CD33 antibodies and methods of treating cancer using same

Abstract

The present disclosure generally relates to immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can bind to CD33 protein. The antibodies of the present technology are useful in methods of detecting and treating Alzheimer's disease or CD33-related cancer in a subject in need thereof.

Description

This application is a divisional application of the patent application for invention with the application date of February 21, 2020, application number 2020800299233, and invention title "CD33 Antibody and Method for Treating Cancer Using the Antibody". Cross - reference to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 809,091, filed on February 22, 2019, the entire content of which is incorporated herein by reference. Technical field

[0002] The present technology generally relates to the preparation of immunoglobulin - related compositions (e.g., antibodies or antigen - binding fragments thereof) that specifically bind to the CD33 protein and the use of such immunoglobulin - related compositions. Specifically, the present technology relates to the preparation of CD33 - binding antibodies and the use of such antibodies in the detection and treatment of CD33 - related cancers and Alzheimer's disease. Background art

[0003] The following provides an explanation of the technical background of the present invention only to assist in understanding the present invention, and does not admit that the explanation describes or constitutes the prior art with respect to the present invention.

[0004] Acute myeloid leukemia (AML) accounts for 25% of pediatric leukemias but more than half of the deaths from childhood leukemia. Compared with acute lymphoblastic leukemia, which is curable in >80% of children, the 5 - year survival rate of pediatric AML is the worst among childhood cancers. In adults, among those patients diagnosed before 60 years of age, AML is curable in 35% - 40% of cases, while only 5% - 15% of those presenting in later years can be cured (Dohner et al., N Engl J Med 373:1136 - 52 (2015)). Summary of the invention

[0005] In one aspect, the present disclosure provides an antibody or an antigen - binding fragment thereof comprising a heavy - chain immunoglobulin variable domain (V H ) and a light - chain immunoglobulin variable domain (V L ), wherein (a) the V H comprises a V H - CDR1 sequence of GYSFTDYN (SEQ ID NO:154), a V H - CDR2 sequence of IDPYKGGT (SEQ ID NO:155), and a V H - CDR3 sequence of AREMITAYYFDY (SEQ ID NO:156), and (b) the V LV containing QDINKY (SEQ ID NO:157) L -CDR1 sequence, V of YAS (SEQ ID NO:158) L -CDR2 sequence and V of LQYDNLLT (SEQ ID NO:159) L -CDR3 sequence.

[0006] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain immunoglobulin variable domain (V H ) and a light-chain immunoglobulin variable domain (V L ), wherein: (a) the V H comprises an amino acid sequence selected from: 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) the V L comprises an amino acid sequence selected from: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0007] In any of the above embodiments, the antibody may further comprise an Fc domain of an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the antibody comprises an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region containing an S228P mutation. In certain embodiments, the antigen-binding fragment is selected from Fab, F(ab’)2, Fab’, scF v and F v . In 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.

[0008] In another aspect, the present 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 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. In certain embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence selected from: SEQ ID NO:16 and SEQ ID NO:14 (chHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34 x CD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34 x CD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34 x CD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5 x mC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5 x hC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6 x mC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6 x hC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6 x mC825); SEQ ID NO:22 and SEQ ID NO:29 (VL4VH6 x hC825); 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).

[0009] In one aspect, the present 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 the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 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 the heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, or 133.

[0010] In another aspect, the present 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 the LC sequence present in any one of SEQ ID NO: 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 the HC sequence present in any one of SEQ ID NO: 16, 20, 22, 136, 139, or 141.

[0011] 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 containing one or more amino acid substitutions selected from N297A and K322A. In certain embodiments, the antibodies of the present technology comprise an IgG4 constant region containing 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 antibodies of the present technology lack α-1,6-fucosylation modification.

[0012] In one aspect, the present 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 NO. 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 NO. 30 - 113 or 142 - 153.

[0013] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a light chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degrading (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0014] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a heavy chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembling degrading (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: 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 fragment 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 NO.30-113 or 142-153.

[0015] In one aspect, the present 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 polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other, and wherein: (a) each of the first polypeptide chain and the fourth polypeptide chain comprises, in the N-terminal to C-terminal direction: (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 flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of a second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of a second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (b) each of the second polypeptide chain and the third polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to the first epitope; and (ii) a heavy chain constant domain of the first immunoglobulin; and wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: 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, TCRγ / δ, NKp46, KIR, or a small molecule DOTA hapten.

[0016] In one aspect, the present disclosure provides a recombinant nucleic acid sequence encoding any one of the antibodies or antigen-binding fragments described herein. In some embodiments, the recombinant nucleic acid sequence is selected from: SEQ ID NO:15, 17, 19, 21, 23, 25, 135, and 137.

[0017] In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0018] In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally 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.

[0019] In some embodiments of the bispecific antibodies or antigen-binding fragments of the present technology, the bispecific antibodies bind to T cells, B cells, myeloid cells, plasma cells or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibodies or antigen-binding fragments bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or the small molecule DOTA hapten.The small molecule DOTA hapten can be selected from DOTA, DOTA-Bn, DOTA-deferoxamine, DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2, Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2, DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2, Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2, Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2, Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2, DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2, (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2, Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2, Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2, Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2 and Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

[0020] In another aspect, the present disclosure provides a method of treating a CD33-related cancer or Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence selected from: SEQ ID NO:16 and SEQ ID NO:14 (chHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34 x CD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34 x CD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34 x CD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5 x mC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5 x hC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6 x mC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6 x hC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6 x mC825); SEQ ID NO:22 and SEQ ID NO:29 (VL4VH6 x hC825); 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), wherein 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.

[0021] In some embodiments, the CD33-related cancer is AML, biphenotypic leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

[0022] Additionally or alternatively, in some embodiments of the method, the antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent. Examples of additional therapeutic agents include one or more of the following: alkylating agents, platinum agents, taxanes, vinca agents, antiestrogenic drugs, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cell-inhibiting alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents.

[0023] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody or antigen-binding fragment of the present technology, wherein the antibody or antigen-binding fragment is configured to localize to a CD33-expressing tumor and is 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 above a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. Positron emission tomography or single photon emission computed tomography can be used to detect the radioactivity level emitted by the antibody or antigen-binding fragment.

[0024] 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-emitting isotope, a beta-particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta-particle-emitting isotopes include 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 Cu. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via an N297A mutation in the Fc region, which results in deglycosylation).

[0025] The present disclosure also provides a kit for detecting and / or treating CD33-related cancer or Alzheimer's disease, the kit comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant), and instructions for use. In certain embodiments, the immunoglobulin-related composition is conjugated to one or more detectable labels. In one embodiment, the one or more detectable labels include a radiolabel, a fluorescent label, or a chromogenic label.

[0026] Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-CD33 immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is conjugated to at least one detectable label selected from a radiolabel, a fluorescent label, or a chromogenic label.

[0027] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the CD33 antigen recognized by the bispecific antibody or antigen-binding fragment of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value.

[0028] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with CD33-related cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to the radiolabeled DOTA hapten and the CD33 antigen target, wherein the complex is configured to localize to a tumor expressing the CD33 antigen target recognized by the bispecific antibody or antigen-binding fragment of the complex.

[0029] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds the radiolabeled DOTA hapten and the CD33 antigen target, wherein the complex is configured to localize to a tumor expressing the CD33 antigen target recognized by the bispecific antibody or antigen-binding fragment of the complex.

[0030] In any of the above embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intraventricularly, 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 comprises 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 optionally comprises an alpha particle-emitting isotope, a beta particle-emitting isotope, or an Auger emitter.

[0031] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with a CD33-related 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 the 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 for treating cancer in a subject in need thereof, 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 the 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 methods of the present technology further comprise administering to the subject an effective amount of a scavenger prior to administering the radiolabeled DOTA hapten.

[0032] Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises 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 optionally includes an isotope that emits alpha particles, an isotope that emits beta particles, 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

[0033] Figure 1 Shows a schematic structure of the CD33 isotypes. The full-length CD33 protein is shown on the left, and the short isotype (a splice variant lacking the extracellular IgV domain) is shown on the right.

[0034] Figure 2 Shows a list of existing anti-CD33 antibodies and their domain specificities, as indicated by their ability to bind to the full-length CD33 protein and / or the short isotype of CD33.

[0035] Figure 3 Shows a schematic of the modular tetravalent IgG-scFv form, which contains an IgG molecule with two binding sites covalently linked to two scFvs, thus providing two additional binding domains.

[0036] Figure 4 Shows FACS data demonstrating the binding of the chimeric HIM34 x CD3 bispecific antibody (chHIM34 BsAb) to CD33(+) AML cell lines U937, THP1, SET2, C1498-CD33, and M-07e without harming the CD33(-) leukemia cell line CMLT1.

[0037] Figure 5(A) shows a schematic of the full-length CD33 protein and the short isotype (a splice variant of CD33). Figure 5(B) shows FACS data demonstrating the binding of chHIM34 BsAb to both the full-length and short isotypes of CD33, while the BsAb based on humanized M195 (BC133) did not bind to the short isotype.

[0038] Figure 6(A) and 6(B) Respectively show T cell-dependent cytotoxicity assays performed on CD33(+) AML cell lines C1498-CD33 and THP1 using chHIM34 BsAb.

[0039] Figure 7(A) shows a bioluminescence image that displays the growth of MOLM13 AML tumors in different groups. Figure 7(B) shows the signal quantification of mice from different groups in Figure 7(A).

[0040] Figure 8 Shows the amino acid sequences (SEQ ID NO:1-7 and 133) of murine and humanized HIM34 heavy chain variable domains. HIM34_VH-1, HIM34_VH-2, HIM_34_VH-3, HIM34_VH-4, HIM34_VH-5, HIM34-VH-6, HIM-34-VH-7 are 7 forms of humanized HIM34 heavy chain variable domains. V H -CDR1, V H -CDR2 and V H -CDR3 sequences are shown in bold.

[0041] Figure 9 Shows the amino acid sequences (SEQ ID NO:8-13) of murine and humanized HIM34 light chain variable domains. HIM34_VL-1, HIM34_VL-2, HIM_34_VL-3, HIM34_VL-4 and HIM34_VL-5 are 5 forms of humanized HIM34 light chain variable domains. V L -CDR1, V L -CDR2 and V L -CDR3 sequences are shown in bold.

[0042] Figures 10(A) and 10(B) show the amino acid sequences and nucleotide sequences (SEQ ID NO:14-17) of the light and heavy chains of chHIM34 BsAb, respectively. The signal peptide is underlined, the variable domains of the chimeric anti-CD33 antibody are indicated in bold font, and the linker sequence is indicated in italics and underlined. The amino acid sequences (SEQ ID NO:114-115) of the V H and V L domains of chHIM34 BsAb are also disclosed.

[0043] Figures 11(A) and 11(B) show the amino acid sequences and nucleotide sequences (SEQ ID NO:18-21) of the light and heavy chains of humanized HIM34 x CD3 BsAb (BC249), respectively. Figures 11(C) and 11(D) show the amino acid sequences and nucleotide sequences (SEQ ID NO:134-137) of the light and heavy chains of humanized HIM34x CD3 BsAb (BC275), respectively. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are indicated in bold font, and the linker sequence is indicated in italics and underlined.

[0044] Figures 12(A) and 12(B) show the amino acid and nucleotide sequences (SEQ ID NO:18, 19, 22, and 23) of the light and heavy chains of the humanized HIM34 x CD3 BsAb (BC267). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are indicated in bold font, and the linker sequences are indicated in italics and underlined.

[0045] Figures 13(A) and 13(B) show the amino acid and nucleotide sequences (SEQ ID NO:24, 25, 22, and 23) of the light and heavy chains of the humanized HIM34 x CD3 BsAb (BC268). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are indicated in bold font, and the linker sequences are indicated in italics and underlined.

[0046] Figure 14(A)-14(F) The amino acid sequences of the light and heavy chains of the humanized HIM34 x C825 (anti-DOTA) BsAb in IgG-scFv form are shown. The light chain amino acid sequence is shown in SEQ ID NO:26 - 29, and the heavy chain amino acid sequence is shown in SEQ ID NO:20 and 22. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are indicated in bold font, and the linker sequences are indicated in italics and underlined.

[0047] Figure 14(G)-14(H) The amino acid sequences of the light and heavy chains of the murine HIM34 x C825 (anti-DOTA) BsAb in IgG-scFv form are shown. The light chain amino acid sequence is shown in SEQ ID NO:138 and 140, and the heavy chain amino acid sequence is shown in SEQ ID NO:139 and 141. The signal peptide is underlined, the variable domains of the murine anti-CD33 antibody are indicated in bold font, and the linker sequences are indicated in italics and underlined.

[0048] Figure 15(A) to Figure 15(BB) The amino acid sequence (SEQ ID NO:30 - 113) of the humanized HIM34 x C825 (anti-DOTA) BsAb in single-chain bispecific tandem fragment variable (scBsTaFv) form is shown. The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are indicated in bold font, and the linker sequences are indicated in italics and underlined.

