoclonal antibody against CD33 protein or its derivative, fragment, analog or homolog, any technique that provides the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, hybridoma techniques (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497), trioma techniques, human B cell hybridoma techniques (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and EBV hybridoma techniques for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). In the practice of the present technology, human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody may be isolated. PCR may be used to amplify sequences encoding portions of antibodies from the population, utilizing primers derived from sequences encoding conserved regions of the antibody, and then DNA encoding the antibody or fragment thereof, e.g., the variable domain, is reconstructed from the amplified sequences.Such amplified sequences can also be fused with DNA encoding other proteins, e.g., bacteriophage coat or bacterial cell surface proteins, for expression and display of the fusion polypeptide in phage or bacteria. The amplified sequences can then be expressed and further selected or isolated, e.g., based on the affinity of the expressed antibody or fragment thereof to an antigen or epitope present on the CD33 protein. Alternatively, hybridomas expressing anti-CD33 monoclonal antibodies can be prepared by immunotreating a subject and then isolating hybridomas from the subject's spleen using routine methods. See, e.g., Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods results in monoclonal antibodies of varying specificity (i.e., for various epitopes) and affinity. The selected monoclonal antibody with the desired properties, e.g., CD33 binding, may be used as expressed by the hybridoma, may be conjugated to a molecule such as polyethylene glycol (PEG) to modify its properties, or the cDNA encoding it may be isolated, sequenced, or manipulated in various ways. To increase the immunogenicity of the CD33 protein, synthetic dendritic trees may be added to the side chains of reactive amino acids, e.g., lysine. Also, CPG dinucleotide technology may be used to increase the immunogenicity of the CD33 protein. Other manipulations include substituting or deleting certain aminoacyl residues that contribute to the instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of the antibody for the CD33 protein.

[0110] Hybridoma technology. In some embodiments, the antibody of the present technology is an anti-CD33 monoclonal antibody produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome comprises human heavy chain transgenes and light chain transgenes, fused to an immortalized cell. Hybridoma technology includes those taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981), which are known in the art. Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0111] Phage display technology. As described above, the antibody of the present technology can be produced by applying recombinant DNA and phage display technology. For example, anti-CD33 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Phages with desired binding properties are selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection with antigen, usually antigen bound or captured to a solid surface or bead. The phages used in these methods are usually filamentous phages, including fd and M13, with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII proteins. Furthermore, to allow rapid efficient identification of monoclonal Fab fragments having the desired specificity for a CD33 polypeptide, e.g., a polypeptide or a derivative, fragment, analog or homolog thereof, methods can be applied for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246: 1275-1281, 1989).Other examples of phage display methods that can be used to generate the antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994, PCT / GB91 / 01134, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, WO96 / 06213, WO92 / 01047 (Medical Research Council al.), WO 97 / 08320 (Morphosys), WO 92 / 01047 (CAT / MRC), WO 91 / 17271 (Affymax) and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. A method useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides through disulfide bonds is described by Lohning, US Pat. No. 6,753,136.As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, e.g., Fab, Fab' and F(ab'). 2 Techniques for recombinantly producing fragments may also be used using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0112] Generally, the hybrid antibodies or hybrid antibody fragments cloned into the display vector can be selected against the appropriate antigen to identify variants that maintain good binding activity since the antibody or antibody fragment is present on the surface of the phage or phagemid particle. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats can be used for this process, such as cloning the antibody fragment library into a soluble phage vector for selection and / or screening (modified T7 or lambda Zap systems).

[0113] Expression of recombinant anti-CD33 antibodies. As described above, the antibodies of the present technology can be produced by application of recombinant DNA technology. The recombinant polynucleotide constructs encoding the anti-CD33 antibodies of the present technology usually contain expression control sequences operably linked to the coding sequences of the anti-CD33 antibody chains, including naturally associated or heterologous promoter regions. As such, another aspect of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-CD33 antibodies of the present technology. For recombinant expression of one or more polypeptides of the present technology, a nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-CD33 antibody is inserted into an appropriate cloning or expression vector (i.e., a vector containing the elements necessary for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA technology well known in the art and as detailed below. Methods for producing a diverse collection of vectors are described by Lerner et al., U.S. Patent Nos. 6,291,160 and 6,680,192.

[0114] In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this disclosure, "plasmid" and "vector" may be used interchangeably since plasmids are the most common form of vectors. However, the technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), that perform equivalent functions. Such viral vectors allow for infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-CD33 antibody and collection and purification of the anti-CD33 antibody, e.g., a cross-reactive anti-CD33 antibody. See generally U.S. Patent Application Publication No. 2002 / 0199213. These expression vectors are usually replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectate lyase, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0115] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having CD33 binding properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector can vary depending on factors such as the choice of the host cell to be transformed, the level of expression of the desired polypeptide, and the like. Common regulatory sequences that are useful as promoters of recombinant polypeptide expression (e.g., anti-CD33 antibodies) include, for example, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, a polynucleotide encoding the anti-CD33 antibody of the present technology is operably linked to the ara B promoter and can be expressed in a host cell. See U.S. Patent No. 5,028,530.The expression vectors of the present technology can be introduced into a host cell to thereby produce a polypeptide or peptide (such as, for example, an anti-CD33 antibody) comprising a fusion polypeptide encoded by a nucleic acid as described herein.

[0116] Another aspect of the present technology relates to a host cell expressing anti-CD33 antibody, which contains a nucleic acid encoding one or more anti-CD33 antibodies. The recombinant expression vector of the present technology can be designed for the expression of anti-CD33 antibody in prokaryotic or eukaryotic cells. For example, anti-CD33 antibody can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for the preparation and screening of polypeptides with predetermined properties, e.g., anti-CD33 antibodies, by expression of stochastically generated polynucleotide sequences have been described previously. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0117] Expression of polypeptides in prokaryotes is most often carried out using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides in E. coli. Fusion vectors add several amino acids to the polypeptides encoded herein, usually at the amino terminus of the recombinant polypeptide. Such fusion vectors usually serve three purposes: (i) to increase the expression of the recombinant polypeptide, (ii) to increase the solubility of the recombinant polypeptide, and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. In fusion expression vectors, a proteolytic cleavage site is often introduced at the junction of the fusion moiety and the recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor X, thrombin, and enterokinase. Common fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide or polypeptide A, respectively, to the target recombinant polypeptide.

[0118] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of separate active peptides or protein domains to obtain multifunctional polypeptides by polypeptide fusion are described by Pack et al., U.S. Pat. Nos. 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, e.g., anti-CD33 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that each individual codon of each amino acid is the one that is preferentially utilized in the expression host, for example, E. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of the nucleic acid sequence of the present technology can be carried out by standard DNA synthesis techniques.

[0119] In another embodiment, the anti-CD33 antibody expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.) and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, anti-CD33 antibodies may be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing polypeptides, such as anti-CD33 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0120] In yet another embodiment, the nucleic acid encoding the anti-CD33 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987).When used in mammalian cells, the control function of expression vectors is often provided by viral regulatory elements.For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells suitable for expressing the anti-CD33 antibodies of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0121] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreatic specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland specific promoters (e.g., whey promoter; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters are also included, such as the mouse hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

[0122] Another aspect of the method relates to a host cell into which the recombinant expression vector of the present technology is introduced.The terms "host cell" and "recombinant host cell" are used interchangeably herein.It is understood that such terms refer not only to the specific target cell, but also to the progeny or potential progeny of such cells.Since certain modifications may occur in subsequent generations due to either mutation or environmental influence, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term herein.

[0123] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-CD33 antibodies can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cell is a non-human cell. The expression vectors of these cells can include expression control sequences, such as origins of replication, promoters, enhancers and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Exemplary expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those of skill in the art.

[0124] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, biolistics or virus-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection (see generally, Sambrook et al., Molecular Cloning). Suitable methods for transducing or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0125] For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selection marker (e.g., resistance to antibiotics) is generally introduced into the host cell together with the gene of interest. Various selection markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selection marker can be introduced into the host cell in the same vector as that encoding the anti-CD33 antibody, or can be introduced in a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selection marker gene survive, while other cells die).

[0126] Host cells comprising the anti-CD33 antibody of the present technology, such as prokaryotic or eukaryotic host cells in culture, can be used to manufacture (i.e., express) recombinant anti-CD33 antibodies. In one embodiment, the method includes culturing the host cells (into which a recombinant expression vector encoding the anti-CD33 antibody has been introduced) in a suitable medium such that the anti-CD33 antibody is produced. In another embodiment, the method further includes isolating the anti-CD33 antibody from the medium or the host cells. Once expressed, the anti-CD33 antibody, e.g., a collection of anti-CD33 antibodies or anti-CD33 antibody-related polypeptides, is purified from the culture medium and the host cells. The anti-CD33 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-CD33 antibody is produced in a host organism by the method of Boss et al. U.S. Pat. No. 4,816,397. Usually, the anti-CD33 antibody chain is expressed with a signal sequence, and thus released into the culture medium. However, if the anti-CD33 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergents. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0127] Polynucleotides encoding anti-CD33 antibodies, such as anti-CD33 antibody coding sequences, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals.See, for example, U.S. Patent Nos. 5,741,957, 5,304,489 and 5,849,992.Suitable transgenes include light and / or heavy chain coding sequences in operable linkage with the promoter and enhancer from mammary gland specific genes, such as casein or β-lactoglobulin.For the production of transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, and the nucleus of such cells is transferred into enucleated oocytes.

[0128] Single chain antibody. In one embodiment, the anti-CD33 antibody of the present technology is a single chain anti-CD33 antibody. According to the present technology, technology can be adopted for the production of single chain antibody specific to CD33 protein (see, for example, U.S. Patent No. 4,946,778). Examples of the technology that can be used to produce single chain Fv and the antibody of the present technology include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0129] Chimeric and humanized antibodies. In one embodiment, the anti-CD33 antibody of the present technology is a chimeric anti-CD33 antibody. In one embodiment, the anti-CD33 antibody of the present technology is a humanized anti-CD33 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody.

[0130] Recombinant anti-CD33 antibodies, for example, chimeric and humanized monoclonal antibodies, including both human and non-human parts, can be produced using standard recombinant DNA technology and are within the scope of the present technology.For some uses, including the in vivo use of the anti-CD33 antibodies of the present technology in humans and the use of these substances in in vitro detection assays, chimeric or humanized anti-CD33 antibodies may be used.Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA technology known in the art. Such useful methods include, but are not limited to, those described in International Application No. PCT / US86 / 02269, U.S. Pat. No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. WO86 / 01533, U.S. Pat. Nos. 4,816,567 and 5,225,539, European Patent No. 125023, Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988.For example, antibodies can be humanized using a variety of techniques including CDR grafting (EP0239400, WO91 / 09967, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP460167), veneering or resurfacing (EP0592106, EP0519596, Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994) and chain shuffling (U.S. Pat. No. 5,565,332).In one embodiment, the cDNA encoding a murine anti-CD33 monoclonal antibody is digested with restriction enzymes specifically selected to remove sequences encoding the Fc constant region and the equivalent portion of the cDNA encoding the human Fc constant region is replaced (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064;

[0131] In one embodiment, the present technology provides for the construction of humanized anti-CD33 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response, while still having effective antibody effector functions. As used herein, in the context of antibodies, the terms "human" and "humanized" refer to any antibody that is predicted to induce a therapeutically acceptable weak immunogenic response in human subjects. In one embodiment, the present technology provides humanized anti-CD33 antibodies, heavy and light chain immunoglobulins.

[0132] CDR antibody. In some embodiments, the anti-CD33 antibody of the present technology is an anti-CD33 CDR antibody. In general, the donor and acceptor antibodies used to generate the anti-CD33 CDR antibody are monoclonal antibodies from different species, and the acceptor antibody is usually a human antibody (to minimize antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. Grafting is performed by removing the single V of the acceptor antibody. H or V L or a graft of a single CDR (or even a portion of a single CDR) within V H and V L The CDRs may be grafted in one or both of the CDRs (or parts thereof). Often, all three CDRs of all variable domains of the acceptor antibody are replaced with the corresponding donor CDRs, although only the number necessary to allow the resulting CDR-grafted antibody to properly bind to the CD33 protein need to be replaced. Methods for making CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; and Winter U.S. Patent No. 5,225,539; and EP0682040. H and V LMethods useful for preparing polypeptides are taught by Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP 0451216; and EP 0120694.

[0133] After selecting suitable framework region candidates from the same family and / or same family members, either or both of the heavy and light chain variable regions are produced by grafting CDRs from the original species into the hybrid framework regions. For any of the above embodiments, the assembly of hybrid antibodies or hybrid antibody fragments with hybrid variable chain regions can be achieved using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on CDRs from the target species and original species) can be produced by oligonucleotide synthesis and / or PCR. Nucleic acids encoding CDR regions can also be isolated from the original species antibody using suitable restriction enzymes and ligated into the target species framework by ligation with suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the original species antibody can be altered by site-directed mutagenesis.

[0134] Since hybrids are constructed by selecting from among multiple candidates corresponding to each framework region, there are many combinations of sequences that are amenable to construction according to the principles described herein.Thus, a library of hybrids can be assembled, with members having different combinations of individual framework regions.Such a library can be an electronic database collection of hybrid sequences or a physical collection of hybrids.

[0135] This process does not usually alter the FRs of the acceptor antibody that flank the grafted CDRs. However, one skilled in the art can sometimes improve the antigen binding affinity of the resulting anti-CD33 CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable positions for substitution include amino acid residues adjacent to or capable of interacting with the CDR (see, e.g., US 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with a donor FR and modify it to be more similar to the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. Doing so may not significantly increase the antigenicity of the resulting modified anti-CD33 CDR-grafted antibody compared to the same antibody with a fully human FR, especially if the resulting FR matches or is at least 90% or more identical to the human consensus FR at that position.

[0136] Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind two targets with distinct structures, such as two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. BsAbs can be made, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, a bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH / VL pair) and a different antigen (or epitope) on its second arm (another VH / VL pair) by molecular function. By this definition, a bispecific binding agent has two distinct antigen binding arms (both in specificity and CDR sequence) and is monovalent for each antigen it binds.

[0137] The bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs) of the present technology have, for example, at least one arm that specifically binds to CD33 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of B cells, T cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, and KIR. In certain embodiments, the BsAbs are capable of binding to tumor cells that express the CD33 antigen on their cell surface. In some embodiments, the BsAbs are engineered to promote tumor cell killing by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen binding site specific for CD33 and a second antigen binding site specific for a small molecule hapten (e.g., DTPA, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA chelates described herein, biotin, fluorescein or those disclosed in Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937-5946).

[0138] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed that utilize either the complete immunoglobulin framework (e.g., IgG), single chain variable fragments (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs, composed of two scFv units in tandem, have proven to be a clinically successful bispecific antibody format. In some embodiments, the BsAb comprises two single chain variable fragments (scFvs) in tandem, designed such that the scFv that binds to a tumor antigen (e.g., CD33) is linked to a scFv that engages T cells (e.g., by binding to CD3). In this way, T cells are recruited to the tumor site so that they can mediate cytotoxic killing of tumor cells. See, e.g., Dreier et al., J. Immunol. 170:4397-4402(2003); Bargou et al., Science 321 :974- 977(2008)). In some embodiments, the BsAb of the present technology comprises two single chain variable fragments (scFvs) in tandem, designed such that the scFv that binds to a tumor antigen (e.g., CD33) is linked to a scFv that engages the small molecule DOTA hapten.

[0139] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues that cross-link more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins by linking two or more different single-chain antibody or antibody fragment segments with the required dual specificity. See, e.g., Coloma et al., Nature Biotech. 15:159-163(1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0140] Bispecific fusion proteins linking two or more different single chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce various fusion proteins. In some specific embodiments, the BsAb of the present technology comprises an immunoglobulin comprising a heavy chain and a light chain and an scFv. In some specific embodiments, the scFv is linked to the C-terminus of the heavy chain of any CD33 immunoglobulin disclosed herein. In some specific embodiments, the scFv is linked to the C-terminus of the light chain of any CD33 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for the in-frame connection of the heavy chain Fd to the scFv is extracted from the V L and V kappa The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and the V domain of the CD33 antibody is H The DNA sequences encoding the bispecific fusion protein are then ligated into a vector containing the DNA sequence encoding the bispecific fusion protein. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell, for expression of the bispecific fusion protein.

[0141] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized in that it does not adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., the first and / or second antigen binding site). In some embodiments, the linker is used in the BsAbs described herein based on certain properties that it imparts to the BsAb, such as increased stability. In some embodiments, the BsAb of the present technology is a G 4 In some specific embodiments, the BsAb of the present technology comprises a (G 4 S) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0142] Self-assembly-disassembly (SADA) conjugates. In some embodiments, the anti-CD33 antibodies of the present technology comprise one or more SADA domains. The SADA domains can be designed and / or tailored to achieve environment-dependent multimerization with beneficial kinetic, thermodynamic, and / or pharmacological properties. For example, it is recognized that SADA domains can be part of a conjugate that allows effective delivery of a payload to a desired target site while minimizing the risk of off-target interactions. The anti-CD33 antibodies of the present technology can comprise a SADA domain linked to one or more binding domains. In some embodiments, such conjugates are characterized by multimerizing to form a complex of a desired size under relevant conditions (e.g., in a solution where the conjugate is present at a concentration or pH above a threshold and / or present at a target site characterized by a relevant level or density of receptors for the payload), and disassembling to a smaller form under other conditions (e.g., in the absence of a relevant environmental multimerization trigger).

[0143] SADA conjugates may have improved characteristics compared to conjugates that do not have a SADA domain. In some embodiments, the improved characteristics of the multimeric conjugates include increased avidity / binding to the target, increased specificity for the target cell or tissue, and / or increased initial serum half-life. In some embodiments, the improved characteristics include that by dissociating into smaller states (e.g., dimers or monomers), the SADA conjugates exhibit reduced non-specific binding, reduced toxicity, and / or improved renal clearance. In some embodiments, the SADA conjugates comprise a SADA polypeptide having an amino acid sequence that exhibits at least 75% identity to a human homomultimerizing polypeptide and exhibit one or more multimerization dissociation constants (K D ) In some embodiments, the SADA conjugate is constructed and arranged to adopt a first multimerization state and one or more higher order multimerization states. In some embodiments, the first multimerization state is less than about 70 kDa in size. In some embodiments, the first multimerization state is a non-multimerized state (e.g., a monomer or a dimer). In some embodiments, the first multimerization state is a monomer. In some embodiments, the first multimerization state is a dimer. In some embodiments, the first multimerization state is a multimerization state (e.g., a trimer or tetramer). In some embodiments, the higher order multimerization state is a homotetramer or higher order homomultimer greater than 150 kDa in size. In some embodiments, the higher order homomultimerization conjugate is constructed and arranged to adopt a first multimerization state and one or more higher order multimerization states. In some embodiments, the first multimerization state is less than about 70 kDa in size ... D In some embodiments, the SADA conjugate is stable in aqueous solution when present at a concentration greater than SADA polypeptide K. D If the total amount of the multimerized protein is less than 100%, the multimerized protein transitions from the higher order multimerization state to the first multimerization state under physiological conditions.

[0144] In some embodiments, the SADA polypeptide is covalently linked to the binding domain via a linker. Any suitable linker known in the art may be used. In some embodiments, the SADA polypeptide is linked to the binding domain via a polypeptide linker. In some embodiments, the polypeptide linker is a Gly-Ser linker. In some embodiments, the polypeptide linker is or comprises a sequence of (GGGGS)n, where n represents the number of repeats of GGGGS units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the binding domain is directly fused to the SADA polypeptide.

[0145] In some embodiments, the SADA domain is a human polypeptide, or a fragment and / or derivative thereof. In some embodiments, the SADA domain is substantially non-immunogenic in humans. In some embodiments, the SADA polypeptide is stable as a multimer. In some embodiments, the SADA polypeptide lacks unpaired cysteine ​​residues. In some embodiments, the SADA polypeptide does not have a large exposed hydrophobic surface. In some embodiments, the SADA domain has, or is predicted to have, a structure that includes helical bundles that can associate in a parallel or antiparallel orientation. In some embodiments, the SADA polypeptide is capable of reversible multimerization. In some embodiments, the SADA domain is a tetramerization domain, a heptamerization domain, a hexamerization domain, or an octamerization domain. In certain embodiments, the SADA domain is a tetramerization domain. In some embodiments, the SADA domain is composed of multimerization domains that are each composed of helical bundles that associate in a parallel or antiparallel orientation. In some embodiments, the SADA domain is selected from one of the following groups of human proteins: p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosomal associated protein 23 (SNAP-23), stefin B (cystatin B), potassium voltage-dependent channel subfamily KQT member 4 (KCNQ4), or cyclin D-associated protein (CBFA2T1). Examples of suitable SADA domains are described in PCT / US2018 / 031235, which is incorporated herein by reference in its entirety. Exemplary SADA domain polypeptide sequences are provided below.

[0146] Human p53 tetramerization domain amino acid sequence (321-359) KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP (SEQ ID NO:125) Human p63 tetramerization domain amino acid sequence (396-450) RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ (SEQ ID NO:126)

[0147] Human p73 tetramerization domain amino acid sequence (348-399) RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (Sequence number 127). Human HNRNPC tetramerization domain amino acid sequence (194-220) QAIKKELTQIKQKVDSLLENLEKIEKE (SEQ ID NO:128)

[0148] Human SNAP-23 tetramerization domain amino acid sequence (23-76) STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL (SEQ ID NO:129) Amino acid sequence of human stefin B tetramerization domain (2-98) MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF (SEQ ID NO:130)

[0149] KCNQ4 tetramerization domain amino acid sequence (611-640) DEISMMGRVVKVEKQVQSIEHKLDLLLGFY (SEQ ID NO:131) CBFA2T1 tetramerization domain amino acid sequence (462-521) TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH (SEQ ID NO:132)

[0150] In some embodiments, the SADA polypeptide is or comprises p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosomal associated protein 23 (SNAP-23), stefin B (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or the tetramerization domain of cyclin D-associated protein (CBFA2T1). In some embodiments, the SADA polypeptide is or comprises a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence set forth in any one of SEQ ID NOs: 125-132.

[0151] Fc Modifications. In some embodiments, the anti-CD33 antibodies of the present technology comprise a variant Fc region, said variant Fc region comprising at least one amino acid modification relative to a wild-type Fc region (or parent Fc region) such that said molecule has an altered affinity for an Fc receptor (e.g., FcγR), based on crystallographic and structural analysis of Fc-Fc receptor interactions, such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000), provided that said variant Fc region does not have substitutions at positions that directly contact an Fc receptor. Examples of positions within the Fc region that directly contact an Fc receptor, e.g., FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop. In some embodiments, the anti-CD33 antibodies of the present technology having a variant Fc region with one or more amino acid modifications have altered affinity for activating and / or inhibitory receptors, wherein said one or more amino acid modifications are an N297 substitution with alanine or a K322 substitution with alanine.