[0049] Figure 15(CC) to Figure 15(FF)Shows the amino acid sequence (SEQ ID NO: 142 - 153) of a murine HIM34 x C825 (anti - DOTA) BsAb in single - chain bispecific tandem fragment variable (scBsTaFv) form. The signal peptide is underlined, the variable domains based on the murine anti - CD33 antibody are indicated in bold font, and the linker sequence is in italic and underlined.

[0050] Figures 16(A) and 16(B) show a summary of the characteristics (including binding kinetics) of chimeric and humanized HIM34 BsAbs.

[0051] Figure 17 Shows FACS analysis of MOLM13 CD33(+) cells in contact with different concentrations of the humanized CD33 BsAb of the present technology.

[0052] Figure 18(A) to Figure 18(B) Shows the cytotoxicity of the humanized BsAb of the present technology against MOLM13 CD33(+) cells in a T - cell - dependent cytotoxicity assay (TDCC).

[0053] Figure 19 Shows the stability of the humanized BsAb of the present technology at 40 °C.

[0054] Figure 20(A) shows a T - cell - engaging bispecific antibody (BsAb) that recruits T cells to AML cells. Figure 20(B) shows that the herein - disclosed BC275 BsAb binds to the membrane - proximal domain (IgC) of the CD33 extracellular region, while BC133 based on the M195 clone (Lintuzumab) and BC269 based on My96 (Gemtuzumab) bind to the membrane - distal domain of the CD33 extracellular region. Figure 20(C) shows the CD33 - binding characteristics of BC275, BC133, and BC269 BsAbs to various CD33(+) and CD33( - ) human cancer cell lines.

[0055] Figure 21(A) to Figure 21(C) Shows the T - cell - dependent cell - mediated cytotoxic effects of BC275, BC133, and BC269 BsAbs on CD33(+) and CD33( - ) human cancer cell lines. Figure 21(D) plots the correlation between the level of CD33 expression on cancer cells and the potency of the test BsAbs in the TDCC assay. The combined data of all three CD3×CD33 BsAbs are shown.

[0056] Figure 22(A) to Figure 22(B)Shows the tumor sizes of immunodeficient NSG mice into which human AML xenografts (10 6 cells) of CD33(+) MOLM13-luciferase with the CC genotype of rs12459419 SNP were intravenously inoculated, and were then treated with BC275, BC133, and BC269 BsAb (CD3×CD33 BsAb) or control BC119 BsAb (CD3×GD2 BsAb). The mice received a single injection of 5×10 6 activated T cells and 0.025 μg BsAb (10 μg / Kg / dose). The timing of T cell and antibody injections is shown in Figure 22(B).

[0057] Figure 23(A) to Figure 23(B) Shows the tumor sizes of immunodeficient NSG mice into which human CD33-transduced NALM6-luciferase leukemia xenografts (0.5×10 6 cells) were intravenously inoculated, and were then treated with BC275, BC133, and BC269 BsAb (CD3×CD33 BsAb) or control BC119 BsAb (CD3×GD2 BsAb). The mice received two injections of activated T cells (2.7×10 6 and 5×10 6 , with a one-week interval between doses) and 10 ng BsAb / 10 6 T cells. The timing of T cell and BsAb injections is shown in Figure 23(B). Figure 23(C) shows the survival curves of animals treated with the three CD3×CD33 BsAbs. The potency ranking of the CD3×CD33 BsAbs is as follows: BC275 > BC269 = BC133.

[0058] Figure 24(A) to Figure 24(D) Shows the tumor sizes of immunodeficient DKO mice into which THP1 human leukemia xenografts with the CC genotype of rs12459419 SNP were subcutaneously inoculated, and were then treated with BC275, BC133, and BC269 BsAb (CD3×CD33 BsAb) or control BC119 BsAb (CD3×GD2 BsAb). The mice received two injections of activated T cells (10 7 , with a one-week interval between doses) and 0.05 μg or 0.5 μg 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 ranking of the CD3×CD33 BsAbs is as follows: BC275 > BC269 > BC133.

[0059] Figure 25(A) to Figure 25(D)Shows the tumor size of immunodeficient DKO mice subcutaneously inoculated with K562 human leukemia xenografts having the TT genotype of the rs12459419 SNP and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or the control BC119 BsAb (CD3×GD2 BsAb). Mice were given three injections of activated T cells (10 7 , one week apart between doses) and 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 potencies of the CD3×CD33 BsAbs ranked as follows: BC275 > BC269 > BC133. DETAILED DESCRIPTION

[0060] It should be understood that certain aspects, modes, embodiments, variations, and features of the methods of the present invention are described below in varying degrees of detail to provide a substantial understanding of the technology of the present invention.

[0061] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to CD33 polypeptides. The immunoglobulin-related compositions of the technology can be used in methods for detecting or treating CD33-related cancers or Alzheimer's disease in a subject in need thereof. Accordingly, various aspects of the methods of the present invention relate to the preparation, characterization, and manipulation of anti-CD33 antibodies. The immunoglobulin-related compositions of the technology can be used alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related composition is a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0062] In practicing the methods of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., New York); 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; U.S. 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 the level of a polypeptide gene expression product (i.e., the level of gene translation) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan and Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., New York, 1999)). Definition

[0063] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and so forth. Generally, 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 those well known and commonly employed in the art.

[0064] Unless the context otherwise indicates or is otherwise apparent, as used herein, the term "about" with respect to a number generally is considered to include numbers that fall within 1%, 5%, or 10% of that number in either direction (greater than or less than) (except in cases where such numbers are less than 0% of a possible value or greater than 100% of a possible value).

[0065] As used herein, "administering" 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 carried out by any suitable route, including but not limited to oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self - administration and administration by another person.

[0066] "Adjuvant" refers to one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. An adjuvant can be administered to a subject before, in combination with, or after administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulating 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.

[0067] As used herein, the term "antibody" collectively refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to IgA, IgD, IgE, IgG, and IgM, their combinations, and similar molecules (such as shark immunoglobulins) produced during an immune response in any vertebrate, such as in mammals (e.g., humans, goats, rabbits, and mice) and non-mammalian species. As used herein, "antibodies" (including intact immunoglobulins) and "antigen-binding fragments" specifically bind to a molecule of interest (or a group of highly similar molecules of interest), while substantially excluding binding to other molecules (e.g., the binding constant for the molecule of interest is at least 10 3 M -1 times greater, at least 10 4 M -1 times greater, or at least 10 5 M -1 greater than the binding constant for other molecules in a biological sample). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0068] More specifically, an antibody refers to a polypeptide ligand that specifically recognizes and binds to an epitope and that comprises at least the immunoglobulin variable region of the light chain or the immunoglobulin variable region of the heavy chain. Antibodies are composed of heavy and light chains, each of which has a variable region, called the heavy chain variable (V H ) region and the light chain variable (V L ) region. The V H region and the V LThe regions are jointly responsible for binding the antigen recognized by the antibody. Generally, an immunoglobulin has heavy (H) and light (L) chains that are interconnected by disulfide bonds. There are two types of light chains, namely lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of the antibody molecule. Each heavy and light chain contains a constant region and a variable region (the regions are also referred to as "domains"). Together, the heavy chain variable region and the light chain variable region specifically bind the antigen. The light chain variable region and the heavy chain variable region contain "framework" regions interrupted by three hypervariable regions (also called "complementary determining regions" or "CDRs"). The ranges of the framework regions and CDRs have been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of the antibody (i.e., the combined framework region of the constitutive light and heavy chains) mainly adopts a β-sheet conformation, and the CDRs form loops connecting the β-sheet structures, and in some cases the loops form part of the β-sheet structure. Thus, the framework region serves to form a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions.

[0069] The CDRs are mainly responsible for binding to the epitope of the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are usually also identified according to the chain in which a particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds to the CD33 protein will have a specific V H region and a V L region sequence, and thus a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs are different between different antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) and antibody fragments. An antibody or its antigen-binding fragment specifically binds to an antigen.

[0070] As used herein, the term "antibody-related polypeptide" means an antigen-binding antibody fragment including a single-chain antibody, which may individually comprise one or more variable regions or a combination of one or more variable regions with all or part of the following polypeptide elements: the hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Also included in the art are any combinations of one or more variable regions and the hinge region, CH1, CH2, and CH3 domains. Antibody-related molecules useful in the methods of the present invention are, for example but not limited to, Fab, Fab', and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments containing V L or V H domains. Examples include: (i) Fab fragments, which are monovalent fragments consisting of V L 、V H 、C L and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of V H and CH1 domains; (iv) Fv fragments consisting of the V L and V H domains of a single arm of an antibody; (v) dAb fragments (Ward et al., Nature 341:544-546, 1989), which consist of V H domains; and (vi) isolated complementarity-determining regions (CDRs). Thus, an "antibody fragment" or "antigen-binding fragment" may comprise a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0071] As used herein, a "bispecific antibody" or "BsAb" refers to an antibody that can simultaneously bind to two targets with different 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 of the bispecific antibody comprises V H and / or V L regions; in some such embodiments, the V H and / or V L regions are those found in a particular monoclonal antibody. In some embodiments, the bispecific antibody contains two antigen-binding portions, each antigen-binding portion comprising V H and / or V L from different monoclonal antibodies.region. In some embodiments, the bispecific antibody contains two antigen-binding portions, one of the two antigen-binding portions comprising an immunoglobulin molecule having a V H and / or V L region, the V H and / or V L region containing CDRs from a first monoclonal antibody; and the other antigen-binding portion comprising an antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.) having a V H and / or V L region, the V H and / or V L region containing CDRs from a second monoclonal antibody.

[0072] As used herein, a "scavenger" is an agent that binds to excess bispecific antibody present in the blood compartment of a subject to facilitate rapid clearance via the kidneys. Use of a scavenger prior to hapten administration (e.g., DOTA) helps to obtain a better tumor-to-background ratio in a pretargeted radioimmunotherapy (PRIT) system. Examples of scavengers include 500kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6):1365-1372 (2012)), 500kD aminodextran-DOTA conjugates, antibodies against the pretargeting antibody, and the like.

[0073] 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 binding. Chemical bonds include, for example, covalent bonds and coordination bonds. Physical binding includes, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic ring stacking.

[0074] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy-chain variable domain (V L ) linked to a light-chain variable domain (V H ) in the same polypeptide chain (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 on the other chain, and two antigen-binding sites are generated. Diabodies are more fully described, for example, in the following documents: EP404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0075] As used herein, the term "single-chain antibody" or "single-chain Fv (scFv)" refers to an antibody fusion molecule of the two domains V L and V H of an Fv fragment. A single-chain antibody molecule can comprise a polymer of multiple individual molecules, such as a dimer, trimer, or other polymer. Additionally, although the two domains V v and V L of an F H fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker such that they can form a single protein chain in which the V L and V H regions pair to form a monovalent molecule (referred to as single-chain F v (scF v ))). 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.

[0076] Any of the above antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as intact antibodies.

[0077] As used herein, an "antigen" refers to a molecule to which an antibody (or an antigen-binding fragment thereof) can selectively bind. A 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). An antigen can also be administered to an animal to generate an immune response in the animal.

[0078] The term "antigen-binding fragment" refers to a fragment of the intact immunoglobulin structure that has a polypeptide portion responsible for binding to an antigen. Examples of antigen-binding fragments that can be used in the present technology include scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2, but are not limited thereto.

[0079] "Binding affinity" means the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigen peptide). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by standard methods known in the art, including those described herein. A low-affinity complex contains an antibody that generally tends to dissociate from the antigen readily, while a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer time.

[0080] As used herein, the term "biological sample" means sample material derived from living cells. Biological samples can include tissue, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissue, cells, and fluids present within a subject. 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, tonsils, thymus, blood, hair, cheek, skin, serum, plasma, CSF, sperm, 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 cancers. Biological samples can be obtained from a subject for diagnosis or research; or can be obtained from non-diseased individuals as controls or for basic research. Samples can be obtained by standard methods, which include, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by fine needle aspiration biopsy.

[0081] As used herein, the term "CDR-grafted antibody" means an antibody in which at least one CDR of a "recipient" antibody is replaced by a CDR "graft" from a "donor" antibody having the desired antigen specificity.

[0082] As used herein, the term "chimeric antibody" means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a murine Fc constant region) is replaced with the Fc constant region of an antibody from another species (e.g., a human Fc constant region) using recombinant DNA techniques. See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 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., 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.

[0083] As used herein, the term "consensus FR" means the framework (FR) antibody regions in consensus immunoglobulin sequences. The FR regions of an antibody do not contact the antigen.

[0084] As used herein, a "control" is an alternative sample used for comparison purposes in an experiment. A control can be "positive" or "negative". For example, in a case where the purpose of an experiment is to determine the relevance of the efficacy of a therapeutic agent in the treatment of 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 treatment or receives a placebo) are typically used.

[0085] 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 disorder described herein or one or more signs or symptoms associated with a disease or disorder described herein. In the case of a therapeutic or prophylactic application, the amount of the composition administered to a subject will vary depending on the composition, the degree, type and severity of the disease, and on the characteristics of the individual (such as general health, age, sex, weight, and drug tolerance). One of ordinary skill in the art will be able to determine an appropriate dosage based 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, a therapeutic composition may be administered to a subject having one or more signs or symptoms of a disease or disorder 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 a disease or disorder are ameliorated or eliminated. A therapeutically effective amount may be administered in one or more administrations.

[0086] As used herein, the term "effector cell" means an immune cell that participates in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytotoxic 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 perform specific immune functions. Effector cells may induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils that are capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR participate in the specific killing of target cells and present antigens to other components of the immune system or bind to cells presenting antigens.

[0087] As used herein, the term "epitope" means a protein determinant capable of specifically binding to an antibody. Epitopes are usually composed of the chemically active surface groups of a molecule (such as amino acids or sugar side chains) and generally have specific three-dimensional structural features, as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that in the presence of a denaturing solvent, binding to a conformational epitope, but not a non-conformational epitope, is lost. In some embodiments, an "epitope" of the CD33 protein is the region of the protein that specifically binds to an anti-CD33 antibody of the present technology. In some embodiments, the epitope is a conformational or non-conformational epitope. To screen for anti-CD33 antibodies that bind to an epitope, conventional cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, edited by Harlow and David Lane (1988). This assay can be used to determine whether an anti-CD33 antibody binds to the same site or epitope as an anti-CD33 antibody of the present technology. Alternatively or additionally, epitope mapping can be performed by methods known in the art. For example, antibody sequences can be mutagenized, such as by alanine scanning, to identify contacting residues. In a different method, peptides corresponding to different regions of the CD33 protein can be used in competition assays with a variety of test antibodies, or with one test antibody and an antibody with a characterized or known epitope.

[0088] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of precursor mRNA to produce mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product (if appropriate expression and function require).

[0089] As used herein, the term "gene" means a DNA segment containing all the information for the regulated biosynthesis of an RNA product, including a promoter, exons, introns, and other untranslated regions that control expression.

[0090] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, which can be aligned for purposes of comparison. When the positions in the sequences being compared are occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences varies with the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when aligned, that percentage of bases (or amino acids) is the same when comparing the two sequences. This alignment and the percentage of homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = 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 having the same or similar biological activity. If two sequences share less than 40% identity or less than 25% identity with each other, then the sequences are considered "unrelated" or "non-homologous".

[0091] As used herein, a "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins in which the hypervariable region residues of the recipient are replaced with hypervariable region residues (donor antibody) from a non-human species (such as mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, a humanized antibody will contain substantially all of at least one, and usually two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FR regions are those of the human immunoglobulin consensus FR sequences, but the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is generally no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally may also include at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of the human immunoglobulin constant region. 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 and Cheung, FEBS Letters 588(2):288-297 (2014).

[0092] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. Hypervariable regions generally contain amino acid residues from "complementary determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V L before and after, and residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in V H before and after (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 "hypervariable loops" (e.g., V Lresidues 26 - 32 (L1), 50 - 52 (L2), and 91 - 96 (L3), and V H residues 26 - 32 (H1), 52A - 55 (H2), and 96 - 101 (H3) in H (Chothia and Lesk J. Mol. Biol. 196:901 - 917 (1987)).

[0093] As used herein, when used in the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "identity" percent refers to when comparing and aligning for maximum correspondence over a comparison window or specific region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below or by manual alignment and visual inspection (e.g., the NCBI website), two or more sequences or subsequences 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., the nucleotide sequence encoding the antibodies described herein or the amino acid sequence of the antibodies described herein)). Such sequences are then referred to as "substantially identical". This term also refers to or can apply to the complement of a test sequence. The term also includes sequences having deletions and / or additions, as well as those having substitutions. In some embodiments, identity exists in a region of at least about 25 amino acids or nucleotides or a length of 50 - 100 amino acids or nucleotides.

[0094] As used herein, the term "intact antibody" or "intact immunoglobulin" means an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The light chain constant region is composed of one domain C L . V H and V L regions can be further subdivided into regions of high variability, called complementarity - determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0095] As used herein, the terms "individual", "patient", or "subject" can be a single organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human.

[0096] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of 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 can be an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) regardless of the method by which it is produced. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific for a single antigenic site. In addition, compared 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 that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. A variety of techniques known in the art can be used to prepare monoclonal antibodies, including, for example, but not limited to, hybridoma, recombinant, and phage display techniques. For example, monoclonal antibodies to be used in accordance with the methods of the present invention can be prepared by the hybridoma method originally described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0097] As used herein, the term "pharmaceutically 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 the administration of a drug. Pharmaceutically acceptable carriers and their formulations are known to those of skill in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th Edition, A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA).

[0098] As used herein, the term "polyclonal antibody" means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. Use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0099] As used herein, the term "polynucleotide" or "nucleic acid" means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA, which may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotide refers to a triple-stranded region containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone has been modified for stability or other reasons.

[0100] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and mean a polymer containing two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). Polypeptides refer 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 genetically encoded amino acids. Polypeptides include amino acid sequences modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Such modifications are well described in basic textbooks and more detailed monographs as well as in long research articles.

[0101] As used herein, "PRIT" or "pretargeted radioimmunotherapy" refers to a multi-step process that addresses the slow blood clearance of tumor-targeting antibodies, which results in unwanted 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 having binding sites for both the hapten and the target antigen is administered first. Then unbound antibody is cleared from the circulation, and subsequently the hapten is administered.

[0102] As used herein, the term "recombinant" when used in reference to, for example, a cell or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or indicates that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene not found within the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise abnormally expressed, under-expressed, or not expressed at all.

[0103] 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.

[0104] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, by the same or different routes of administration. More specifically, sequential use means that the entire administration of one active ingredient precedes the commencement of the administration of one or more other active ingredients. Thus, one active ingredient can be administered minutes, hours, or days before the administration of one or more other active ingredients. In this case, there is no co-therapy.

[0105] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or an antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize and bind to other molecules. As used herein, the terms "specifically binds to" a particular molecule (e.g., a polypeptide or an epitope on a polypeptide), "specifically binds" the particular molecule, or has "specificity" for the particular molecule can be determined, for example, by the K of the molecule for the molecule to which it binds d to be approximately 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 -12Exhibited by M. The term "specifically binds" can also refer to a binding where a molecule (e.g., an antibody or an antigen-binding fragment thereof) binds to a specific polypeptide (e.g., a CD33 polypeptide) or an epitope on a specific polypeptide, with substantially no binding to any other polypeptide or polypeptide epitope.

[0106] As used herein, the term "co-treatment" refers to the administration of at least two active ingredients by the same route and simultaneously or substantially simultaneously.

[0107] As used herein, the term "therapeutic agent" is intended to mean a compound that produces a desired therapeutic effect in a subject in need thereof when present in an effective amount.

[0108] As used herein, "treating" ("treating" or "treatment") encompasses treating a subject, such as a human, of a disease or disorder described herein, and includes: (i) inhibiting the disease or disorder, i.e., preventing its development; (ii) alleviating 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 symptoms associated with the disease, for example, are alleviated, reduced, cured, or in remission.

[0109] It should also be understood that the various modes of treatment of the disorders described herein are intended to mean "substantially", which includes complete treatment but also less than complete treatment, and in which some biologically or medically relevant result is achieved. The treatment can be a continuous extended treatment for a chronic disease, or a single or several administrations for the treatment of an acute condition.

[0110] One or more amino acid sequence modifications 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 antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the desired antibody as long as the resulting antibody has the desired properties. Modifications also include changes in the protein glycosylation pattern. The sites of most interest for performing substitution mutagenesis include the hypervariable regions, but FR alterations are also contemplated. "Conservative substitutions" are shown in the following table.

[0111] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. In particular, several hypervariable regions (e.g., 6 - 7 regions) are mutated to generate all possible amino acid substitutions at each site. The antibody variants so generated are displayed monovalently from filamentous phage particles as fusions with the gene III product of M13 packaged in each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity), as disclosed herein. To identify candidate hypervariable regions for modification, alanine-scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. According to the techniques detailed herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, this set of variants is screened as described herein, and antibodies having similar or superior properties in one or more relevant assays can be selected for further development. CD33

[0112] CD33 (also known as Siglec-3, SIGLEC3, gp67, p67) is a 67 kDa plasma membrane protein that binds to sialic acid and is a member of the sialic acid-binding Ig-like lectin (SIGLEC) protein family. Siglec proteins are thought to be involved in a variety of biological processes such as hematopoiesis, neuronal development, and immunity (Vinson et al., J. Biol. Chem. 271:9267 - 9272 (1996)). Studies have also shown 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 and one IgC2 domain) ( Figure 1 ). The intracellular portion of CD33 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). In the immune response, CD33 can act as an inhibitory receptor upon ligand-induced tyrosine phosphorylation by recruiting one or more cytoplasmic phosphatases that block signal transduction by dephosphorylating signaling molecules.

[0113] CD33 is known to be expressed on myeloid cells. CD33 expression has also been reported on many malignant cells. Anti-CD33 agents are generally divided into four groups: naked antibodies, antibody-toxin conjugates, radionuclide conjugates, and bispecific antibodies. Although CD33 has been targeted for the treatment of cancers such as acute myeloid leukemia, there is currently no effective CD33-targeted therapy on the market. Existing anti-CD33 agents particularly have poor tumor antigen binding affinity and short in vivo half-life. In addition, none of these antibodies can target the short isoform of CD33 (splice variant of CD33)( Figure 2 ).

[0114] CD33 is expressed on most leukemia cells, but nearly 50% of leukemia cells lack the external IgV domain due to the rs12459419 polymorphism (J Clin Oncol. 35(23):2674 - 2682(2017)). These splice variants, which account for nearly 50% of AML patients, do not benefit from current anti-CD33-IgV domain antibody-based therapies such as gemtuzumab ozogamicin, lintuzumab, AMV564). Immunoglobulin-related compositions of the present technology

[0115] This technology describes methods and compositions for generating and using anti-CD33 immunoglobulin-related compositions (e.g., anti-CD33 antibodies or their antigen-binding fragments). The anti-CD33 immunoglobulin-related compositions of the present disclosure can be used for diagnosing or treating CD33-related cancers or Alzheimer's disease. Anti-CD33 immunoglobulin-related compositions within the scope of this technology include, for example but not limited to, monoclonal antibodies, chimeric antibodies, humanized, bispecific antibodies, and diabodies that specifically bind to a target polypeptide, its homologs, derivatives, or fragments. The present disclosure also provides antigen-binding fragments of any anti-CD33 antibody disclosed herein, wherein the antigen-binding fragments are selected from Fab, F(ab)'2, Fab’, scF v and F v . The technology discloses anti-CD33 bispecific antibody forms that address the existing problems of poor tumor antigen binding affinity and short in vivo half-life in addition to binding both the full-length CD33 protein and the short isoform of CD33. Importantly, the short isoform of CD33 is expressed in half of AML patients. In one aspect, the technology provides chimeric and humanized variants of HIM34, including multispecific immunoglobulin-related compositions (e.g., bispecific antibody agents).

[0116] In one aspect, the present disclosure provides a composition comprising a heavy-chain immunoglobulin variable domain (V H ) and a light-chain immunoglobulin variable domain (V LAn antibody or an antigen-binding fragment thereof, wherein (a) said V H V comprising 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) said V L V comprising QDINKY (SEQ ID NO:157) L -CDR1 sequence, V of YAS (SEQ ID NO:158) L -CDR2 sequence and V of LQYDNLLT (SEQ ID NO:159) L -CDR3 sequence.

[0117] In one aspect, the present technology provides an antibody or an antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ), wherein (a) said V H comprises an amino acid sequence selected from: 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) said V L comprises an amino acid sequence selected from: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0118] In any of the above embodiments, the antibody further comprises an Fc domain of any isotype, said isotype such as but not limited to IgG (including IgG1, IgG2, IgG3 and IgG4), IgA (including IgA1 and IgA2), IgD, IgE or IgM and IgY. Non-limiting examples of constant region sequences include:

[0119] Human IgD constant region, Uniprot: P01880 (SEQ ID NO:116) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0120] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO:117) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO:118) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0122] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO:119) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0123] Human IgM constant region, Uniprot: P01871 (SEQ ID NO:120) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0124] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO:121) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0125] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO:122) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0126] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 123) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0127] Human Igκ constant region, Uniprot: P01834 (SEQ ID NO: 124) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0128] In some embodiments, the immunoglobulin-related compositions of the present technology comprise heavy chain constant regions that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NOs: 116-123. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise light chain constant regions that are 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 compositions of the present technology bind to the IgC2 domain of CD33. In some embodiments, the epitope is a conformational epitope.

[0129] In another aspect, the present disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or an antigen-binding fragment thereof) that comprises a heavy chain (HC) amino acid sequence containing 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.

[0130] Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence containing 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.