[0152] Glycosylation modifications. In some embodiments, the anti-CD33 antibodies of the present technology have an Fc region with variant glycosylation compared to a parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose, and in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells. In some embodiments, the antibodies of the present technology may have glycosylation sites that are modified relative to a suitable reference antibody that binds to an antigen of interest (e.g., CD33) without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, a "glycosylation site" includes any particular amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is specifically and covalently attached.

[0153] Oligosaccharide side chains are usually linked to the backbone of the antibody by either N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. For example, Fc-glycoforms (hCD33-IgGln) that lack certain oligosaccharides, including fucose and terminal N-acetylglucosamine, can be produced in certain CHO cells and exhibit enhanced ADCC effector function. In some embodiments, the carbohydrate content of the immunoglobulin-related constructs disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, for example, U.S. Patent No. 6,218,149, EP0359096B1, U.S. Patent Publication No. US2002 / 0028486, International Publication No. WO03 / 035835, U.S. Patent Publication No. 2003 / 0115614, U.S. Patent No. 6,218,149, U.S. Patent No. 6,472,511, all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some specific embodiments, the present technology includes deleting the glycosylation site of the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0154] Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be made by any method known to those of skill in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTIII), by expressing the molecule comprising an Fc region in various organisms or cell lines derived from various organisms, or by modifying the carbohydrate(s) after the molecule comprising an Fc region is expressed. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473, U.S. Patent No. 6,602,684, U.S. Patent Application No. 10 / 277,370, U.S. Patent Application No. 10 / 113,929, International Publication Nos. WO00 / 61739A1, WO01 / 292246A1, WO02 / 311140A1, WO02 / 30954A1, POTILLEGENT™ technology (Biowa, Inc. Princeton, NJ), GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, e.g., International Publication No. WO 00 / 061739, U.S. Patent Application Publication No. 2003 / 0115614, Okazaki et al., 2004, JMB, 336: 1239-49.

[0155] Fusion proteins. In one embodiment, the anti-CD33 antibody of the present technology is a fusion protein. The anti-CD33 antibody of the present technology can be used as an antigenic tag when fused to a second protein. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other heterologous functional regions. The fusion does not necessarily have to be direct, but can occur through a linker sequence. Furthermore, the fusion protein of the present technology can also be genetically engineered to improve the characteristics of the anti-CD33 antibody. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the anti-CD33 antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties can be added to the anti-CD33 antibody to facilitate purification. Such regions can be removed prior to final preparation of the anti-CD33 antibody. The addition of peptide moieties to facilitate handling of a polypeptide is a well-known and routine technique in the art. The anti-CD33 antibody of the present technology can be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In selected embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in pQE vectors (QIAGEN, Inc., Chatsworth, Calif.), among others, many of which are commercially available. For example, hexahistidine provides convenient purification of the fusion protein, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0156] Thus, any of these above fusion proteins may be engineered using the polynucleotides or polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0157] Fusion proteins with disulfide-bonded dimeric structures (such as IgG) can be more effective at binding and neutralizing other molecules compared to monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995. Similarly, EP-AO 464533 (Canadian counterpart 2045869) discloses fusion proteins that include various portions of the constant region of an immunoglobulin molecule together with another human protein or fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnosis, and thus may result in, for example, improved pharmacokinetic properties. See EP-A0232262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected, and purified. For example, the Fc portion may interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunotherapy. In drug discovery, for example, human proteins such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0158] Labeled anti-CD33 antibody. In one embodiment, the anti-CD33 antibody of the present technology is coupled with a label moiety, i.e., a detectable group. The particular label or detectable group conjugated to the anti-CD33 antibody is not a critical aspect of the technology, so long as it does not significantly interfere with the specific binding of the anti-CD33 antibody of the present technology to the CD33 protein. The detectable group can be any material that has a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, the label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the implementation of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other contrast agents such as microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O (for positron emission tomography), 99m T.C., 111In (for single photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference in its entirety for all purposes. Also, Handbook of Fluorescent Probes and Research Chemicals (6 th Ed., Molecular Probes, Inc., Eugene OR.

[0159] The label may be directly or indirectly coupled to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on factors such as the required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation and disposal regulations.

[0160] Non-radioactive labels are often attached by indirect means. In general, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule that is either inherently detectable or covalently bound to a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Several ligands and anti-ligands can be used. When the ligand has a neutral anti-ligand, e.g., biotin, thyroxine, and cortisol, it can be used with a labeled naturally occurring anti-ligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-CD33 antibody.

[0161] Molecules can also be directly conjugated to signal generating compounds, for example, by conjugation with enzymes or fluorophores. Enzymes of interest as labels are primarily hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, for example, luciferin and 2,3-dihydrophthalazinediones, such as luminol. For a summary of various labeling or signal generating systems that can be used, see U.S. Patent No. 4,391,904.

[0162] Means for detecting the label are well known to those skilled in the art. Thus, for example, when the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. When the label is a fluorescent label, it can be detected by exciting the fluorescent dye with the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually, by photographic film, by using electronic detectors such as charge-coupled devices (CCDs) or photomultipliers, etc. Similarly, enzymatic labels can be detected by providing the enzyme with the appropriate material and detecting the resulting reaction product. Finally, simple colorimetric labels can be simply detected by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0163] Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of labeled antibodies, such as anti-CD33 antibodies. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, no components need to be labeled, and the presence of labeled antibodies is detected by simple visual inspection.

[0164] B. Identification and Characterization of Anti-CD33 Antibodies of the Present Technology Methods for identifying and / or screening anti-CD33 antibodies of the present technology. Methods useful for identifying and screening antibodies against CD33 polypeptides for those with the desired specificity for the CD33 protein (e.g., those that bind to the IgC2 domain of CD33) include any immunologically mediated technique known in the art. Components of the immune response can be detected in vitro by a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes are incubated with radioactively labeled target cells, and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes are incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells are incubated with whole protein antigen, and the presentation of that antigen on MHC can be detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells are incubated with a reagent that crosslinks their Fc-epsilon receptors, and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983) and (5) enzyme-linked immunosorbent assay (ELISA).

[0165] Similarly, the products of an immune response in either a model organism (e.g., a mouse) or a human subject can also be detected by a variety of methods well known to those of skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container across the scratch site to capture the migrating cells (Peters et al., Blood, 72: 1310-5, 1988), (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactants can be detected by a variety of methods, such as ELISA, (4) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (5) the migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container across the scratch site to capture the migrating cells (Peters et al., Blood, 72: 1310-5, 1988), (6) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactants can be detected by a variety of methods, such as ELISA, (7) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (8) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (9) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (10) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (11) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA, (12) the production of antibodies in response to vaccination can be readily detected by standard methods currently 3 H-thymidine, (4) phagocytosis of granulocytes, macrophages and other phagocytes in PBMCs can be measured by placing PBMCs into wells with labeled particles (Peters et al., Blood, 72: 1310-5, 1988), and (5) differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs expressing these markers.

[0166] In one embodiment, the anti-CD33 antibody of the present technology is selected by using the display of CD33 peptide on the surface of replicable genetic package.See, for example, U.S. Patent No. 5,514,548, U.S. Patent No. 5,837,500, U.S. Patent No. 5,871,907, U.S. Patent No. 5,885,793, U.S. Patent No. 5,969,108, U.S. Patent No. 6,225,447, U.S. Patent No. 6,291,650, U.S. Patent No. 6,492,160, EP585287, EP605522, EP616640, EP1024191, EP589877, EP774511, EP844306.Methods useful for producing / selecting filamentous bacteriophage particles containing phagemid genomes that code for binding molecules with desired specificity are described. See, for example, EP 774511, US 5,871,907, US 5,969,108, US 6,225,447, US 6,291,650, US 6,492,160. In some embodiments, the anti-CD33 antibodies of the present technology are selected using display of CD33 peptides on the surface of yeast host cells. A method useful for isolating Fv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0167] In some embodiments, the anti-CD33 antibody of the present technology is selected using ribosome display.A method useful for identifying ligands in peptide libraries using ribosome display is described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994 and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997. In certain embodiments, the anti-CD33 antibody of the present technology is selected using tRNA display of CD33 peptide. A useful method for in vitro selection of ligands using tRNA display is described by Merryman et al., Chem. Biol., 9: 741-46, 2002.

[0168] In one embodiment, the anti-CD33 antibody of the present technology is selected using RNA display.A method useful for selecting peptides and proteins using RNA display libraries is described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997, and Nemoto et al., FEBS Lett., 414: 405-8, 1997.A method useful for selecting peptides and proteins using non-natural RNA display libraries is described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0169] In some embodiments, the anti-CD33 antibody of the present technology is expressed in the periplasm of gram-negative bacteria and mixed with labeled CD33 protein. See WO02 / 34886. The clone expressing a recombinant polypeptide with affinity to CD33 protein will have an enriched concentration of labeled CD33 protein bound to the anti-CD33 antibody, and the cells can be isolated from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and US Patent Publication No. 2004 / 0058403.

[0170] It is contemplated that after selection of the desired anti-CD33 antibody, the antibody may be mass-produced by any technique known to those skilled in the art, such as, for example, prokaryotic or eukaryotic expression. For example, but not limited to, an anti-CD33 antibody that is an anti-CD33 hybrid antibody or fragment may be produced using conventional techniques to construct an expression vector encoding an antibody heavy chain, in which the CDRs and, optionally, the minimal portion of the variable region framework necessary to retain the original species' antibody binding specificity (as genetically engineered according to the techniques described herein) are derived from the original species' antibody, and the remainder of the antibody is derived from the target species immunoglobulin engineered as described herein, thereby generating a vector for expression of the hybrid antibody heavy chain.

[0171] Measurement of CD33 binding. In some embodiments, CD33 binding assay refers to an assay format in which CD33 protein and anti-CD33 antibody are mixed under suitable conditions for binding between CD33 protein and anti-CD33 antibody, and the amount of binding between CD33 protein and anti-CD33 antibody is evaluated. The amount of binding is compared to a suitable control, which may be the amount of binding in the absence of CD33 protein, the amount of binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding may be evaluated by any suitable method. Binding assay methods include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for direct measurement of CD33 protein binding with anti-CD33 antibody include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assay, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of the candidate anti-CD33 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-CD33 antibody, the candidate anti-CD33 antibody is useful as an anti-CD33 antibody of the present technology. In some embodiments, the CD33 protein is a short isoform of CD33 (a splice variant lacking the IgV domain).

[0172] Use of the anti-CD33 antibody of the present technology General. The anti-CD33 antibodies of the present technology are useful in methods known in the art for localizing and / or quantifying CD33 protein (e.g., for use in measuring the level of CD33 protein in an appropriate physiological sample, for use in diagnostic methods, for use in imaging of polypeptides, etc.). The antibodies of the present technology are useful for isolating CD33 protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-CD33 antibodies of the present technology can facilitate the purification of natural immunoreactive CD33 protein from biological samples, such as mammalian serum or cells, as well as the purification of recombinantly produced immunoreactive CD33 protein expressed in a host system. In addition, the anti-CD33 antibodies can be used to detect immunoreactive CD33 protein (e.g., in plasma, cell lysates, or cell supernatants) to evaluate the amount and pattern of expression of the immunoreactive polypeptide. The anti-CD33 antibodies of the present technology can be used diagnostically to monitor immunoreactive CD33 protein levels in tissues as part of a clinical trial procedure, for example, to determine the effectiveness of a given treatment regimen. As described above, detection can be facilitated by coupling (i.e., physically linking) the anti-CD33 antibodies of the present technology to a detectable substance.