[0131] In some embodiments, the immunoglobulin-related compositions of the present technology comprise HC amino acid sequences and LC amino acid sequences selected from SEQ ID NO:16 and SEQ ID NO:14 (chHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34 x CD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34 x CD3 BsAb); SEQ ID NO:22 and SEQ ID NO:18 (BC267-hHIM34 x CD3 BsAb); SEQ ID NO:22 and SEQ ID NO:24 (BC268-hHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5 x mC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5 x hC825); SEQ ID NO:22 and SEQ ID NO:26 (VL3VH6 x mC825); SEQ ID NO:22 and SEQ ID NO:27 (VL3VH6 x hC825); SEQ ID NO:22 and SEQ ID NO:28 (VL4VH6 x mC825); SEQ ID NO:22 and SEQ ID NO:29 (VL4VH6 x hC825); 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).

[0132] In any of the above embodiments of the immunoglobulin-related composition, 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.

[0133] 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.

[0134] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one CD33 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology are at about 10 -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 The dissociation constant (K D ) of M binds to at least one CD33 polypeptide. In certain embodiments, the immunoglobulin-related composition 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.

[0135] In certain embodiments, the immunoglobulin-related composition 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 the 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 the 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 composition provided herein are replaced with another amino acid. The replacement can be a "conservative replacement" as defined herein.

[0136] In some embodiments, the immunoglobulin-related composition 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 the 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 the HC sequence present in any one of SEQ ID NOs: 16, 20, 22, 136, 139, or 141.

[0137] In one aspect, the present disclosure provides an immunoglobulin-related composition 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 composition of the present disclosure comprises an amino acid sequence selected from SEQ ID NOs. 30 - 113 or 142 - 153.

[0138] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a light chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembling degrading (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0139] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a heavy chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembling degrading (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0140] In certain embodiments of the bispecific antigen-binding fragment 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 NO. 30-113 or 142-153.

[0141] In one aspect, the present 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 polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other, and wherein: (a) each of the first polypeptide chain and the fourth polypeptide chain comprises, in the N-terminal to C-terminal direction: (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 flexible peptide linker comprising the amino acid sequence (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 the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (b) each of the second polypeptide chain and the third polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to the first epitope; and (ii) a heavy chain constant domain of the first immunoglobulin; and wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: 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, TCRγ / δ, NKp46, KIR, or a small molecule DOTA hapten.

[0142] In certain embodiments, the immunoglobulin-related composition comprises an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition comprises an IgG4 constant region containing an S228P mutation.

[0143] In some aspects, the anti-CD33 immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some aspects, the anti-CD33 immunoglobulin-related compositions (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 aspects, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some aspects, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or slow release.

[0144] In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Recombinant nucleic acid sequences encoding any of the antibodies described herein are also disclosed. In some embodiments, the nucleic acid sequence is selected from SEQ ID NO: 15, 17, 19, 21, 23, 25, 135, and 137.

[0145] In another aspect, the present technology provides a host cell expressing any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0146] The immunoglobulin-related compositions of the present technology (e.g., anti-CD33 antibodies) can be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more CD33 polypeptides, or can be specific for both one or more CD33 polypeptides and a heterologous composition (such as a heterologous polypeptide or a solid support material). See, for example, WO 93 / 17715; WO 92 / 08802; WO 91 / 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; 6,106,835; Kostelny et al., J. Immunol. 148:1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

[0147] The immunoglobulin-related compositions of the present technology can be further recombinantly fused at the N-terminus or C-terminus with a heterologous polypeptide, or chemically conjugated (including covalently and non-covalently) with a polypeptide or other composition. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated with molecules that can be used as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, for example, WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Patent No. 5,314,995; and EP 0 396 387.

[0148] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen-binding fragment can optionally be conjugated with an agent selected from the group consisting of isotopes, dyes, chromogens, 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, the functional groups on the immunoglobulin-related composition typically associate with the functional groups on the agent. Alternatively, the functional groups on the agent associate with the functional groups on the immunoglobulin-related composition.

[0149] The functional groups on the agent and the functional groups on the immunoglobulin-related composition can associate directly. For example, a functional group on the agent (such as a thiol group) can associate with a functional group on the immunoglobulin-related composition (such as a thiol group) to form a disulfide bond. Alternatively, the functional groups can associate through a crosslinker (i.e., a linker). Some examples of crosslinkers are described below. The crosslinker can be attached to the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions 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 composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with the agent depends on the number of functional groups present on the agent.

[0150] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated with one agent. In one embodiment, the conjugate comprises at least one agent chemically bonded (such as conjugated) with at least one immunoglobulin-related composition. The agent can be chemically bonded to the immunoglobulin-related composition by any method known to those skilled in the art. For example, a functional group on the agent can be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, thiol, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0151] The agent can also be chemically bonded to the immunoglobulin-related composition through a crosslinking agent (such as dialdehyde, carbodiimide, dimaleimide, etc.). The crosslinking agent can be obtained, for example, from Pierce Biotechnology, Inc. in Rockford, Illinois. The website of Pierce Biotechnology, Inc. can provide assistance. Additional crosslinking agents include the platinum crosslinking agents described in the following documents: U.S. Patent Nos. 5,580,990; 5,985,566; and 6,133,038 of Kreatech Biotechnology, B.V. in Amsterdam, the Netherlands.

[0152] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homo-bifunctional crosslinking agents are generally used to crosslink the same functional groups. Examples of homo-bifunctional crosslinking agents 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., bismaleimidohexane). Such homo-bifunctional crosslinking agents are also available from Pierce Biotechnology, Inc.

[0153] In other cases, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The aforementioned website of Pierce Biotechnology, Inc. can also assist those skilled in the art in selecting a suitable crosslinking agent that can be cleaved by, for example, enzymes in cells. Thus, the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfo-succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (such as sulfo-succinimidyl 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).

[0154] In another embodiment, the conjugate comprises at least one agent physically conjugated to at least one immunoglobulin-related composition. Any method known to those skilled in the art can be used to physically conjugate the agent to the immunoglobulin-related composition. For example, the immunoglobulin-related composition and the agent can be mixed by any method known to those skilled in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be placed in a container and agitated, such as by shaking the container, to mix the immunoglobulin-related composition and the agent.

[0155] The immunoglobulin-related composition can be modified by any method known to those skilled in the art. For example, as described above, the immunoglobulin-related composition can be modified by a crosslinking agent or a functional group. A. Method for preparing the anti-CD33 antibody of the present technology

[0156] Overview. First, a target polypeptide is selected, and an antibody of the present technology against the target polypeptide can be produced. For example, an antibody can be produced against the full-length CD33 protein, the CD33 protein lacking the external IgV domain, or a portion of the extracellular domain of the CD33 protein (e.g., the IgC2 domain). Techniques for producing antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, xenogeneic or human-mouse, hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from the CD33 protein that contains an extracellular domain (e.g., the IgC2 domain) capable of eliciting an immune response.

[0157] It should be understood that recombinant engineered antibodies and antibody fragments (e.g., antibody-related polypeptides) against the CD33 protein and its fragments are suitable for use in accordance with the present disclosure.

[0158] Anti-CD33 antibodies that can be subjected to the techniques described herein include monoclonal antibodies and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods for the high-yield production of antibody Fv-containing polypeptides such as Fab′ and F(ab′)2 antibody fragments have been described. See U.S. Patent No. 5,648,237.

[0159] Generally, the antibody is obtained from the origin species. More specifically, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain, or both of the origin species antibody that is specific for the target polypeptide antigen is obtained. The origin species is any species that can be used to produce the antibodies or antibody libraries of the present technology, such as rats, mice, rabbits, chickens, monkeys, humans, etc.

[0160] Phage or phagemid display technology is a technology that can be used to derive the antibodies of the present technology. The techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. The expression of the sequences encoding the antibodies of the present technology can be carried out in Escherichia coli.

[0161] Due to the degeneracy of nucleic acid coding sequences, other sequences encoding amino acid sequences that are substantially identical to those of naturally occurring proteins can be used in the practice of the present technology. These sequences include, but are not limited to, nucleic acid sequences including all or part of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons for functionally equivalent amino acid residues within the coding sequence, thereby producing silent changes. It should be understood that the nucleotide sequences of the immunoglobulins according to the present technology tolerate sequence homology variations of up to 25% calculated by standard methods (“Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pages 127-149, 1998, Alan R. Liss, Inc.), provided that such variants form effective antibodies that recognize the CD33 protein. For example, one or more amino acid residues within the polypeptide sequence can be replaced by another amino acid having similar polarity, which acts as a functional equivalent, thereby resulting in a silent change. The substituents of the amino acids within the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral 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, such as by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc. Additionally, the nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to produce and / or disrupt the translation, initiation, and / or termination of the sequence, or to generate variations in the coding region and / or form new restriction endonuclease sites or disrupt previously existing such sites to facilitate further in vitro modification. Any mutagenesis techniques known in the art can be used, including but not limited to site-directed mutagenesis in vitro (J. Biol. Chem. 253:6551), use of Tab linkers (Pharmacia), etc.

[0162] Preparation of polyclonal antiserum and immunogen. Methods for generating the antibodies or antibody fragments of the present technology generally include immunizing a subject (usually a non-human subject, such as a mouse or a rabbit) with a purified CD33 protein or a fragment thereof or with cells expressing a CD33 protein or a fragment thereof. Suitable immunogenic preparations can contain, for example, recombinantly expressed CD33 protein or chemically synthesized CD33 peptides. Using standard techniques for the preparation of polyclonal and monoclonal antibodies, the extracellular domain of the CD33 protein or a portion or fragment thereof (e.g., the IgC2 domain) can be used as an immunogen to generate anti-CD33 antibodies that bind to the CD33 protein or a portion or fragment thereof.

[0163] The full-length CD33 protein or a fragment thereof can be used as a fragment as an immunogen. In some embodiments, the CD33 fragment contains the IgC2 domain of CD33 such that the antibodies generated against the peptide form specific immune complexes with the CD33 protein, which includes short isoforms of CD33 (CD33 splice variants lacking the IgV domain).

[0164] The IgC2 domain of CD33 is 84 amino acids in length. In some embodiments, the antigenic CD33 peptide contains at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, or 80 amino acid residues. Depending on the use and according to methods well known to those skilled in the art, sometimes longer antigenic peptides are required rather than shorter ones. Polymers of a given epitope are sometimes more effective than monomers.

[0165] If desired, the immunogenicity of the CD33 protein (or a fragment thereof) can be enhanced by fusion or conjugation with a hapten such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. The CD33 protein can also be combined with a conventional adjuvant such as Freund's complete or incomplete adjuvant to enhance the immune response of the subject to the polypeptide. Various adjuvants for enhancing the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants (such as Bacille Calmette-Guerin and Corynebacterium parvum), or similar immunostimulatory compounds. These techniques are standard in the art.

[0166] In describing the present technology, an immune response can be described as a "primary" or "secondary" immune response. A primary immune response, also referred to as a "protective" immune response, is an immune response that occurs in an individual due to an initial exposure (e.g., initial "immunization") to a specific antigen (e.g., the CD33 protein). In some embodiments, immunization can be carried out by vaccinating an individual with a vaccine containing the antigen. For example, the vaccine can be a CD33 vaccine containing an antigen from one or more sources of the CD33 protein. Over time, the primary immune response may wane or weaken and may even disappear or at least become attenuated to the point of being undetectable. Accordingly, the present technology also relates to a "secondary" immune response, also referred to herein as a "memory immune response". The term secondary immune response refers to an immune response that is elicited in an individual after a primary immune response has been generated.

[0167] Accordingly, a secondary immune response can be elicited, for example, to enhance an existing immune response that has weakened or attenuated, or to re-generate a previous immune response that has disappeared or can no longer be detected. A secondary or memory immune response can be a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs after stimulation of memory B cells that were generated upon first presentation of the antigen. A delayed type hypersensitivity (DTH) reaction is a type of CD4 + T cell-mediated cellular secondary or memory immune response. First exposure to an antigen primes the immune system, and additional exposure(s) result(s) in DTH.

[0168] After appropriate immunization, anti-CD33 antibodies can be prepared from the serum of a subject. If desired, antibody molecules directed against the CD33 protein can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as polypeptide A chromatography to obtain the IgG fraction.

[0169] Monoclonal antibodies. 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 murine anti-CD33 monoclonal antibody. To prepare monoclonal antibodies against the CD33 protein or its derivatives, fragments, analogs, or homologs, any technique that produces antibody molecules by continuous cell line culture can be used. Such techniques include, but are not limited to, hybridoma technology (see, e.g., Kohler and Milstein, 1975. Nature 256:495-497); trioma technology; human B-cell hybridoma technology (see, e.g., Kozbor et al., 1983. Immunol. Today 4:72) and EBV hybridoma technology to produce human monoclonal antibodies (see, e.g., Cole et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the present technology and can be produced by using human hybridomas (see, e.g., Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030) or by in vitro transformation of human B cells 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 nucleic acid population encoding antibody regions can be isolated. PCR using primers derived from sequences encoding conserved regions of the antibody is used to amplify the sequences encoding antibody portions from the population, and then DNA encoding the antibody or its fragments (such as variable domains) is reconstructed from the amplified sequences. Such amplified sequences can also be fused with DNA encoding other proteins - such as phage coat or bacterial cell surface proteins, - for expression and display of fusion polypeptides on phage or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibody or its fragment for the antigen or epitope present on the CD33 protein. Alternatively, hybridomas expressing anti-CD33 monoclonal antibodies can be prepared by immunizing a subject and subsequently isolating hybridomas from the subject's spleen using conventional methods. See, e.g., Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73:3-46). Screening the hybridomas using standard methods will yield monoclonal antibodies with different specificities (i.e., against different epitopes) and affinities.Selected monoclonal antibodies with desired properties (e.g., CD33 binding) can be used as expressed by the hybridoma, can be conjugated to molecules such as polyethylene glycol (PEG) to alter their properties, or the cDNA encoding the monoclonal antibody can be isolated, sequenced, and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains such as lysine to enhance the immunogenic properties of the CD33 protein. Additionally, CPG-dinucleotide technology can be used to enhance the immunogenic properties of the CD33 protein. Other manipulations include making specific aminoacyl residue substitutions or deletions that promote 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.