[0173] Detection of CD33 protein. An exemplary method for detecting the presence or absence of immunoreactive CD33 protein in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with the anti-CD33 antibody of the present technology that can detect immunoreactive CD33 protein, thereby detecting the presence of immunoreactive CD33 protein in the biological sample. Detection can be achieved by a detectable label attached to the antibody. The term "labeled," with respect to anti-CD33 antibodies, is intended to encompass direct labeling of an antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of an antibody by reactivity with another compound, such as a secondary antibody, which is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected using fluorescently labeled streptavidin.

[0174] In some embodiments, the anti-CD33 antibody disclosed herein is conjugated to one or more detectable labels.For such use, the anti-CD33 antibody can be detectably labeled by covalent or non-covalent attachment of chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or other labels. Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6 phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0175] Examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y,67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Examples include Pd. 111 In is 125 I or 131 I is an exemplary isotope when in vivo imaging is used, since it avoids the problem of dehalogenation by the liver of I-labeled CD33-binding antibodies. Furthermore, this isotope has a more convenient gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, I isotope coupled to a monoclonal antibody using 1-(P-isothiocyanatobenzyl)-DPTA. 111 In shows little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870(1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe is one example.

[0176] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0177] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn and iron. The detection method of the present technology can be used to detect immunoreactive CD33 protein in biological samples in vitro as well as in vivo. In vitro techniques for detecting immunoreactive CD33 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay and immunofluorescence. In addition, in vivo techniques for detecting immunoreactive CD33 protein include introducing labeled anti-CD33 antibody into a subject. For example, anti-CD33 antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains CD33 protein molecules obtained from a test subject.

[0178] Immunoassays and imaging. The anti-CD33 antibodies of the present technology can be used to assay immunoreactive CD33 protein levels in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase and radioisotopes or other radioactive substances, such as iodine ( 125 I, 121 I,131 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 112 In) and technetium ( 99 mTc) and fluorescent labels, such as fluorescein, rhodamine and green fluorescent protein (GFP) and biotin.

[0179] In addition to assaying immunoreactive CD33 protein level in biological samples, the anti-CD33 antibody of the present technology can be used for in vivo imaging of CD33.Antibodies useful for this method include those that can be detected by X-ray photography, NMR or ESR.For X-ray photography, suitable labels include radioisotopes such as barium or cesium that emit detectable radiation but are not obviously harmful to subjects.For NMR and ESR, suitable markers include those that have detectable characteristic spins, such as deuterium, which can be incorporated into anti-CD33 antibody by labeling the nutrients of related scFv clones.

[0180] A suitable detectable imaging moiety, such as a radioisotope (e.g., 131 I, 112 In, 99 An anti-CD33 antibody labeled with a radiopaque substance or a material detectable by nuclear magnetic resonance (mTc) is introduced into the subject (e.g., parenterally, subcutaneously or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will dictate the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is usually about 5-20 millicuries. 99 The labeled anti-CD33 antibody then accumulates at the location of cells that contain specific target polypeptide.For example, the labeled anti-CD33 antibody of the present technology accumulates in the subject in the cells and tissues where CD33 protein is localized. Thus, the present technology provides a method for diagnosing a medical condition, comprising: (a) assaying expression of immunoreactive CD33 protein in an individual's cells or bodily fluids by measuring binding of an anti-CD33 antibody of the present technology; and (b) comparing the amount of immunoreactive CD33 protein present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive CD33 protein compared to the standard indicates the medical condition.

[0181] Affinity purification. The anti-CD33 antibody of the present technology can be used to purify immunoreactive CD33 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins such as polyacrylamide and latex beads. The technology of coupling antibodies to such solid supports is well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)). The simplest way to bind antigen to an antibody-support matrix is ​​to collect beads in a column and run the antigen solution through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a slow flow rate. The immobilized antibody captures the antigen as it flows past. Alternatively, the antigen may be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or shaking the slurry, which allows maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through the column for recovery of the beads. The beads are washed using a suitable washing buffer, and then. The pure or substantially pure antigen is eluted.

[0182] The antibody or polypeptide of interest can be conjugated to a solid support such as beads.Furthermore, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugating polypeptide to support, if necessary.Therefore, any of the conjugation methods and means disclosed herein for conjugating polypeptide to solid support can be applied to conjugating the first support to the second support, and the first and second solid supports can be the same or different.

[0183] Suitable linkers that can be cross-linking agents for use in conjugating polypeptides to solid supports include various substances that can react with functional groups present on the surface of the support, or with the polypeptide, or with both. Reagents that are useful as cross-linking agents include homobifunctional and, in particular, heterobifunctional reagents. Useful bifunctional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Cross-linking agents can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photolabile cross-linking agents such as 3-amino-(2-nitrophenyl)propionic acid can be used as a means to cleave the polypeptide from the solid support (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996) and U.S. Patent No. 5,643,722). Other cross-linking reagents are known in the art (see, for example, Wong (1991), supra; and Hermanson (1996), supra).

[0184] The antibody or polypeptide can be immobilized on a solid support such as beads by a covalent amide bond formed between a carboxyl functionalized bead and the amino terminus of the polypeptide, or vice versa by a covalent amide bond formed between an amino functionalized bead and the carboxyl terminus of the polypeptide. Additionally, a bifunctional trityl linker can be attached to the support, for example, a 4-nitrophenyl active ester on a resin such as a Wang resin, by an amino group on the resin or a carboxyl group on the resin by an amino resin. Using the bifunctional trityl approach, the solid support may require treatment with a volatile acid such as formic acid or trifluoroacetic acid to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide may be deposited on the bottom of a well of the solid support or as a bead-free patch on the flat surface of the solid support. After adding the matrix solution, the polypeptide can be released into the MS.

[0185] Hydrophobic trityl linkers can also be utilized as acid-labile linkers by using a volatile acid or a suitable matrix solution, such as a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid lability can also be modified. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be modified to a suitable p-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine bonds are made to the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by breaking the hydrophobic attraction or, if necessary, by cleaving the trityl ether or tritylamine bond under acidic conditions, including typical MS conditions where a matrix such as 3-HPA acts as an acid.

[0186] Orthogonally cleavable linkers can also be useful for binding a first solid support, such as a bead, to a second solid support, or for binding a polypeptide of interest to a solid support. Using such linkers, a first solid support (e.g., a bead) can be selectively cleaved from a second solid support without cleaving the polypeptide from the support, and then the polypeptide can be cleaved from the bead later. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to bind the bead to the second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide to the support. If necessary, for example, the link between the first and second supports can be left intact, and the link of the polypeptide to the solid support can be cleaved first. Trityl linkers can provide covalent or hydrophobic conjugation, but regardless of the nature of the conjugation, the trityl group is easily cleaved in acidic conditions.

[0187] For example, beads can be attached to a second support by a linking group that can be selected to have a length and chemical properties that promote high density binding of beads to solid support or high density binding of polypeptides to beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing a variety of functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythrol, and tris-hydroxy-aminomethane. Non-covalent association. By non-covalent interaction, the antibody or polypeptide can be conjugated to the solid support, or the first solid support can also be conjugated to the second solid support. For example, magnetic beads made of ferromagnetic material that can be magnetized can be attracted to the magnetic solid support and can be released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic moieties, which can respectively allow the interaction of the ionic or hydrophobic moieties with the polypeptide, for example, the polypeptide containing the attached trityl group, or with the second solid support that has hydrophobicity.

[0188] The solid support may also be provided with a member of a specific binding pair and thus be conjugated to a polypeptide or a second solid support that contains a complementary binding moiety. For example, a bead coated with avidin or streptavidin may be bound to a polypeptide that incorporates a biotin moiety, or to a second solid support coated with biotin or a derivative of biotin, such as iminobiotin.

[0189] It should be understood that any of the binding members disclosed herein or otherwise known in the art may be reversed. Thus, biotin may be incorporated, for example, into either the polypeptide or the solid support, and conversely, avidin or other biotin-binding moieties are incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and antigens that specifically interact with each other, and other such pairs known to those skilled in the art.

[0190] A. Diagnostic Uses of the Anti-CD33 Antibodies of the Present Technology General. The anti-CD33 antibodies of the present technology are useful in diagnostic methods. As such, the present technology provides a method of using the antibodies in diagnosing CD33 activity in a subject. The anti-CD33 antibodies of the present technology can be selected to have any level of epitope binding specificity and extremely high binding affinity to CD33 protein. In general, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed to remove non-specifically bound materials in an immunoassay without removing the target polypeptide. Thus, the anti-CD33 antibodies of the present technology useful in diagnostic assays are usually about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 1011 M -1 or 10 12 M -1 Additionally, anti-CD33 antibodies used as diagnostic reagents desirably have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 hours, at least five (5) hours, or at least one (1) hour.

[0191] Anti-CD33 antibodies can be used to detect immunoreactive CD33 protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay and immunometric assays. Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 2002). See U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074 and 4,098,876. Biological samples can be obtained from any tissue or body fluid of a subject. In certain embodiments, the subject is in the early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of CD33 protein in the sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF) and biopsied body tissue.

[0192] Immunoassay or sandwich assay is one format for the diagnostic method of the present technology. See US Patent Nos. 4,376,110, 4,486,530, 5,914,241 and 5,965,375. Such assay uses one antibody, for example, anti-CD33 antibody or a population of anti-CD33 antibodies immobilized on a solid phase and another anti-CD33 antibody or a population of anti-CD33 antibodies in solution. Usually, the solution anti-CD33 antibody or the population of anti-CD33 antibodies is labeled. When an antibody population is used, the population can contain antibodies that bind to different epitope specificities in the target polypeptide. Thus, the same population can be used for both solid phase and solution antibodies. When an anti-CD33 monoclonal antibody is used, a first and a second CD33 monoclonal antibody with different binding specificities are used for solid and solution phase. The solid phase (also called "capture") and solution (also called "detection") antibodies can be contacted with the target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the CD33 protein with the anti-CD33 antibody, the sample is usually incubated for a period that varies from about 10 minutes to about 24 hours, usually about 1 hour. A washing step is then performed to remove components of the sample that are not specifically bound to the anti-CD33 antibody used as a diagnostic reagent. If the solid phase and solution antibodies are bound in separate steps, washing may be performed after either or both binding steps. After washing, binding is usually quantified by detecting a label linked to the solid phase by binding of the labeled solution antibody. Usually, for a given pair of antibodies or populations of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. The concentration of immunoreactive CD33 protein in the sample being tested is then read from the calibration curve (i.e., the standard curve) by interpolation.The analyte can be measured from the amount of labeled solution antibody bound at equilibrium, or by kinetic measurements of the amount of labeled solution antibody bound at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of CD33 protein in the sample.

[0193] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway, NJ), and the like. Immobilization can be by absorption or by covalent attachment. Anti-CD33 antibodies can be linked to a linker molecule such as biotin for attachment to a surface-bound linker such as avidin.

[0194] In some embodiments, the present disclosure provides an anti-CD33 antibody of the present technology conjugated to a diagnostic agent.The diagnostic agent may include a radioactive or non-radioactive label, a contrast agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label may be a gamma-, beta-, alpha-, Auger electron-, or positron-radioactive isotope.The diagnostic agent is a molecule that is conjugated to an antibody portion, i.e., an antibody or antibody fragment or subfragment, and administered, and is useful in diagnosing or detecting disease by determining the location of cells that contain the antigen.

[0195] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). US Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents, and is incorporated herein by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load the antibody component with radiometals or paramagnetic ions, it may be necessary to react it with a reagent that has a long tail to which multiple chelating groups are attached to bind the ions. Such tails can be polymers such as polylysine, polysaccharides or other derivatized or derivatizable chains with pendant groups to which chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes and similar groups known to be useful for this purpose, can be attached. Chelates can be coupled to the antibodies of the present technology using standard chemistry. Chelates are usually linked to the antibody by a group that allows for the formation of bonds with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used with diagnostic isotopes for radioimaging. The same chelates, when complexed with non-radioactive metals such as manganese, iron and gadolinium, are useful for MRI when used with the anti-CD33 antibodies of the present technology.