[0170] Hybridoma technology. In some embodiments, the antibodies of the technology are anti-CD33 monoclonal antibodies produced by hybridomas, the hybridomas comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgenic and a light chain transgenic that are fused to immortalized cells. Hybridoma technology includes those known in the art and taught in the following: 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). Other methods for generating hybridomas and monoclonal antibodies are well known to those of skill in the art.

[0171] Phage display technology. As pointed out above, antibodies of the present technology can be generated by applying recombinant DNA technology and phage display technology. For example, various phage display methods known in the art can be used to prepare anti-CD33 antibodies. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry polynucleotide sequences encoding the functional antibody domains. Phages with desired binding properties are selected from a library or combinatorial antibody library (such as human or murine) by direct selection with an antigen (usually an antigen bound or captured on a solid surface or bead). The phages used in these methods are typically filamentous phages, including fd and M13 that have Fab, Fv, or disulfide-stabilized Fv antibody domains, which are recombinantly fused to phage gene III or gene VIII proteins. In addition, the method is suitable for the construction of Fab expression libraries (see, for example, Huse et al., Science 246:1275-1281, 1989) to rapidly and efficiently identify monoclonal Fab fragments with the desired specificity for CD33 polypeptides, such as polypeptides or their derivatives, fragments, analogs, or homologs.Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in the following references: Huston et al., Proc. Natl. Acad. Sci U.S.A., 85:5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 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; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al.); WO 97 / 08320 (Morphosys); WO92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax) and 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. Lohning's U.S. Patent No. 6,753,136 has described a method for displaying polypeptides on the surface of phage particles by attaching polypeptides via disulfide bonds. As described in the above references, after phage selection, the antibody-encoding regions can be isolated from the phage and used to produce full antibodies (including human antibodies) or any other desired antigen-binding fragments and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast, and bacteria).For example, techniques for generating Fab, Fab′ and F(ab′)2 fragments by recombination can also be utilized using methods known in the art, such as those disclosed in the following references: WO 92 / 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.

[0172] Generally, a hybrid antibody or hybrid antibody fragment cloned into a display vector can be selected against an appropriate antigen to identify variants that retain good binding activity, since the antibody or antibody fragment will be present on the surface of phage or phagemid particles. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector forms can be used for this process, such as cloning an antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and / or screening.

[0173] Expression of recombinant anti-CD33 antibodies. As described above, the antibodies of the present technology can be produced by applying recombinant DNA techniques. A recombinant polynucleotide construct encoding an anti-CD33 antibody of the present technology generally includes an expression control sequence operably linked to the coding sequence of the anti-CD33 antibody chain, the expression control sequence including a native or heterologous promoter region. Thus, another aspect of the present technology includes a vector containing one or more nucleic acid sequences encoding an anti-CD33 antibody 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 a suitable cloning or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the polypeptide coding sequence to be inserted) by recombinant DNA techniques well known in the art and detailed below. U.S. Patent Nos. 6,291,160 and 6,680,192 to Lerner et al. have described methods for generating a variety of vector populations.

[0174] Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In the present disclosure, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors. However, the technology is intended to include such other forms of expression vectors that are not technically plasmids but perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors allow infection of a subject and expression of the construct in that subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. 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 for collection and purification of the anti-CD33 antibody (e.g., cross-reactive anti-CD33 antibody). See generally U.S. 2002 / 0199213. These expression vectors generally replicate in the host organism either as episomes or as part of the host chromosomal DNA. Usually, the expression vectors contain selectable markers, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectate lyase, that can be used to direct the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0175] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having CD33 binding properties, said recombinant expression vector being in a form suitable for the expression of said nucleic acid in a host cell, which means that said recombinant expression vector comprises one or more regulatory sequences selected according to the host cell for expression, said one or more regulatory sequences being operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory sequences in such a way as to permit 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, California (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). Those skilled in the art will understand that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired polypeptide, etc. Typical regulatory sequences that can be used as promoters for the expression of recombinant polypeptides (e.g., anti-CD33 antibodies) include, for example, but are not limited to, the promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, in particular, the promoters from alcohol dehydrogenase, iso-cytochrome C, and the enzymes responsible for maltose and galactose utilization. In one embodiment, the polynucleotide encoding the anti-CD33 antibody of the present technology is operably linked to the ara B promoter and is expressible in a host cell. See U.S. Patent 5,028,530. The expression vector of the present technology can be introduced into a host cell to produce a polypeptide or peptide encoded by the nucleic acid described herein, including a fusion polypeptide (e.g., an anti-CD33 antibody, etc.).

[0176] Another aspect of the present technology relates to host cells expressing anti-CD33 antibodies, which contain nucleic acids encoding one or more anti-CD33 antibodies. The recombinant expression vectors of the present technology can be designed to express anti-CD33 antibodies in prokaryotic or eukaryotic cells. For example, anti-CD33 antibodies can be expressed in bacterial cells (such as Escherichia coli), insect cells (using baculovirus expression vectors), fungal cells (such as yeast, yeast cells), or mammalian cells. Suitable host cells are further described in the following literature: Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using the T7 promoter regulatory sequence and T7 polymerase. Methods for preparing and screening polypeptides (such as anti-CD33 antibodies) with predetermined properties by expressing randomly generated polynucleotide sequences have been previously described. 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; 6,569,641.

[0177] Expression of polypeptides in prokaryotes is most commonly carried out in Escherichia coli using vectors containing constitutive or inducible promoters that direct the expression of fused or unfused polypeptides. Fusion vectors add many amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors generally have three purposes: (i) to increase the expression of the recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to facilitate the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Typically, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion portion and the recombinant polypeptide so that the recombinant polypeptide can be separated from the fusion portion after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A to the target recombinant polypeptide, respectively.

[0178] 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). U.S. Patent Nos. 6,294,353; 6,692,935 to Pack et al. have described methods for targeting the assembly of different active peptide or protein domains via polypeptide fusions to produce multifunctional polypeptides. One strategy for maximizing the expression of a recombinant polypeptide (e.g., an anti-CD33 antibody) in E. coli is to express the polypeptide in a host bacterium in which the ability to proteolytically cleave the recombinant polypeptide is impaired. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into the expression vector such that the individual codons for each amino acid are those preferentially used in the expression host (e.g., E. coli) (see, e.g., Wada et al., 1992. Nucl. Acids Res. 20:2111-2118). This alteration of the nucleic acid sequence of the present technology can be carried out by standard DNA synthesis techniques.

[0179] In another embodiment, the anti-CD33 antibody expression vector is a yeast expression vector. Examples of vectors for expression in 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, a baculovirus expression vector can be used to express the anti-CD33 antibody in insect cells. Baculovirus vectors that can be used to express polypeptides (e.g., anti-CD33 antibody) 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).

[0180] In yet another embodiment, a nucleic acid encoding an anti-CD33 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, for example but 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 functions of the expression vector are typically provided by viral regulatory elements. For example, commonly used promoters are derived from polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that can be used to express the anti-CD33 antibody of the present technology, see, for example, 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, New York, 1989.

[0181] In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of a nucleic acid in a specific 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), the lymphocyte-specific promoter (Calame and Eaton, Adv. Immunol. 43:235-275, 1988), the promoters of the T cell receptor (Winoto and Baltimore, EMBO J. 8:729-733, 1989) and immunoglobulin (Banerji et al., 1983. Cell 33:729-740; Queen and Baltimore, Cell 33:741-748, 1983.), the neuron-specific promoter (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), the pancreas-specific promoter (Edlund et al., 1985. Science 230:912-916), and the mammary gland-specific promoter (e.g., the whey acidic protein promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Also encompassed are developmentally regulated promoters, such as the murine hox promoters (Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989).

[0182] Another aspect of the methods of the invention relates to host cells into which a recombinant expression vector of the technology has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in succeeding generations due to mutation or environmental influences, such progeny may in fact be different from the parental cell, but are still included within the scope of the term as used herein.

[0183] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-CD33 antibodies can be expressed in bacterial cells such as Escherichia 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, New York, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and the suitable host cell lines include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for 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 termination sequences. Queen et al., Immunol. Rev. 89:49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. Co et al., J Immunol. 148:1149, 1992. Other suitable host cells are known to those skilled in the art.

[0184] The vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene gun, or virus-based transfection. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (generally see Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in the following references: Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) and other laboratory manuals. Depending on the type of cell host, the vector containing the DNA segment of interest can be transferred into the host cell by well-known methods.

[0185] For stable transfection of mammalian cells, it is known that depending on the expression vector and transfection technique used, only a small fraction of cells will integrate exogenous DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., resistance to an antibiotic) is typically introduced into the host cell along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding the anti-CD33 antibody, or it can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive while other cells die).

[0186] Host cells (such as prokaryotic or eukaryotic host cells in culture) comprising the anti-CD33 antibody of the present technology can be used to produce (i.e., express) recombinant anti-CD33 antibody. In one embodiment, the method comprises culturing the host cell (into which a recombinant expression vector encoding the anti-CD33 antibody has been introduced) in a suitable medium, thereby producing the anti-CD33 antibody. In another embodiment, the method further comprises the step of isolating the anti-CD33 antibody from the medium or the host cell. Once expressed, the anti-CD33 antibody is purified from the medium and the host cell, e.g., a collection of anti-CD33 antibody or anti-CD33 antibody-related polypeptides. The anti-CD33 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. In one embodiment, the anti-CD33 antibody is produced in a host organism by the method of U.S. Patent No. 4,816,397 to Boss et al. Generally, the anti-CD33 antibody chain is expressed along with a signal sequence and is thereby released into the medium. However, if the anti-CD33 antibody chain is not naturally secreted by the host cell, the anti-CD33 antibody chain can be released by treatment with a mild detergent. 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, etc. (see generally Scopes, Protein Purification (Springer-Verlag, New York, 1982)).

[0187] Polynucleotides encoding anti-CD33 antibodies, such as the coding sequences of anti-CD33 antibodies, 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 the coding sequences of light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes such as casein or β-lactoglobulin. For the production of transgenic animals, the transgene can be microinjected into fertilized oocytes, or the transgene can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells can be transferred into enucleated oocytes.

[0188] Single-chain antibodies. In one embodiment, the anti-CD33 antibody of the present technology is a single-chain anti-CD33 antibody. According to the present technology, the technology can be suitable for producing single-chain antibodies specific for the CD33 protein (see, for example, U.S. Patent No. 4,946,778). Examples of techniques that can be used to produce single-chain Fvs and antibodies of the present technology include those described in the following documents: 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.

[0189] 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 antibody and the acceptor antibody are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), and in this case the resulting CDR-grafted antibody is called a "humanized" antibody.

[0190] Recombinant anti-CD33 antibodies that contain human and non-human portions (such as chimeric monoclonal antibodies and humanized monoclonal antibodies) can be prepared using standard recombinant DNA techniques and are within the scope of the present technology. For certain uses, including in vivo use of anti-CD33 antibodies of the present technology in humans and use of these agents in in vitro assay determinations, chimeric or humanized anti-CD33 antibodies can be used. Such chimeric monoclonal antibodies and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, for example but not limited to, the methods described in the following documents: International Application No. PCT / US86 / 02269; U.S. Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86 / 01533; U.S. Patent No. 4,816,567; 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. Science 239:1534; Morrison, Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Patent No. 5,807,715; and Beidler et al., 1988. J. Immunol. 141:4053-4060.For example, a variety of techniques can be used to humanize antibodies, including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Patent Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.A., 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. Patent No. 5,565,332). In one embodiment, the cDNA encoding a murine anti-CD33 monoclonal antibody is digested with a specifically selected restriction endonuclease to remove the sequence encoding the Fc constant region and replace an equivalent portion of the cDNA encoding the human Fc constant region (see Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 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 Nos. 6,180,370; U.S. Patent Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422).

[0191] In one embodiment, the present technology provides the construction of a humanized anti-CD33 antibody that is 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, the terms "human" and "humanized" with respect to an antibody refer to any antibody that is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides humanized anti-CD33 antibodies, heavy chain, and light chain immunoglobulins.

[0192] CDR antibodies. In some embodiments, the anti-CD33 antibody of the present technology is an anti-CD33 CDR antibody. Generally, the donor and recipient antibodies used to generate the anti-CD33 CDR antibody are monoclonal antibodies from different species; generally, the recipient antibody is a human antibody (to minimize its antigenicity in humans), and in this case the resulting CDR-grafted antibody is called a "humanized" antibody. The graft can have a single CDR (or even a part of a single CDR) within a single V H or V L or can have multiple CDRs (or parts thereof) within one or both of the V H and V L . Generally, all three CDRs in all variable domains of the recipient antibody will be replaced by the corresponding donor CDRs, but only the required number need to be replaced to enable the resulting CDR-grafted antibody to bind sufficiently to the CD33 protein. Methods for generating CDR-grafted and humanized antibodies are taught in the following documents: U.S. Patent No. 5,585,089 to Queen et al.; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; and U.S. 5,225,539 to Winter; and EP 0682040. Methods for preparing V H and V L polypeptides are taught in the following documents: Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP0368684; EP0451216; and EP0120694.

[0193] After selecting suitable framework region candidates from the same family and / or the same family members, one or both of the heavy and light chain variable regions are generated by grafting the CDRs from the source species into the heterologous framework region. Assembly of heterologous antibodies or heterologous antibody fragments with heterologous variable chain regions with respect to any of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the heterologous variable domains described herein (i.e., based on the framework of the target species and the CDRs from the source species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the source species antibody using appropriate restriction enzymes and ligated into the target species framework by ligation with an appropriate ligase. Alternatively, the framework region of the variable chain of the source species antibody can be altered by site-directed mutagenesis.

[0194] Since the hybrids are constructed from selections between multiple candidates corresponding to each framework region, there are many sequence combinations suitable for construction according to the principles described herein. Thus, libraries of hybrids can be assembled, the members of which have different combinations of individual framework regions. Such libraries can be an electronic database collection of sequences or a physical collection of hybrids.

[0195] This process generally does not alter the FR of the recipient antibody flanking the grafted CDR. However, those skilled in the art can sometimes improve the antigen-binding affinity of the resulting anti-CD33 CDR-grafted antibody by substituting certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable substitution positions include amino acid residues adjacent to the CDR or amino acid residues capable of interacting with the CDR (see, e.g., US 5,585,089, especially columns 12-16). Alternatively, those skilled in the art can start with the donor FR and modify it to make it more similar to the recipient FR or the human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR conforms to the human consensus FR at that position or is at least 90% or more identical to such consensus FR, 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.

[0196] Bispecific antibody (BsAb). A bispecific antibody is an antibody that can simultaneously bind two targets with different 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). BsAbs can be prepared, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, by molecular function, a bispecific binder 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 (a different VH / VL pair). By this definition, a bispecific binder has two different antigen-binding arms (both the specificity and the CDR sequences are different) and is monovalent for each antigen it binds.

[0197] The bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs) of the present technology have at least one arm that specifically binds to, for example, 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 a B cell, T cell, myeloid cell, plasma cell, or mast cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, and KIR. In certain embodiments, the BsAb is capable of binding to tumor cells that express the CD33 antigen on the cell surface. In some embodiments, the BsAb has been engineered to promote the killing of tumor cells by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen-binding site that is specific for CD33 and a second antigen-binding site that is specific for a small molecule hapten (e.g., DTPA, IMP288, DOTA, DOTA-Bn, DOTA deferoxamine, other DOTA chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D. A. et al., 1994, Cancer Res. 54(22):5937-5946).

[0198] Using molecular engineering, a variety of bispecific fusion proteins can be generated. For example, BsAbs have been constructed that utilize intact immunoglobulin frameworks (such as IgG), single-chain variable fragments (scFv), or combinations thereof. In some embodiments, the bispecific fusion protein is bivalent and comprises, for example, an scFv having a single binding site for one antigen and a Fab fragment having a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent and comprises, for example, an scFv having a single binding site for one antigen and another scFv fragment having a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent and comprises, for example, an immunoglobulin (such as IgG) having two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs composed of two tandem scFv units have been shown to be a clinically successful form of bispecific antibody. In some embodiments, the BsAb comprises two tandem single-chain variable fragments (scFv) that are designed such that the scFv that binds to a tumor antigen (e.g., CD33) is linked to an scFv that engages a T cell (e.g., by binding 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 tandem single-chain variable fragments (scFv) that are designed such that the scFv that binds to a tumor antigen (e.g., CD33) is linked to an scFv that engages a small molecule DOTA hapten.

[0199] Recent methods for generating BsAbs include engineering recombinant monoclonal antibodies that have additional cysteine residues such that they crosslink more firmly than 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 that link two or more different single-chain antibody or antibody fragment segments with the desired dual specificity. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). A variety of bispecific fusion proteins can be generated using molecular engineering.

[0200] Bispecific fusion proteins that link two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some specific embodiments, the BsAb according to the present technique comprises an immunoglobulin containing a heavy chain and a light chain, as well as 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. By PCR reaction, an appropriate linker sequence necessary for in-frame ligation of the heavy chain Fd to the scFv is introduced into the V L and V κ domains. Then the DNA fragment encoding the scFv is ligated into a staging vector containing the DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and ligated into a vector containing the DNA sequence of the V H region of the CD33 antibody. The resulting vector can be used to transfect a suitable host cell, such as a mammalian cell, to express the bispecific fusion protein.

[0201] 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 by its tendency not to adopt a rigid three-dimensional structure, but rather to provide flexibility to the polypeptide (e.g., the first and / or second antigen-binding sites). In some embodiments, a linker is employed in the BsAb described herein based on the specific properties conferred to the BsAb, such as increased stability. In some embodiments, the BsAb of the present technique comprises a G4S linker. In some specific embodiments, the BsAb of the present technique comprises a (G4S) n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0202] Self-Assembling 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 customized to achieve environmentally dependent multimerization with beneficial kinetic, thermodynamic, and / or pharmacological properties. For example, it has been recognized that the SADA domain can be part of a conjugate that allows for efficient delivery of a payload to a target site of interest while minimizing the risk of off-target interactions. The anti-CD33 antibodies of the present technology can comprise SADA domains linked to one or more binding domains. In some embodiments, such conjugates are characterized in that they multimerize under relevant conditions (e.g., in a solution where the conjugate is present above a threshold concentration or pH and / or when present at a target site characterized by a relevant level or density of a receptor for the payload) to form a complex of a desired size and dissociate into smaller forms under other conditions (e.g., in the absence of a relevant environmental multimerization trigger).

[0203] Compared to conjugates without SADA domains, SADA conjugates can have improved characteristics. In some embodiments, the improved characteristics of the multimeric conjugates include: increased affinity / binding to a target, increased specificity for a target cell or tissue, and / or an extended initial serum half-life. In some embodiments, the improved characteristics include that the SADA conjugate exhibits reduced non-specific binding, reduced toxicity, and / or improved renal clearance by dissociating into smaller states (e.g., dimers or monomers). In some embodiments, the SADA conjugate comprises a SADA polypeptide having an amino acid sequence that shows at least 75% identity to the amino acid sequence of a human homologous multimerization polypeptide and is characterized by one or more multimerization dissociation constants (K D ).

[0204] In some embodiments, the SADA conjugate is constructed and arranged such that it adopts a first multimerization state and one or more higher-order multimerization states. In some embodiments, the size of the first multimerization state is less than about 70 kDa. In some embodiments, the first multimerization state is a non-multimerized state (e.g., monomer or 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 multimerized state (e.g., trimer or tetramer). In some embodiments, the higher-order multimerization state is a homotetramer or higher-order homotetramer with a size greater than 150 kDa. In some embodiments, when the higher-order homotetrameric conjugate is present at a concentration higher than the K D of the SADA polypeptide, the conjugate is stable in an aqueous solution. In some embodiments, when the concentration of the SADA conjugate is lower than the K DUpon this, the conjugate transitions from one or more higher-order multimeric states to a first multimeric state under physiological conditions.

[0205] In some embodiments, the SADA polypeptide is covalently linked to the binding domain via a linker. Any suitable linker known in the art can 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 repetitive 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.

[0206] 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 comprising a helical bundle, which 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, each of which is composed of a helical bundle that associates in a parallel or antiparallel orientation. In some embodiments, the SADA domain is selected from one of the following human proteins: p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosome-associated protein 23 (SNAP-23), Stefin B (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4) or cyclin D-related protein (CBFA2T1). Examples of suitable SADA domains are described in PCT / US2018 / 031235, which is hereby incorporated by reference in its entirety. Polypeptide sequences of exemplary SADA domains are provided below.

[0207] Amino acid sequence of the human p53 tetramerization domain (321 - 359) KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP (SEQ ID NO:125)

[0208] Amino acid sequence of human p63 tetramerization domain (396 - 450) RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ (SEQ ID NO:126)

[0209] Amino acid sequence of human p73 tetramerization domain (348 - 399) RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (SEQ ID NO:127).

[0210] Amino acid sequence of human HNRNPC tetramerization domain (194 - 220) QAIKKELTQIKQKVDSLLENLEKIEKE (SEQ ID NO:128)

[0211] Amino acid sequence of human SNAP-23 tetramerization domain (23 - 76) STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL (SEQ ID NO:129)

[0212] Amino acid sequence of human Stefin B tetramerization domain (2 - 98) MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF (SEQ ID NO:130)

[0213] Amino acid sequence of KCNQ4 tetramerization domain (611 - 640) DEISMMGRVVKVEKQVQSIEHKLDLLLGFY (SEQ ID NO:131)

[0214] Amino acid sequence of CBFA2T1 tetramerization domain (462 - 521) TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH (SEQ IDNO:132)

[0215] In some embodiments, the SADA polypeptide is or comprises the tetramerization domain of p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosome-associated protein 23 (SNAP-23), Stefin B (cysteine protease inhibitor B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or cyclin D-related 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 the sequences shown in any one of SEQ ID NOs: 125-132.

[0216] Fc modification. In some embodiments, the anti-CD33 antibody of the present technology comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region (or parental Fc region) such that the affinity of the molecule for Fc receptors (e.g., FcγR) is altered, provided that based on crystallographic and structural analyses of Fc-Fc receptor interactions (such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000)), the variant Fc region has no substitutions at positions that directly contact the Fc receptor. Examples of positions in the Fc region that directly contact the Fc receptor (such as 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).

[0217] In some embodiments, the anti-CD33 antibody of the present technology has an altered affinity for activating and / or inhibitory receptors, wherein the variant Fc region has one or more amino acid modifications, and wherein the one or more amino acid modifications are substitution of N297 with alanine or substitution of K322 with alanine.

[0218] Glycosylation modification. In some embodiments, the anti-CD33 antibody of the present technology has an Fc region that contains variant glycosylation compared to the parental Fc region. In some embodiments, the variant glycosylation includes the absence of fucose; in some embodiments, the variant glycosylation is due to expression in GnT1-deficient CHO cells.

[0219] In some embodiments, relative to a suitable reference antibody that binds to a target antigen (e.g., CD33), the antibodies of the present technology can have modified glycosylation sites without altering the functionality of the antibody, such as the binding activity to the antigen. As used herein, "glycosylation site" includes any specific amino acid sequence in the antibody that will specifically and covalently attach to an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together).

[0220] Oligosaccharide side chains are generally linked to the antibody backbone via N-linkage or O-linkage. 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 hydroxy amino acid, such as serine or threonine. For example, an Fc-glycoform (hCD33-IgGln) lacking certain oligosaccharides (including fucose) and terminal N-acetylglucosamine can be produced in specific CHO cells and exhibits enhanced ADCC effector function.

[0221] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions 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 in the technology of the present invention. See, for example, U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US2002 / 0028486; International Patent Application Publication No. WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; the above patents are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or its related portion or component) is modified by deleting one or more endogenous carbohydrate moieties. In some specific embodiments, the present technology includes deleting the glycosylation site in the Fc region of the antibody by modifying asparagine at position 297 to alanine.

[0222] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or attenuating effector function. Engineered glycoforms can be generated by any method known to those skilled in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes (e.g., N-acetylglucosaminyltransferase III (GnTIII)), by expressing a molecule containing an Fc region in various organisms or cell lines from various organisms, or by modifying one or more carbohydrates after a molecule containing an Fc region has been expressed. Methods for generating engineered glycoforms are known in the art and include but are not limited to those described in the following references: 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 Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publication WO 00 / 61739A1; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT TM TECHNOLOGY (Biowa, Inc., Princeton, NJ); GLYCOMAB TM glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of these references is hereby incorporated by reference in its entirety. See, for example, International Patent Application Publication WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0223] Fusion proteins. In one embodiment, the anti-CD33 antibody of the present technology is a fusion protein. When fused to a second protein, the anti-CD33 antibody of the present technology can be used as an antigen tag. Examples of domains that can be fused to a polypeptide include not only heterologous signal sequences but also other heterologous functional regions. The fusion need not be direct but can be via a linker sequence. Moreover, the fusion proteins of the present technology can also be engineered to improve the characteristics of the anti-CD33 antibody. For example, regions of additional amino acids (especially charged amino acids) can be added to the N-terminus of the anti-CD33 antibody to improve stability and persistence during purification from host cells or subsequent handling and storage. Additionally, a peptide moiety can be added to the anti-CD33 antibody to facilitate purification. Such regions can be removed prior to the final preparation of the anti-CD33 antibody. Adding peptide moieties to facilitate the handling of polypeptides is a conventional technique well known in the art. The anti-CD33 antibody of the present technology can be fused to a tag sequence such as a peptide that facilitates the purification of the fusion polypeptide. In a selected embodiment, the tagged amino acid sequence is a hexahistidine peptide, especially such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, California), and many such hexahistidine peptides are commercially available. As described by Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989, for example, hexahistidine enables the convenient purification of the fusion protein. Another peptide tag that can be used for purification is the "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984.