[0196] Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are for use with a variety of metals and radiometals, such as radionuclides of gallium, yttrium and copper, respectively. Such metal chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Other examples of DOTA chelates include: (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH 2 ;(ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH 2 ;(iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH 2 ;(iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH 2 ;(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH 2 ;(ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 ;(x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH 2 ;(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 ;(xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2;(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH 2 ;(xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH 2 ;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 ;(xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH 2 ; and (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH 2 Examples include:

[0197] For RAIT 223 Other ring-type chelates, such as macrocyclic polyethers, which are of interest for stably binding nuclides such as Ra, are also contemplated.

[0198] B. Therapeutic Uses of the Anti-CD33 Antibodies of the Present Technology The immunoglobulin-related constructs (e.g., antibodies or antigen-binding fragments thereof) of the present technology are useful for the treatment of CD33-associated cancers and Alzheimer's disease. Such treatments can be used in patients identified as having pathologically high levels of CD33 (e.g., diagnosed by the methods described herein) or in patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method of treating CD33-associated cancer or Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to, AML, mixed leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloblastic crisis of chronic myelogenous leukemia, or acute lymphoblastic leukemia.

[0199] The composition of the present technology can be used with other therapeutic agents useful in the treatment of CD33-related cancer.For example, the antibody of the present technology can be administered separately, sequentially or simultaneously with at least one additional therapeutic agent selected from the group consisting of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents and targeted biological therapeutic agents (e.g., therapeutic peptides described in US6306832, WO2012007137, WO2005000889, WO2010096603, etc.).In some embodiments, at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapy agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, elbrigatran, cyclosporine ... In some embodiments, the therapeutic agent may be cyclosporine, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceae acetogenins, or combinations thereof.

[0200] The compositions of the present technology may be administered to a subject in need thereof as a single bolus, or the dosing regimen may include multiple administrations administered at various times after the appearance of a tumor. The method of treating Alzheimer's disease may further include administering to the subject at least one additional therapy selected from donepezil, galantamine, memantine, rivastigmine, memantine extended release and donepezil (namzalic), aducanumab, solanezumab, insulin, verubecestat, AADvac1, CSP-1103, and intepirdine, either sequentially, separately, or simultaneously. Administration can be performed by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally or subcutaneously), rectally, intracranially, intratumorally, intrathecally, or topically. Administration includes self-administration and administration by another. It should also be understood that the various modes of treatment of medical conditions as described include complete treatment as well as less than complete treatment, and are intended to mean "substantial" in which some biologically or medically relevant result is achieved.

[0201] In some embodiments, the antibodies of the present technology include pharmaceutical formulations that can be administered to a subject in need thereof in one or more doses. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response). Typically, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect ranges from about 0.000001 mg / kilogram of body weight / day to about 10,000 mg / kilogram of body weight / day. Typically, the dosage range is from about 0.0001 mg / kilogram of body weight / day to about 100 mg / kilogram of body weight / day. For administration of anti-CD33 antibodies, dosages range from about 0.0001 to 100 mg / kg of subject's body weight, more usually 0.01 to 5 mg / kg every week, every two weeks, or every three weeks. For example, dosages can be in the range of 1 mg / kg or 10 mg / kg every week, every two weeks, or every three weeks, or 1 to 10 mg / kg every week, every two weeks, or every three weeks. In one embodiment, a single dose of the antibody ranges from 0.1 to 10,000 micrograms / kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment dosing regimens involve administration once every two weeks or once a month or once every three to six months. The anti-CD33 antibody may be administered multiple times. The interval between single doses may be hourly, daily, weekly, monthly or yearly. The interval may be irregular as indicated by measuring blood levels of the antibody in the subject. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-CD33 antibody may be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration vary depending on whether the treatment is preventive or therapeutic.In preventive application, a relatively low dosage is administered for a long period of time at relatively infrequent intervals.In therapeutic application, a relatively high dosage at relatively short intervals is sometimes required until the progression of disease is reduced or terminated, or until the subject shows partial or complete remission of the symptoms of disease.Then, the patient can be administered a preventive administration regimen.

[0202] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, comprising: (a) administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present technology, the antibody being configured to localize to tumors expressing CD33 and labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissues and calculating the average radioactivity level present in non-tumor (normal) tissues ± standard deviation by computer. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0203] In some embodiments, the subject has been diagnosed with or is suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0204] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle radioisotope, a beta particle radioisotope, an Auger emitter, or any combination thereof. Examples of beta particle radioisotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67Examples of alpha particle radioisotopes include: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At and 255 Examples of Auger emitters include: 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl and 203 Pb. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., by N297A mutation in the Fc region resulting in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio by computer. In some embodiments, the immunoconjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0205] PRIT. In one aspect, the present disclosure provides a method for detecting a tumor in a subject in need thereof, comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and the CD33 antigen, wherein the conjugate is configured to localize in a tumor that expresses the CD33 antigen that is recognized by the bispecific antibody of the conjugate; and (b) detecting the presence of a solid tumor in the subject by detecting a radioactivity level emitted by the conjugate that is higher than a reference value. In some embodiments, the subject is a human.

[0206] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, comprising: (a) administering to the subject an effective amount of a complex comprising radiolabeled DOTA hapten and the bispecific antibody of the present technology that binds to radiolabeled DOTA hapten and CD33 antigen, wherein the complex is configured to localize in the tumor that expresses the CD33 antigen that is recognized by the bispecific antibody of the complex; (b) detecting the radioactive level emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy when the radioactive level emitted by the complex is higher than reference value.In some embodiments, the subject is human.

[0207] Examples of DOTA haptens include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH 2 ;(ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH 2 ;(iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH 2 ;(iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2;(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH 2 ;(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH 2 ;(ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 ;(x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH 2 ;(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 ;(xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 ;(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH 2 ;(xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ;(xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH 2 ;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 ;(xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH 2 ;(xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH 2and (xx) DOTA. The radiolabel can be an alpha particle radioisotope, a beta particle radioisotope or an Auger emitter. Examples of radiolabels include: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th, or 64 Cu is an example.

[0208] In some embodiments of the methods disclosed herein, the radioactivity level emitted by the complex is detected using positron emission tomography or single photon emission computed tomography.In addition or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with or suspected of having Alzheimer's disease or CD33-related cancer, such as AML, mixed leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloblastic crisis of chronic myelogenous leukemia, or acute lymphoblastic leukemia. Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, subcutaneously, intraventricularly, orally, intratumorally, or intranasally. In certain embodiments, the complex is administered into the cerebrospinal fluid or blood of the subject.

[0209] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected between 2 and 120 hours after the complex is administered. In certain embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is expressed as a percentage of the injected dose per gram of tissue (%ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and computing the average level of radioactivity present in the non-tumor (normal) tissue ± standard deviation. In some embodiments, the reference value is the standard uptake value (SUV). See Thie JA, J Nucl Med. 45(9):1431-4(2004). In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0210] In another aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD33-associated cancer, comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, the anti-DOTA bispecific antibody configured to localize to tumors expressing a CD33 antigen target, and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a period of time sufficient to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is removed from the bloodstream following administration of the anti-DOTA bispecific antibody. In some embodiments, a radiolabeled DOTA hapten is administered after a period of time that may be sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0211] The radiolabeled DOTA hapten can be administered any time between 1 minute and 4 or more days following administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time after 4 or more days following administration of the anti-DOTA bispecific antibody.

[0212] Additionally or alternatively, in some embodiments, the method further comprises administering an effective amount of a clearance agent to the subject prior to administration of the radiolabeled DOTA hapten.The clearance agent can be any molecule (dextran or dendrimer or polymer) that can be conjugated with C825-hapten.In some embodiments, the clearance agent is less than or equal to 2000kD, 1500kD, 1000kD, 900kD, 800kD, 700kD, 600kD, 500kD, 400kD, 300kD, 200kD, 100kD, 90kD, 80kD, 70kD, 60kD, 50kD, 40kD, 30kD, 20kD, 10kD or 5kD. In some embodiments, the clearance agent is a 500 kD aminodextran-DOTA conjugate (such as 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu) or 500 kD dextran-DOTA-Bn(In)).

[0213] In some embodiments, the clearance agent and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody of the present technology.For example, in some embodiments, the anti-DOTA bispecific antibody of the present technology is administered according to a regimen that includes at least one cycle of (i) administration of the anti-DOTA bispecific antibody of the present technology (so that related tumor cells may be saturated), (ii) administration of the radiolabeled DOTA hapten and optionally a clearance agent, (iii) any further administration of the radiolabeled DOTA hapten and / or the clearance agent without further administration of the anti-DOTA bispecific antibody.In some embodiments, the method can include multiple such cycles (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0214] Additionally or alternatively, in some embodiments of the method, the anti-DOTA bispecific antibody and / or radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, subcutaneously, intraventricularly, intratumorally, orally, or intranasally. In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with CD33-associated cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to a CD33 antigen target, wherein the conjugate is configured to localize to a tumor that expresses the CD33 antigen target recognized by the bispecific antibody of the conjugate.The conjugate can be administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally.In some embodiments, the subject is a human.

[0215] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, the anti-DOTA bispecific antibody being configured to localize to a tumor expressing a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten being configured to bind to the anti-DOTA bispecific antibody. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a period sufficient to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is removed from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period that may be sufficient to allow clearance of unbound anti-DOTA bispecific antibody. In some embodiments, the subject is a human.

[0216] Thus, in some embodiments, the methods further comprise administering to the subject an effective amount of a clearance agent prior to administration of the radiolabeled DOTA hapten, which may be administered any time between 1 minute and 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time after 4 or more days following administration of the anti-DOTA bispecific antibody.

[0217] The clearance agent may be a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu) or 500 kD dextran-DOTA-Bn(In), etc.). In some embodiments, the clearance agent and radiolabeled DOTA hapten are administered without further administration of an anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one cycle of (i) administration of an anti-DOTA bispecific antibody of the present technology (so that associated tumor cells may be saturated), (ii) administration of a radiolabeled DOTA hapten and optionally a clearance agent, (iii) any further administration of a radiolabeled DOTA hapten and / or a clearance agent without further administration of an anti-DOTA bispecific antibody. In some embodiments, the method may include multiple such cycles (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0218] Also provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a complex comprising radiolabeled DOTA hapten and the bispecific antibody of the present technology that recognizes and binds to the CD33 antigen target, wherein the complex is configured to localize in the tumor that expresses the CD33 antigen target that is recognized by the bispecific antibody of the complex.The therapeutic efficacy of such a complex can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio by computer. In some embodiments, the complex has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0219] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-CD33 antibody described herein provides a therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the anti-CD33 antibodies described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (the lethal dose for 50% of the population) or LD 100 The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dose range for use in humans. The dosage of the anti-CD33 antibody described herein is within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range according to the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the subject's condition. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0220] Formulation of pharmaceutical compositions. According to the methods of the present technology, the anti-CD33 antibody may be incorporated into a pharmaceutical composition suitable for administration. The pharmaceutical composition generally comprises a recombinant or substantially purified antibody and a pharma- ceutical acceptable carrier in a form suitable for administration to a subject. The pharma- ceutical acceptable carrier is determined in part by the particular composition to be administered, as well as by the particular method used to administer the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody constructs (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18). th (See, e.g., U.S. Pat. No. 6,339,341, ed., 1990.) Pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0221] The terms "pharmaceutical acceptable", "physiologically acceptable" and grammatical variations thereof are used interchangeably when referring to compositions, carriers, diluents and reagents, and indicate that the material can be administered to a subject without causing undesirable physiological effects to the extent that they would prevent administration of the composition. For example, "pharmaceutical acceptable excipients" generally refer to excipients that are safe and non-toxic and useful in the preparation of the desired pharmaceutical composition, including excipients that are acceptable for veterinary use, as well as for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. "Pharmaceutical acceptable salts and esters" refer to salts and esters that are pharmaceutical acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids, e.g., methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-CD33 antibody, e.g., C 1-6Examples of suitable salts include alkyl esters. When two acidic groups are present, the pharma- ceutically acceptable salts or esters may be mono-acid-mono-salts or esters or di-salts or esters; similarly, when two or more acidic groups are present, some or all of such groups may be salified or esterified. The anti-CD33 antibodies named in this technology may exist in unsalted or unesterified form, or salified and / or esterified form, and the naming of such anti-CD33 antibodies is intended to include both the original (unsalted and unesterified) compound and its pharma- ceutically acceptable salts and esters. Also, certain embodiments of the technology may exist in two or more stereoisomeric forms, and the naming of such anti-CD33 antibodies is intended to include all single stereoisomers and all mixtures (racemic or not) of such stereoisomers. Those skilled in the art will have no difficulty in determining the appropriate timing, sequence and dosage of administration of a particular drug and composition of the technology.