[0224] Thus, any of these above-mentioned fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology. Additionally, in some embodiments, the fusion proteins described herein exhibit an increased in vivo half-life.

[0225] Compared to proteins or protein fragments secreted as monomers alone, fusion proteins having a disulfide-linked dimer structure (due to IgG) can bind and neutralize other molecules more efficiently. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0226] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins which comprise various portions of the constant regions of immunoglobulin molecules as well as another human protein or fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnosis and can thus result in, for example, improved pharmacokinetic properties. See EP-A 0232262. Alternatively, it may be desirable to delete or modify the Fc portion after expression, detection and purification of the fusion protein. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may interfere with therapy and diagnosis. In drug discovery, for example, human proteins such as hIL-5 have been fused to the Fc portion 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.

[0227] Labeled anti-CD33 antibody. In one embodiment, the anti-CD33 antibody of the present technology is conjugated to a label portion, i.e., a detectable group. The particular label or detectable group conjugated to the anti-CD33 antibody is not a critical aspect of the present 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 having detectable physical or chemical properties. Such detectable labels have been well developed in the fields of immunoassays and imaging. In general, almost any label that can be used in such methods can be applied to the present technology. Thus, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads TM ), 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 imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C, 15 O (for positron emission tomography), 99m TC, 111In (for single photon emission tomography), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes 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. Patent 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 and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Edition, Molecular Probes, Inc., Eugene OR).

[0228] The label can be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As described above, a variety of labels can be used, and the choice of label depends on factors such as the desired sensitivity, ease of conjugation to the compound, stability requirements, available instrumentation, and disposal regulations.

[0229] Non-radioactive labels are generally attached by an indirect method. Typically, a ligand molecule (such as biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule, which is inherently detectable or covalently bound to a signal system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Many ligands and anti-ligands can be used. In cases where the ligand (such as biotin, thyroxine, and cortisol) has a natural anti-ligand, the ligand can be used in combination with the labeled naturally occurring anti-ligand. Alternatively, any hapten or antigenic compound can be used in combination with an antibody such as an anti-CD33 antibody.

[0230] The molecule can also be directly conjugated to a signal-producing compound, for example, by conjugation to an enzyme or fluorophore. The enzymes used as labels will mainly be hydrolases, especially phosphatases, esterases, and glycosidases, or oxidoreductases, especially peroxidases. Fluorescent compounds that can be used as label moieties include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds that can be used as label moieties include, but are not limited to, for example, fluorescein and 2,3-dihydrophthalazinedione, such as luminol. For a review of the various labels or signal-producing systems that can be used, see U.S. Patent No. 4,391,904.

[0231] Means for detecting labels are well known to those skilled in the art. Thus, for example, where the label is a radioactive label, the detection means includes a scintillation counter or film, as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of an appropriate wavelength and detecting the resulting fluorescence. The fluorescence can be detected in a visual form, with the aid of film, by using an electronic detector such as a charge-coupled device (CCD) or a photomultiplier tube, etc. Similarly, an enzyme label can be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. Finally, a simple colorimetric label can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, the conjugate is often pink, while the various conjugated beads exhibit the color of the beads.

[0232] Some assay formats do not require the use of labeled components. For example, an agglutination assay can be used to detect the presence of a target antibody such as an anti-CD33 antibody. 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 the target antibody is detected by simple visual inspection. B. Identifying and characterizing the anti-CD33 antibody of the present technology

[0233] Methods for identifying and / or screening anti-CD33 antibodies of the present technology. Methods for identifying and screening antibodies having the desired specificity for the CD33 protein (e.g., those that bind to the IgC2 domain of CD33) among antibodies against the CD33 polypeptide include any immunologically mediated techniques known in the art. Components of an immune response can be detected in vitro by various methods well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells, and lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with an antigen and antigen-presenting cells, and the synthesis and secretion of cytokines can be measured by standard methods (Windhagen A et al., Immunity, 2:373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen, and the presentation of this antigen on MHC can be detected by a T lymphocyte activation assay or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with a reagent (crosslinking their Fc-ε receptors) and histamine release can be measured by an enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); (5) enzyme-linked immunosorbent assay (ELISA).

[0234] Similarly, the products of an immune response in 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 ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories such as ELISA; (2) the migration of immune cells to the site of inflammation can be detected by scratching the skin surface and placing a sterile container to capture the migrating cells at the site of the scratch (Peters et al., Blood, 72:1310-5, 1988); (3) 3 the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be measured using

[0235] 3H-thymidine; (4) the phagocytic ability of granulocytes, macrophages, and other phagocytic cells in PBMCs can be measured by placing PBMCs in a well together with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) the 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.

[0236] In one embodiment, anti-CD33 antibodies of the present technology are selected using the display of CD33 peptides on the surface of replicable genetic packages. See, for example, U.S. Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP1024191; EP 589 877; EP 774 511; EP 844 306. Methods for generating / selecting filamentous phage particles that contain a phagemid genome encoding a binding molecule with the desired specificity have been described. See, for example, EP 774 511; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0237] In some embodiments, anti-CD33 antibodies of the technology are selected using ribosome display. Methods for using ribosome display to identify ligands in peptide libraries have been 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.

[0238] In certain embodiments, anti-CD33 antibodies of the technology are selected using tRNA display of CD33 peptides. Methods for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. Biol., 9:741-46, 2002.

[0239] In one embodiment, anti-CD33 antibodies of the technology are selected using RNA display. Methods for selecting peptides and proteins using RNA display libraries have been described by Roberts et al., Proc. Natl. Acad. Sci. USA, 94:12297-302, 1997; and Nemoto et al., FEBS Lett., 414:405-8, 1997. Methods for selecting peptides and proteins using unnatural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13:506-12, 2003.

[0240] In some embodiments, anti-CD33 antibodies of the technology are expressed in the periplasm of Gram-negative bacteria and mixed with labeled CD33 protein. See WO 02 / 34886. In clones expressing recombinant polypeptides having affinity for CD33 protein, the concentration of labeled CD33 protein bound to the anti-CD33 antibody increases, and the cells are allowed to separate from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22:9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0241] After selecting the desired anti-CD33 antibody, it is contemplated that the antibody can be produced in large quantities by any technique known to those of skill in the art, such as prokaryotic or eukaryotic cell expression, etc. Anti-CD33 antibodies, which are, for example but not limited to, anti-CD33 hybrid antibodies or fragments, can be produced by constructing an expression vector encoding the antibody heavy chain using conventional techniques, retaining in the antibody heavy chain the minimal portions of the CDRs and (if desired) variable region framework required for the antibody binding specificity of the originating species, which are engineered according to the techniques described herein and are derived from the originating species antibody, and the remainder of the antibody being derived from the target species immunoglobulin that can be manipulated as described herein, thereby generating a vector for expressing the hybrid antibody heavy chain.

[0242] Measurement of CD33 binding. In some embodiments, a CD33 binding assay refers to an assay format in which CD33 protein and an anti-CD33 antibody are mixed under conditions suitable for binding between the CD33 protein and the anti-CD33 antibody and for assessing the amount of binding between the CD33 protein and the anti-CD33 antibody. The amount of binding is compared to a suitable control, which can 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 can be evaluated by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, proximity scintillation assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring CD33 protein bound to an anti-CD33 antibody are, 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, which are, for example, radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of a candidate anti-CD33 antibody is at least 1% higher than the binding observed in the absence of the candidate anti-CD33 antibody, then the candidate anti-CD33 antibody can be used 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). Use of the anti-CD33 antibody of the present technology

[0243] Overview. The anti-CD33 antibodies of the present technology can be used in methods known in the art related to the localization and / or quantification of CD33 protein (e.g., for measuring the level of CD33 protein in a suitable biological sample, for diagnostic methods, for polypeptide imaging, etc.). The antibodies of the present technology can be used to isolate CD33 protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-CD33 antibodies of the present technology can facilitate the purification of native 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 expression abundance and pattern of immunoreactive polypeptides. The anti-CD33 antibodies of the present technology can be used to diagnostically monitor the level of immunoreactive CD33 protein in tissues as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. As described above, detection can be facilitated by conjugating (i.e., physically linking) the anti-CD33 antibodies of the present technology with a detectable substance.

[0244] Detection of CD33 protein. Exemplary methods for detecting the presence of immunoreactive CD33 protein in a biological sample involve obtaining a biological sample from a test subject and contacting the biological sample with an anti-CD33 antibody of the present technology capable of detecting immunoreactive CD33 protein, thereby detecting the presence of immunoreactive CD33 protein in the biological sample. Detection can be accomplished by a detectable label attached to the antibody.

[0245] The term "labeled" with respect to the anti-CD33 antibody is intended to encompass directly labeling the antibody by conjugating (i.e., physically linking) a detectable substance with the antibody, as well as indirectly labeling the antibody through reactivity with another compound that is directly labeled (such as a secondary antibody). Examples of indirect labeling include detecting a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin such that it can be detected with a fluorescently labeled streptavidin.

[0246] In some embodiments, the anti-CD33 antibodies disclosed herein are conjugated with one or more detectable labels. For such uses, the anti-CD33 antibodies can be detectably labeled by covalent or non-covalent attachment of a chromogenic agent, an enzymatic agent, a radioisotopic agent, an isotopic agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, a nuclear magnetic resonance contrast agent or other label.

[0247] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0248] 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 Pd, etc. 111 In is an exemplary isotope for in vivo imaging because it avoids the problem of 125 I or 131 I-labeled CD33-binding antibodies being dehalogenated by the liver. In addition, this isotope has a gamma emission energy that is more favorable 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, 111 In conjugated with monoclonal antibodies having 1-(P-isothiocyanatobenzyl)-DPTA exhibits little uptake in non-tumor tissues (especially the liver) and enhances the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioisotope labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe.

[0249] Examples of suitable fluorescent labels include 152Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, phycocyanin labeling, allophycocyanin labeling, green fluorescent protein (GFP) labeling, o-phthalaldehyde labeling, and fluorescamine labeling. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0250] Examples of chemiluminescent labels include luminol labeling, isoluminol labeling, aromatic acridinium ester labeling, imidazole labeling, acridinium salt labeling, oxalate ester labeling, luciferin labeling, luciferase labeling, and aequorin labeling. Examples of magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0251] The detection methods of the present technology can be used to detect immunoreactive CD33 protein in biological samples both in vitro and in vivo. In vitro techniques for detecting immunoreactive CD33 protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, radioimmunoassay, and immunofluorescence. In addition, in vivo techniques for detecting immunoreactive CD33 protein include introducing a labeled anti-CD33 antibody into a subject. For example, an anti-CD33 antibody can be labeled with a radioactive label, and the presence and location of the radioactive label in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains CD33 protein molecules from a test subject.

[0252] Immunoassays and imaging. The anti-CD33 antibodies of the present technology can be used to determine the level of immunoreactive CD33 protein in biological samples (such as 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 that can be used to detect 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 agents (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.

[0253] In addition to measuring immunoreactive CD33 protein levels in biological samples, the anti-CD33 antibodies of the present technology can also be used for in vivo imaging of CD33. Antibodies that can be used in this method include those that can be detected by radiography, NMR, or ESR. For radiography, suitable labels include radioactive isotopes such as barium or cesium, which emit detectable radiation but cause no significant harm to the subject. Labels suitable for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into the anti-CD33 antibody by labeling the nutrients used for the relevant scFv cloning.

[0254] An anti-CD33 antibody labeled with an appropriate detectable imaging moiety (such as a radioactive isotope (e.g., 131 I, 112 In, 99 mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance) is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into a subject. It will be understood in the art that the size of the subject and the imaging system used will determine the amount of the imaging moiety required to produce a diagnostic image. In the case of a radioactive isotope moiety, for a human subject, the amount of radioactivity injected is typically in the range of about 5 to 20 millicuries 99 mTc. The labeled anti-CD33 antibody will then accumulate at the cellular locations containing the specific target polypeptide. For example, the labeled anti-CD33 antibody of the present technology will accumulate in the cells and tissues in the subject in which the CD33 protein has been localized.

[0255] Accordingly, the present technology provides a method for diagnosing a medical condition, which involves: (a) determining the expression of immunoreactive CD33 protein by measuring the binding of the anti-CD33 antibody of the present technology in the cells or body fluids of an individual; (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 a medical condition.

[0256] Affinity purification. The anti-CD33 antibodies 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), acrylic resins, and polymers such as polyacrylamide and latex beads. Techniques for coupling the antibody to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology", 4th Edition, Blackwell Scientific Publications, Oxford, UK, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34, Academic Press, New York (1974)).