[0222] Examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution and 5% human serum albumin.Non-aqueous vehicles such as liposomes and hardened oils can also be used.The use of such media and compounds for pharmacoactive substances is well known in the art.Unless any conventional media or compound is incompatible with anti-CD33 antibody, its use in the composition is considered.Supplementary active compounds can also be incorporated into the composition. The pharmaceutical composition of the present technology is formulated to be compatible with its intended route of administration.The anti-CD33 antibody composition of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal or intramuscular routes, or as an inhalant.Anti-CD33 antibodies can also be administered in combination with other agents that are at least partially effective in treating various CD33-related cancers or Alzheimer's disease.

[0223] Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, hydrogenated oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial compounds such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating compounds such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetate, citrate or phosphate, and compounds for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0224] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including compounds that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0225] Sterile injectable solutions can be prepared by incorporating the anti-CD33 antibody of the present technology in the required amount in a suitable solvent with one or a combination of the components listed above as necessary, followed by sterilization by filtration.Generally, dispersions are prepared by incorporating the anti-CD33 antibody into a sterile vehicle that contains a basic dispersion medium and other components required from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the method of preparation is vacuum drying and lyophilization, which obtains a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile filtered.The antibody of the present technology can be administered in the form of depot injection or indwelling preparation, which can be formulated in such a way that it allows sustained or pulsatile release of active ingredient.

[0226] Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, anti-CD33 antibodies may be incorporated with excipients and used in the form of tablets, lozenges or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, swished, and expectorated or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch, a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide, a sweetening compound such as sucrose or saccharin, or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

[0227] For administration by inhalation, the anti-CD33 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated in the formulation is used. Such penetrants are generally known in the art, and include, for example, surfactants, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, anti-CD33 antibodies are formulated into ointments, salves, gels or creams as generally known in the art. Anti-CD33 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0228] In one embodiment, the anti-CD33 antibody is prepared with a carrier that protects the anti-CD33 antibody from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be clear to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells with monoclonal antibodies against viral antigens, can also be used as pharma- ceutical acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0229] C.Kit The present technology provides a kit for detecting and / or treating CD33-associated cancer or Alzheimer's disease, comprising at least one immunoglobulin-related construct (e.g., any antibody or antigen-binding fragment described herein) or a functional variant (e.g., a substitution variant) of the present technology. The above-mentioned components of the kit of the present technology may be packaged in a suitable container and labeled for the diagnosis and / or treatment of CD33-associated cancer or Alzheimer's disease. The above-mentioned components may be stored in unit or multi-dose containers, such as sealed ampoules, vials, bottles, syringes and test tubes, as aqueous, preferably sterile solutions, or as lyophilized, preferably sterile formulations for reconstitution. The kit may further comprise a second container that holds a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharma-ceutically acceptable excipients of the pharmaceutical composition and saline solution. Additionally, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The container may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle). The kit may further include more containers containing pharma- ceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture media for one or more of the suitable hosts. The kit may include instructions customarily included in commercial packages of therapeutic or diagnostic agents, which contain information regarding, for example, indications, usage, dosage, manufacture, administration, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic agents.

[0230] The kit is useful for detecting the presence of immunoreactive CD33 protein in any body fluid, including, but not limited to, biological samples, such as serum, plasma, lymph, cyst fluid, urine, stool, cerebrospinal fluid, ascites, or blood, and including biopsy samples of body tissue. For example, the kit may include one or more humanized, chimeric, or bispecific anti-CD33 antibodies (or antigen-binding fragments thereof) of the present technology that can bind to CD33 protein in biological samples, a means for determining the amount of CD33 protein in the sample, and a means for comparing the amount of immunoreactive CD33 protein in the sample with a standard. One or more of the anti-CD33 antibodies may be labeled. The kit components (e.g., reagents) may be packaged in suitable containers. The kit may further include instructions for using the kit to detect immunoreactive CD33 protein.

[0231] For antibody-based kits, the kits can include, for example, 1) a first antibody, e.g., a humanized, chimeric, or bispecific anti-CD33 antibody of the present technology (or an antigen-binding fragment thereof) attached to a solid support that binds to CD33 protein, and, optionally, 2) a second, different antibody that binds either to CD33 protein or the first antibody and is conjugated to a detectable label.

[0232] The kit may also include, for example, a buffer, a preservative, or a protein stabilizing agent. The kit may further include components necessary for detecting the detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package with instructions for interpreting the results of the assay performed using the kit. The kit of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagent contained in the kit for treating CD33-associated cancer in a subject in need thereof, for example, for detecting CD33 protein in vitro or in vivo. In certain embodiments, the use of the reagent may follow the method of the present technology. EXAMPLES

[0233] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization and use of the exemplary anti-CD33 antibodies of the present technology. The following examples demonstrate the production of the chimeric, humanized and bispecific antibodies of the present technology and the characterization of their binding specificity and in vitro and in vivo biological activity.

[0234] Example 1 Structure and Binding Affinities of Chimeric and Humanized Anti-CD33 Antibodies of the Disclosure Humanization of mouse HIM34. The CDRs of the heavy and light chains of HIM34 are integrated into the human framework IGHV1-2 VH. * 02IGHJ4 * 01. Human framework IGKV1-33 of VL * 01-IGKJ2 *Based on the homology with huHIM34.01, each was grafted onto a human IgG1 framework. From the six heavy and five light chain designs, 30 versions of huHIM34 were gene synthesized and expressed in DG44 cells. The amino acid sequences of the mouse, chimeric, and humanized HIM34 variable heavy chains are shown in SEQ ID NOs:2-7 and 114, and in Figures 8 and 10(B). The amino acid sequences of the mouse, chimeric, and humanized HIM34 variable light chains are shown in SEQ ID NOs:9-13 and 115, and in Figures 9 and 10(A).

[0235] Generation of CD33-BsAb bispecific antibody. CD33-BsAb was designed using an IgG-scFv format (Figure 3). CD33-BsAb was generated using the IgG-scFv (G 4 S) 3 The humanized OKT3 scFv was constructed by fusing the C-terminus of the light chain of chimeric or humanized anti-CD33 antibodies via a linker. N297A and K322A mutations were introduced into the Fc region of the antibody to eliminate FcR and complement binding activity, respectively (Shields RL et al., Journal of Biological Chemistry 276:6591-6604(2001); Idusogie EE et al., Journal of Immunology 164:4178-4184(2000)). The DNA constructs were then transfected into CHO-S cells and stable clones were selected for high-level antibody production. For large-scale antibody purification, the selected stable clones were expanded in shaker flasks. Bispecific antibodies were purified from the supernatant using one-step protein A affinity chromatography.

[0236] Figure 10 shows the amino acid sequences of the light and heavy chains of the chimeric CD33-BsAb (chHIM34 BsAb), corresponding to SEQ ID NO: 14 and SEQ ID NO: 16, respectively. Figures 11(A)-11(B), 11(C)-11(D), 12(A)-12(B), and 13(A)-13(B) show the amino acid sequences of the light and heavy chains of four humanized CD33-BsAbs (hHIM34 BsAbs), BC249, BC275, BC267, and BC268, respectively. Figures 11(A)-11(B) show the amino acid sequences of the light and heavy chains of BC249, corresponding to SEQ ID NO: 18 and SEQ ID NO: 20, respectively, and based on the humanized HIM34 variable light chain VL-3 and variable heavy chain VH-5. Figures 11(C)-11(D) show the amino acid sequences of the light and heavy chains of BC275, corresponding to SEQ ID NO: 134 and SEQ ID NO: 136, respectively, and based on humanized HIM34 variable light chain VL-4 and variable heavy chain VH-5. Figures 12(A)-12(B) show the amino acid sequences of the light and heavy chains of BC267, which correspond to SEQ ID NO: 18 and SEQ ID NO: 22, respectively, and are based on humanized HIM34 variable light chain VL-3 and variable heavy chain VH-6. Figures 13(A)-13(B) show the amino acid sequences of the light and heavy chains of BC268, which correspond to SEQ ID NO: 24 and SEQ ID NO: 22, respectively, and are based on humanized HIM34 variable light chain VL-4 and variable heavy chain VH-6.

[0237] Comparison of binding kinetics between chimeric and humanized anti-CD33 antibodies by surface plasmon resonance. To investigate whether the humanized anti-CD33 HIM34 antibody binds to CD33 with similar affinity as the chimeric form, a surface plasmon resonance assay was performed using a Biacore T-200. Biacore T-200 biosensor, CM5 sensor chip, and related reagents were purchased from GE Healthcare. CD33 as the active surface and blank as the reference were immobilized on the CM5 sensor chip at approximately 1000 RU (response units) using the Amino Coupling kit (GE Healthcare). CD33 antibody and related control antibodies were diluted to different concentrations (31–500 nM) in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20) before analysis. Samples were injected over the sensor surface for 1 min at a flow rate of 30 μL / min. After completion of the association phase, dissociation was monitored for 3 min at the same flow rate in HBS-EP buffer or for 30 min at 37 °C for antibodies with slow koff. At the end of each cycle, the surface was regenerated with 10 mM NaOH for 30 s at a flow rate of 50 μL / min. Biosensor curves obtained by injecting samples on the active surface were subtracted from control curves obtained by injecting samples on a reference surface prior to kinetic analysis. Data were analyzed using Biacore T-200 evaluation software. As shown in Figures 16(A)-16(B), the humanized anti-CD33 HIM34 antibody showed a koff similar to that of the chimeric version. on and k off was bonded with.

[0238] These results demonstrate that the antibody or antigen-binding fragment of the present technology specifically binds to CD33 antigen with high binding affinity.Therefore, the immunoglobulin-related construct disclosed herein is useful in detecting CD33 protein in samples.

[0239] Example 2 Biological activity of the T cell-engaging CD33-BsAb antibody of this technology Chimeric CD33-BsAb binds to AML cell lines. Binding of chHIM34 BsAb to target cells was tested by FACS immunostaining. chHIM34 BsAb bound to CD33(+) AML cell lines U937, THP1, SET2, C1498-CD33 and M-07e, but had no effect on CD33(-) leukemia cell line CMLT1 (Figure 4).

[0240] Chimeric CD33-BsAb binds to both splice variants and full-length CD33. Full-length CD33 molecules have two extracellular domains, a membrane-distal IgV domain and a membrane-proximal IgC2 domain (Figure 1). There are no FDA-approved antibodies that bind to CD33 splice variants lacking the IgV domain (Figure 2). Importantly, 50% of AML patients have spliced ​​CD33 proteins and therefore cannot benefit from current CD33 antibody drugs. The chimeric CD33-BsAb of the present disclosure, chHIM34 BsAb, binds to both full-length and splice variants of CD33, whereas the humanized M195-based BsAb (BC133) does not bind to splice variants of CD33 (Figures 5(A)-5(B)). Figure 19 shows the stability of the humanized BSAb of the present technology (hHIM34xCD3) at 40°C using HPLC. Among all the clones tested, the hHIM34 L4H5(BC275) clone shows the highest stability at 40°C.

[0241] Chimeric CD33-BsAb redirects T cells to kill CD33(+) AML cell lines. To assess whether chimeric CD33-BsAb can redirect T cells to kill leukemic cells, the cytotoxicity of T cells against CD33(+) AML cells was tested in a standard 4-h 51Cr release assay. In the presence of chHIM34 BsAb, substantial killing of AML cell lines was observed, with EC50s as low as 1 fM (for C1498-CD33) to 200 fM (for THP1) (Figure 6(A)-6(B)). Comparison of binding kinetics of chimeric and humanized HIM34 bispecific antibodies by surface plasmon resonance. The affinity of humanized HIM34 BsAb for CD33 was tested using surface plasmon resonance technology. The results are shown in the table below.

[0242] [Table 2]

[0243] Mean fluorescence intensity (MFI) is a reliable criterion for predicting the in vitro potency of the CD33 antibody of the present technology. To test the binding of humanized HIM34 BsAb, MOLM13 CD33(+) cells were contacted with different concentrations of humanized anti-CD33 BsAb disclosed herein. The flow cytometry results are shown in Figure 17. The cytotoxicity of humanized BsAb against MOLM13 cells was tested by T cell-dependent cytotoxicity assay (TDCC). As shown in Figures 18(A)-18(B), clones with better binding to CD33 (higher MFI) showed more potent effects (lower EC50). These results demonstrate that MFI is a reliable criterion for predicting the in vitro potency of the CD33 antibody of the present technology.

[0244] These results demonstrate that the antibody or its antigen-binding fragment of the present technology specifically targets CD33 positive cancer cells.Therefore, the immunoglobulin-related construct disclosed herein is useful in detecting and treating CD33-related cancer in subjects who need it.

[0245] Example 3 In vivo therapeutic study using CD33-BSAb The prognosis of patients with FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations (FLT3 / ITD) is generally poor. In pediatric AML, the negative impact of these mutations is more pronounced (Levis and Small, Leukemia 17:1738-52 (2003)). To evaluate whether chimeric CD33-BsAb can redirect T cells to AML with FLT3 / ITD mutations, we performed in vivo experiments using NOD.Cg-Prkdcscid Il2rgtm1 WjlTg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ (NSGS) mice. Mice were randomly divided into four groups, and all mice received 1 million MOLM13 cells containing the firefly luciferase gene. Group 1: activated T cells (ATC) only, group 2: ATC+chHIM34 BsAb (0.1 μg / dose), group 3: ATC+control BsAb (0.1 μg / dose), and group 4: ATC+chHIM34 BsAb (0.01 μg / dose). Treatment was initiated on day 7, when leukemia was established. For 3 weeks, mice were weakly injected with 10 million ATC mixed with BsAb. BsAb was administered twice a week, once mixed with ATC and once alone. A total of 5 doses of BsAb were injected. Interleukin-2 at 1000 IU was administered subcutaneously twice a week to support T cell growth in vivo. Bioluminescence imaging was performed weekly to assess leukemia burden. As shown in Figures 7(A)-7(B), administration of ATC in the presence of 100 ng / dose or 10 ng / dose of chHIM34 BsAb cured leukemic mice, but the control BsAb + ATC at 100 ng / dose had no antitumor effect, similar to the ATC-only group.

[0246] Thus, the immunoglobulin-related constructs disclosed herein are useful in the detection and treatment of CD33-associated cancers in subjects in need thereof.

[0247] Example 4 Comparative activity of the anti-CD33 BsAb of this technology and existing CD33 antibodies The BC275 (CD3×CD33) BsAb disclosed herein binds to the membrane proximal domain (IgC) of the extracellular region of CD33. See Figures 20(A)-20(B). Other existing examples of CD3×CD33 BsAbs, BC133 (lintuzumab) based on the M195 clone and BC269 (gemtuzumab) based on My96, bind to the membrane distal domain of the extracellular region of CD33 (Figure 20(B)). Various CD33(+) and CD33(-) human cancer cell lines were contacted with the three bispecific antibodies to evaluate binding to CD33. Secondary antibodies were used to detect the binding of the BsAbs to the human cancer cell lines. As shown in Figure 20(C), all three BsAbs bind to CD33(+) cells but do not react with CD33(-) cells. Activated human T cells were mixed with various CD33(+) or CD33(-) (NALM6) cancer cells, and the T cell-dependent cell-mediated cytotoxicity (TDCC) of the three CD3×CD33 BsAbs was evaluated. As shown in Figures 21(A)-21(C), all three BsAbs potently lysed CD33(+) leukemia cells, but not CD33(-) NALM6 cells. These results demonstrate that CD3×CD33 BsAbs selectively target CD33(+) cancers. Furthermore, there was a positive correlation between the expression level of CD33 on cancer cells and the potency of the BsAbs in the TDCC assay (pooled data for all three CD3×CD33 BsAbs are shown in Figure 21(D)).

[0248] To evaluate and compare the efficacy of the three BsAbs in vivo, several animal studies were performed. In the first study, immunodeficient NSG mice were transfected with CD33(+)MOLM13-luciferase (genotype CC at rs12459419 SNP) human AML xenografts (10 6 Mice were inoculated intravenously with 5 × 10 6A single injection of 0.025 μg of the test BsAb (10 μg / Kg / dose) with activated T cells was performed. The timing of T cell and antibody injections is shown in Figure 22(B). In the control group, mice received T cells and BC119 (CD3×GD2 BsAb). Injections of BsAb alone were continued for four more times. Leukemia growth was monitored using an IVIS bioluminescence imager. All three CD3×CD33 BsAbs redirected T cells to treat mice inoculated with AML cells, whereas mice receiving control BsAb and T cells showed leukemia progression (Figures 22(A)-22(B)).

[0249] We then inoculated immunodeficient NSG mice with human CD33-transduced NALM6-luciferase leukemia xenografts (0.5 × 10 6 Mice were inoculated intravenously with 10 ng BsAb / 10 6 Activated T cells (2.7 x 10 6 pcs and 5×10 6 In the control group, mice were administered T cells and BC119 (CD3×GD2 BsAb). The timing of T cell and BsAb injections is shown in Figure 23(B). Leukemia growth was monitored using an IVIS bioluminescence imaging device. To support T cell engraftment, all mice were subcutaneously injected with interleukin 2 (1000 IU) twice during the first week of treatment. Figure 23(C) shows the survival curves of mice treated with the three CD3×CD33 BsAbs. The most potent BsAb was BC275 (a CD33 antibody of this technology), which promoted a higher degree of survival compared to the conventional BC269 and BC133 BsAbs (which showed comparable efficacy).

[0250] The next experiment was performed in immunodeficient DKO mice subcutaneously inoculated with THP1 human leukemia xenografts (genotype CC for rs12459419 SNP). Treatment was started 14 days later (Figures 24(A)-24(D)). Mice were injected twice (107 In the control group, mice were administered T cells and BC119 (CD3×GD2 BsAb). The timing of T cell and antibody injections is shown in Figure 24(A). Leukemia progression was monitored by measuring tumor size. To support T cell engraftment, all mice were subcutaneously injected with interleukin-2 (1000 IU) twice a week for the first 3 weeks of T cell injection. The most potent BsAb was BC275 (a CD33 antibody of this technology), which showed the highest degree of tumor reduction compared to the conventional BsAbs BC269 and BC133 (i.e., BC275>BC269>BC133).

[0251] Animal experiments were performed on immunodeficient DKO mice subcutaneously inoculated with K562 human leukemia xenografts (genotype TT for rs12459419 SNP). Treatment was started 7 days later (Figures 25(A)-25(D)). Mice were injected with activated T cells supplemented with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose) three times (10 7 In the control group, mice were administered T cells and BC119 (CD3×GD2 BsAb). The timing of T cell and antibody injections is shown in Figure 25(A). Leukemia progression was monitored by measuring tumor size. To support T cell engraftment, all mice were subcutaneously injected with interleukin-2 (1000 IU) twice per week for the first 3 weeks of T cell injection. The most potent BsAb was BC275 (a CD33 antibody of the present technology), which showed the highest degree of tumor reduction compared to the conventional BsAbs BC269 and BC133 (i.e., BC275>BC269>BC133). Thus, the immunoglobulin-related constructs disclosed herein are useful in the detection and treatment of CD33-associated cancers in subjects in need thereof.

[0252] Example 5 Use of the present anti-CD33 BsAb antibody in PRIT IgG-based CD33-C825 BsAb. CD33(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or by other routes. Treatment begins after tumor formation (depending on tumor type and injection route). Treatment consists of one or more cycles. Each cycle involves administration of the test BsAb (250 μg intravenously), followed 24-48 hours later by injection of a clearance agent (DOTA-dextran or DOTA-dendrimer, dose 5-15% of the BsAb dose, see Cheal SM et al., Mol Cancer Ther 13:1803-12, 2014). 4 hours later, DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi) was injected intravenously. 225 Ac is DOTA- 177 It is more potent than Lu and may require fewer cycles to eradicate tumors.

[0253] Tetramerized BsAb. CD33(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or by other routes, and treatment begins after tumor formation (depending on tumor type and injection route). Treatment consists of one or more cycles. Each cycle consists of administration of BsAb (250 μg intravenously) followed 24–48 hours later by DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 In general, the treatment consisted of intravenous injection of DOTA-Ac (1 μCi). 225 Ac is DOTA- 177 It is more potent than Lu and may require fewer cycles to eradicate tumors. These results demonstrate that the antibody or antigen-binding fragment of the present technology can detect tumors and inhibit tumor growth and / or metastasis progression.Therefore, the immunoglobulin-related constructs disclosed herein are useful for detecting and treating CD33-related cancer in subjects in need thereof.

[0254] Example 6 Use of the Anti-CD33 BsAb of the Present Technology in the Treatment of Alzheimer's Disease Various rodent models of Alzheimer's disease (AD) have been developed (Nazem et al., J Neuroinflammation 17;12:74 (2015)). Because the HIM34 antibody binds to human CD33 and does not cross-react with mouse CD33, we will generate a knock-in mouse model of human CD33 (hCD33). The hCD33 mice will then be crossed with the Alzheimer's disease transgenic mice of Nazem et al. (2015), and the offspring will be used to examine the therapeutic effect of the CD33 antibody or antigen-binding fragment of this technology in vivo. In one experiment, an anti-CD33 BsAb disclosed herein comprising a C825 anti-DOTA binding moiety was injected intracranially or into the cerebrospinal fluid, followed by administration of a radiolabeled metal-DOTA conjugate (e.g., 177 The mice will be intravenously injected with anti-CD33 BsAb (Lu-DOTA-Bn). Mouse behavioral analysis as well as histopathological evaluation of brain tissue over time will be used as endpoints. Animals treated with the anti-CD33 BsAb are expected to show improvement in one or more symptoms of Alzheimer's disease (AD) compared to symptoms observed in untreated controls. Thus, the immunoglobulin related constructs disclosed herein are useful for treating Alzheimer's disease in a subject in need thereof.

[0255] Equivalent The present technology should not be limited with respect to the specific embodiments described in this application, but are intended as single illustrations of individual aspects of the present technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present technology. It should be understood that the present technology is not limited to specific methods, reagents, compound compositions, or biological systems, which of course may vary. It should also be understood that the terms used herein are for the purpose of merely describing specific embodiments, and are not intended to be limiting.

[0256] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure also is described in terms of any individual members or subgroups of members of the Markush group. As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. The recited ranges can be easily recognized as being fully described and effective with at least equal halves, thirds, quarters, fifths, tenths, etc. of the same range broken down. As a non-limiting example, each range discussed herein can be easily broken down into a lower third, middle third, and upper third, etc. Also, as will be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than" and the like refers to a range that includes the recited numbers and can then be broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0257] All patents, patent applications, provisional applications, and publications referenced or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

Claims

1. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L and wherein the antibody or antigen-binding fragment thereof comprises (a) V H is GYSFTDYN (SEQ ID NO: 154) V H - CDR1 sequence, IDPYKGGT (SEQ ID NO: 155) H - CDR2 sequence, and V of AREMITAYYFDY (SEQ ID NO: 156) H - comprising the CDR3 sequence, and (b) V L is QDINKY (SEQ ID NO: 157) V L - CDR1 sequence, V of YAS (SEQ ID NO: 158) L - CDR2 sequence, and LQYDNLLT (SEQ ID NO: 159) V L - an antibody or antigen-binding fragment thereof, comprising the CDR3 sequence.

2. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L and wherein the antibody or antigen-binding fragment thereof comprises (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133; and / or (b) V L An antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

3. 3. The antibody or antigen-binding fragment of claim 1 or 2, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE.

4. The antibody of claim 3, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

5. The antibody of claim 3, comprising an IgG4 constant region comprising an S228P mutation.

6. Fab, F(ab') 2 , Fab', scF v and F v The antigen-binding fragment of claim 1 or 2, selected from the group consisting of:

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, which binds to the IgC2 domain of CD33.

8. The antibody according to any one of claims 1 to 5 or 7, which is a monoclonal antibody, a chimeric antibody, a humanized antibody or a bispecific antibody.

9. An antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:141, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:140, or a variant thereof having one or more conservative amino acid substitutions.

10. each, SEQ ID NO:16 and SEQ ID NO:14 (chHIM34xCD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34xCD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34xCD3 BsAb); SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5xmC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5xhC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6xmC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6xhC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6xmC825); SEQ ID NO: 22 and SEQ ID NO: 29 (VL4VH6xhC825); SEQ ID NO:139 and SEQ ID NO:138 (mouse VL-mouse VH x mC825); and SEQ ID NO: 141 and SEQ ID NO: 140 (mouse VL-mouse VH x hC825) The antibody of claim 9, comprising an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of:

11. (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to any one of SEQ ID NOs: 9, 10, 11, 12, or 13, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, or 133. An antibody comprising:

12. (a) an LC sequence that is at least 95% identical to an LC sequence present in any one of SEQ ID NOs: 14, 18, 24, 26, 27, 28, 29, 134, 138, or 140; and / or (b) an HC sequence that is at least 95% identical to an HC sequence present in any one of SEQ ID NOs: 16, 20, 22, 136, 139, or 141; An antibody comprising:

13. The antibody according to any one of claims 9 to 12, which is a chimeric antibody, a humanized antibody or a bispecific antibody.

14. An antibody according to any one of claims 9 to 13, which binds to the IgC2 domain of CD33.

15. The antibody of any one of claims 9 to 14, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

16. The antibody of any one of claims 9 to 14, comprising an IgG4 constant region comprising an S228P mutation.

17. A bispecific antibody or antigen-binding fragment comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 30-113 or 142-153.

18. 18. The antibody or antigen-binding fragment of claim 17, comprising an amino acid sequence selected from any one of SEQ ID NOs: 30-113 or 142-153.

19. A recombinant nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 18.

20. A recombinant nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 135 and 137.

21. A host cell or vector comprising a recombinant nucleic acid sequence according to claim 19 or claim 20.

22. 9. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

23. 19. A composition comprising the antibody of any one of claims 9 to 18 and a pharma- ceutically acceptable carrier, wherein the antibody is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

24. The antibody of any one of claims 1 to 5, 7 or 8, which lacks α-1,6-fucose modification.

25. The antibody according to any one of claims 9 to 16, which lacks α-1,6-fucose modification.

26. 14. The bispecific antibody of claim 8 or 13, which binds to T cells, B cells, myeloid cells, plasma cells or mast cells.

27. 20. The bispecific antibody or antigen-binding fragment of claim 8, 13, 17 or 18, which binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR or the small molecule DOTA hapten.

28. 1. A method of treating a CD33-associated cancer or Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of an antibody comprising an HC amino acid sequence and an LC amino acid sequence, each of which comprises: each, SEQ ID NO:16 and SEQ ID NO:14 (chHIM34xCD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34xCD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34xCD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34xCD3 BsAb); SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5xmC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5xhC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6xmC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6xhC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6xmC825); SEQ ID NO: 22 and SEQ ID NO: 29 (VL4VH6xhC825); SEQ ID NO:139 and SEQ ID NO:138 (mouse VL-mouse VH x mC825); SEQ ID NO: 141 and SEQ ID NO: 140 (mouse VL-mouse VH x hC825) comprising a sequence selected from the group consisting of The method, wherein the antibody specifically binds to CD33.

29. 1. A method of treating a CD33-associated cancer or Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of a bispecific antibody or antigen-binding fragment comprising an amino acid sequence selected from any one of SEQ ID NOs: 30-113 or 142-153.

30. 30. The method of claim 28 or 29, wherein the CD33-associated cancer is acute myeloid leukemia (AML), mixed leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloblastic crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

31. The method of any one of claims 28 to 30, wherein the antibody or antigen-binding fragment is administered to the subject separately, sequentially or simultaneously with a further therapeutic agent.

32. 32. The method of claim 31 , wherein the additional therapeutic agent is one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen agent, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, antimetabolite, an endocrine / hormonal agent, a bisphosphonate therapy.

33. 1. A method for detecting a tumor in a subject in vivo, comprising: (a) administering to a subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody is configured to localize to tumors expressing CD33 and is labeled with a radioisotope; (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody or antigen-binding fragment that is higher than a reference value; The method includes:

34. 34. The method of claim 33, wherein the subject has been diagnosed with or is suspected of having cancer or Alzheimer's disease.

35. 35. The method of claim 33 or 34, wherein the level of radioactivity emitted by the antibody or antigen-binding fragment is detected using positron emission tomography or single photon emission computed tomography.

36. 36. The method of any one of claims 33 to 35, further comprising administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 18 conjugated to a radionuclide.

37. 37. The method of claim 36, wherein the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof.

38. Beta particle radioisotopes, 86 Y. 90 Y. 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67 38. The method of claim 37, wherein the metal is selected from the group consisting of Cu.

39. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 18 and instructions for use.

40. 40. The kit of claim 39, wherein the antibody or antigen-binding fragment of any one of claims 1 to 18 is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label and a chromogenic label.

41. The kit according to claim 39 or 40, further comprising a secondary antibody that specifically binds to the antibody according to any one of claims 1 to 18.

42. 20. The bispecific antibody or antigen-binding fragment of claim 8, 13, 17 or 18, wherein the bispecific antibody binds to a radiolabeled DOTA hapten and a CD33 antigen.

43. 1. A method for selecting a subject for pretargeted radioimmunotherapy, comprising: (a) administering to a subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment of claim 42, said conjugate configured to localize to tumors expressing CD33; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy if the level of radioactivity emitted by the complex is higher than a reference value; The method includes:

44. A method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD33-associated cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of claim 42, wherein the conjugate is configured to localize to tumors expressing CD33.

45. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of claim 42, wherein the conjugate is configured to localize to tumors expressing CD33.

46. 1. A method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD33-associated cancer, comprising: (a) administering an effective amount of the bispecific antibody or antigen-binding fragment of claim 42, wherein the bispecific antibody or antigen-binding fragment is configured to localize to CD33 expressed in a tumor; (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten configured to bind to the bispecific antibody or antigen-binding fragment. The method includes:

47. 1. A method of treating cancer in a subject in need thereof, comprising: (a) administering an effective amount of the bispecific antibody or antigen-binding fragment of claim 42, wherein the bispecific antibody or antigen-binding fragment is configured to localize to CD33 expressed in a tumor; (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, the radiolabeled DOTA hapten configured to bind to the bispecific antibody or antigen-binding fragment. The method includes:

48. 48. The method of claim 46 or 47, further comprising administering to the subject an effective amount of a clearance agent prior to administration of the radiolabeled DOTA hapten.

49. The method of any one of claims 43 to 48, wherein the subject is a human.

50. 46. ​​The method of any one of claims 43 to 45, wherein the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intraventricularly, orally, intratumorally, or intranasally.

51. 51. The method of any one of claims 43 to 50, wherein the radiolabeled DOTA hapten comprises an alpha particle radioisotope, a beta particle radioisotope or an Auger emitter.

52. The radiolabeled DOTA hapten is 213 Bi, 211 At, 225 A c, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y. 90 Y. 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 G.A. 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 G.A. 227 Th, or 64 The method of any one of claims 43 to 51, comprising Cu.

53. A bispecific antigen-binding fragment comprising a first polypeptide chain, the first polypeptide chain comprising, from N-terminus to C-terminus: i. a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. Amino acid sequence (GGGGS) 6 A flexible peptide linker comprising iii. a light chain variable domain of a first immunoglobulin; iv. Amino acid sequence (GGGGS) 4 A flexible peptide linker comprising v. a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; vi. Amino acid sequence (GGGGS) 6 A flexible peptide linker comprising vii. a light chain variable domain of a second immunoglobulin; viii. a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and ix. Self-assembly disassembly (SADA) polypeptides Including, The heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13. Bispecific antigen-binding fragment.

54. A bispecific antigen-binding fragment comprising a first polypeptide chain, the first polypeptide chain comprising, from N-terminus to C-terminus: i. a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. Amino acid sequence (GGGGS) 6 A flexible peptide linker comprising iii. a heavy chain variable domain of a first immunoglobulin; vi. Amino acid sequence (GGGGS) 4 A flexible peptide linker comprising v. a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; vi. Amino acid sequence (GGGGS) 6 A flexible peptide linker comprising vii. a light chain variable domain of a second immunoglobulin; viii. a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and ix. Self-assembly disassembly (SADA) polypeptides Including, The heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13. Bispecific antigen-binding fragment.

55. 55. The antigen-binding fragment of claim 53 or 54, wherein the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain.

56. 56. The antigen-binding fragment of claim 55, wherein the SADA polypeptide comprises the tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, or CBFA2T1.

57. 57. The antigen-binding fragment of any one of claims 53 to 56, comprising an amino acid sequence selected from SEQ ID NOs: 30 to 113 or 142 to 153.

58. 1. A bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other; a. each of the first and fourth polypeptide chains comprises, from the N-terminus to the C-terminus i. a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. a light chain constant domain of a first immunoglobulin; iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising iv. a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light and heavy chain variable domains of the second immunoglobulin are capable of specifically binding to a second epitope, and have the amino acid sequence (GGGGS) 6 a light chain variable domain or a heavy chain variable domain of a second immunoglobulin linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, b. each of the second and third polypeptide chains is arranged in the N-terminus to C-terminus i. a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. the heavy chain constant domain of the first immunoglobulin Including, The heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:133, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13. Bispecific antibodies.

59. 59. The bispecific antibody of claim 58, wherein the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

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