[0257] The simplest method of binding the antigen to the antibody-support matrix is to collect the beads in a column and pass the antigen solution downward 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 low flow rate. The immobilized antibody captures the antigen as it flows by. Alternatively, the antigen can be brought into contact with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or shaking the slurry so as to achieve maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through a column to collect the beads. The beads are washed with a suitable wash buffer and then the pure or substantially pure antigen is eluted.

[0258] The antibody or polypeptide of interest can be conjugated to a solid support such as beads. Additionally, if desired, a first solid support such as beads can 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 polypeptides to supports. Thus, any conjugation methods and means disclosed herein for conjugating polypeptides to solid supports can also be used to conjugate a first support to a second support, where the first and second solid supports can be the same or different.

[0259] Suitable linkers (which can be crosslinking agents) for conjugating polypeptides to solid supports include a variety of agents that can react with functional groups present on the support surface or with the polypeptide or both. Reagents that can be used as crosslinking agents include homobifunctional reagents and, in particular, heterobifunctional reagents. Useful bifunctional crosslinking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. A crosslinking agent can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, a photo-labile crosslinking agent such as 3-amino-(2-nitrophenyl)propionic acid can be used as a means for cleaving 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 crosslinking reagents are well known in the art. (See, for example, Wong (1991), supra; and Hermanson (1996), supra).

[0260] An antibody or polypeptide can be immobilized on a solid support such as a bead by forming a covalent amide bond between a carboxyl-functionalized bead and the amino terminus of the polypeptide, or conversely by forming a covalent amide bond between an amino-functionalized bead and the carboxyl terminus of the polypeptide. Additionally, a bifunctional trityl linker can be attached to the support via an amino resin through an amino or carboxyl group on the resin, for example, a 4-nitrophenyl active ester attached to a resin such as Wang resin. When using the bifunctional trityl method, the solid support may need to be treated with a volatile acid such as formic acid or trifluoroacetic acid to ensure that the polypeptide is cleaved and can be removed. In this case, the polypeptide can be deposited as a bead-free patch at the bottom of the pores of the solid support or on the flat surface of the solid support. After adding the matrix solution, the polypeptide can be desorbed into the MS.

[0261] A hydrophobic trityl linker can also be used as an acid-labile linker by cleaving the amino-linked trityl from the polypeptide using a volatile acid or a suitable matrix solution (such as a matrix solution containing 3-HPA). The acid lability can also be altered. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be changed to a suitable para-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., a trityl ether bond and a tritylamine bond can be formed with the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by disrupting the hydrophobic attraction under acidic conditions or by cleaving the trityl ether bond or the tritylamine bond, the acidic conditions including (if desired) under typical MS conditions where a matrix such as 3-HPA is used as an acid.

[0262] Orthogonally cleavable linkers can also be used to couple a first solid support (e.g., beads) to a second solid support, or to couple a polypeptide of interest to a solid support. Using such linkers, the first solid support (e.g., beads) can be selectively cleaved from the second solid support without cleaving the polypeptide from the support; the polypeptide can then be cleaved from the beads at a later time. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to couple the beads to the second solid support, and an acid-cleavable bifunctional trityl can be used to immobilize the polypeptide to the support. If desired, the linkage between the polypeptide and the solid support can be cleaved first, e.g., leaving the linkage between the first and second supports intact. The trityl linker can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl is readily cleaved under acidic conditions.

[0263] For example, the beads can be coupled to the second support via a linking group, and the linking group can be chosen to have a length and chemical nature that promotes high-density binding of the beads to the solid support or of the polypeptide to the beads. Such a linking group can have, for example, a "dendritic" structure, thereby providing multiple functional groups for each attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythritol, and tris(hydroxymethyl)aminomethane.

[0264] Non-covalent binding associations. Antibodies or polypeptides can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support, by non-covalent interactions. For example, magnetic beads made of a ferromagnetic material that can be magnetized can be attracted to a magnetic solid support and released from the support by removing the magnetic field. Alternatively, the solid support can have ionic or hydrophobic moieties, which can allow the ionic or hydrophobic moieties to interact with a polypeptide (e.g., a polypeptide containing an attached trityl) or with a second solid support having hydrophobic characteristics, respectively.

[0265] The solid support can also have a member of a specific binding pair and can thus be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, beads coated with avidin or streptavidin can bind to a polypeptide incorporating a biotin moiety, or to a second solid support coated with biotin or a biotin derivative such as iminobiotin.

[0266] It should be recognized that any binding member disclosed herein or otherwise known in the art can be reversed. Thus, for example, biotin can be incorporated into a polypeptide or a solid support, and conversely, avidin or other biotin-binding moieties can be incorporated into a support or a polypeptide. 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 the antigens with which they specifically interact, and other such pairs known to those of skill in the art. A. Diagnostic use of the anti-CD33 antibody of the present technology

[0267] Overview. The anti-CD33 antibodies of the present technology can be used in diagnostic methods. Accordingly, the present technology provides methods for diagnosing CD33 activity in a subject using the antibodies. Anti-CD33 antibodies of the present technology can be selected such that they have any level of epitope-binding specificity and a very high binding affinity for the CD33 protein. Generally, the higher the binding affinity of an antibody, the more stringent the washing conditions can be performed in an immunoassay to remove non-specifically bound material without removing the target polypeptide. Thus, the anti-CD33 antibodies of the present technology for diagnostic assays typically have a binding affinity of about 10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 or 10 12 M -1 In addition, anti-CD33 antibodies intended to be used as diagnostic reagents are desired to have a sufficient kinetic association rate to reach equilibrium within at least 12 h, at least five (5) h, or at least one (1) hour under standard conditions.

[0268] Anti-CD33 antibodies can be used to detect immunoreactive CD33 protein in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow and Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Patent 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. A biological sample can be obtained from any tissue or body fluid of a subject. In certain embodiments, the subject is in an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of CD33 protein in a sample obtained from the subject. In certain embodiments, the sample is selected from urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsy body tissue.

[0269] An immunoassay or sandwich assay is a form of the diagnostic method of the present technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use an antibody (e.g., an anti-CD33 antibody or a population of anti-CD33 antibodies) immobilized to a solid phase and another anti-CD33 antibody or a population of anti-CD33 antibodies in solution. Typically, the solution anti-CD33 antibody or population of anti-CD33 antibodies is labeled. If a population of antibodies is used, the population can contain antibodies that bind specifically to different epitopes within the target polypeptide. Thus, the same population can be used for both the solid phase and solution antibodies. If anti-CD33 monoclonal antibodies are used, a first and a second CD33 monoclonal antibody with different binding specificities are used for the solid phase and solution phases. The solid phase (also referred to as the "capture") and solution (also referred to as the "detection") antibodies can be contacted with the target antigen in any 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 incubated for a period of time, which typically ranges from about 10 minutes to about 24 hours and is usually about 1 hour. Then a washing step is performed to remove components in the sample that did not specifically bind to the anti-CD33 antibody used as the diagnostic reagent. When the solid phase antibody and solution antibody bind in separate steps, the washing can be performed after either or both binding steps. After washing, the binding is quantified, typically by detecting the label attached to the solid phase via the binding of the labeled solution antibody. Typically, for a given antibody pair or population of antibodies and given reaction conditions, a calibration curve is made from samples containing known concentrations of the target antigen. Then the concentration of immunoreactive CD33 protein in the sample being tested is read by interpolation from the calibration curve (i.e., the standard curve). The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the labeled solution antibody bound at a series of time points before equilibrium is reached. The slope of this curve is a measure of the concentration of CD33 protein in the sample.

[0270] Suitable supports for the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also include particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX TM (Amersham Pharmacia Biotech, Piscataway, New Jersey), etc. Immobilization can be achieved by absorption or by covalent attachment. Optionally, the anti-CD33 antibody can be linked to a linker molecule (such as biotin) to attach to a surface-bound linker (such as avidin).

[0271] In some embodiments, the present disclosure provides anti-CD33 antibodies of the present technology conjugated to diagnostic agents. Diagnostic agents can include radioactive or non-radioactive labels, contrast agents (such as for magnetic resonance imaging, computed tomography, or ultrasound), and radioactive labels can be γ, β, α, Auger electron, or positron-emitting isotopes. A diagnostic agent is a molecule conjugated and administered with an antibody moiety, i.e., an antibody or an antibody fragment or sub-fragment, and can be used to diagnose or detect a disease by localizing cells containing the antigen.

[0272] Useful diagnostic agents include, but are not limited to, radioactive isotopes, dyes (such as using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated with MRI enhancers, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from radioactive isotopes, enhancers for magnetic resonance imaging, and fluorescent compounds. To load the antibody component with a radioactive metal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail attached with a variety of chelating groups for binding the ion. Such a tail can be a polymer, such as polylysine, polysaccharide, or other derivatized or derivatizable chains, which have side groups capable of binding to chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamine, crown ether, bis-thiosemicarbazone, polyoxime, and similar groups known for this purpose. The chelate can be conjugated to the antibody of the present technology using standard chemical methods. The chelate is typically linked to the antibody through a group capable of forming a bond with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates with antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal-chelator combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs for use with diagnostic isotopes in radioimaging. The same chelates can be used for MRI when complexed with non-radioactive metals (such as manganese, iron, and gadolinium) and used with the CD33 antibody of the present technology.

[0273] Macrocyclic chelates such as NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylamine tetraacetic acid) are used together with various metals and radioactive metals (such as radionuclides of gallium, yttrium, and copper). Such metal-chelate complexes can be stabilized by sizing the ring to fit the metal of interest. Examples of other DOTA chelates include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HS G)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Ts cg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;(xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; and (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.;

[0274] Other cyclic chelators of interest for stable binding of radionuclides, such as 223 Ra for RAIT, are also contemplated, such as macrocyclic polyethers. B. Therapeutic use of the anti-CD33 antibody of the present technology

[0275] The immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology can be used to treat CD33-related cancers and Alzheimer's disease. Such treatment can be used in patients identified as having pathologically high levels of CD33 (e.g., those diagnosed by the methods described herein) or patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating a CD33-related cancer or Alzheimer's disease in a subject in need thereof, the method 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 with the antibodies of the present technology include, but are not limited to: AML, biphenotypic leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

[0276] The compositions of the present technology can be used in combination with other therapeutic agents useful for treating CD33-related cancers. For example, the antibodies 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, antiestrogenic drugs, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, alkaloids that inhibit cells, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents, and targeted biotherapeutic agents (e.g., therapeutic peptides described in US 6306832, WO 2012007137, WO2005000889, WO 2010096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic 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, temozolomide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprorelin, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, lapatinib, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, nitrogen mustard, bleomycin, microtubule poisons, annonaceous acetogenins, or combinations thereof.

[0277] The compositions of the present technology can optionally be administered to a subject in need thereof as a single bolus. Alternatively, the dosing regimen can include multiple administrations at different times after the appearance of the tumor.

[0278] The method for treating Alzheimer's disease can further include administering to the subject sequentially, separately, or simultaneously at least one additional therapy selected from the group consisting of donepezil, galantamine, memantine, rivastigmine, memantine extended release and donepezil (Namzaric), aducanumab, solanezumab, insulin, verubecestat, AADvac1, CSP-1103, and intepirdine.

[0279] Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal, intracranial, intratumoral, intrathecal or topical. Administration includes self-administration and administration by another person. It should also be understood that the various modes of treatment of the medical conditions described herein are intended to mean "substantial", which includes complete treatment but also less than complete treatment, and in which some biologically or medically relevant result is achieved.

[0280] In some embodiments, the antibodies of the technology comprise a pharmaceutical formulation, which can be administered to a subject in need thereof in one or more doses. The dosage regimen can be adjusted to provide the desired response (e.g., a therapeutic response).

[0281] Generally, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect ranges from about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Generally, the dosage range is from about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For the administration of anti-CD33 antibodies, the dosage range is from about 0.0001 to 100 mg / kg of subject body weight weekly, bi-weekly or tri-weekly, and more typically from 0.01 to 5 mg / kg of subject body weight. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight weekly, bi-weekly or tri-weekly, or in the range of 1-10 mg / kg weekly, bi-weekly or tri-weekly. In one embodiment, the single dosage of the antibody ranges from 0.1-10,000 micrograms per kilogram of body weight. In one embodiment, the concentration of the antibody in the vehicle ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens call for administration once every two weeks or once a month or once every 3 to 6 months. The anti-CD33 antibody can be administered at multiple occasions. The interval between individual dosages can be hourly, daily, weekly, monthly or annually. The interval can also be irregular as indicated by measuring the blood level of the antibody in the subject. In some methods, the dosage is adjusted to achieve the following serum antibody concentrations in the subject: 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 can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency vary according to the half-life of the antibody in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high dosages are sometimes required to be given at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement of the disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0282] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize to a tumor expressing CD33 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactive level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as percent injected dose per gram (%ID / g). The reference value can be calculated by measuring the radioactive level present in non-tumor (normal) tissue and calculating the mean radioactive level ± standard deviation present in non-tumor (normal) tissue. In some embodiments, the ratio of the radioactive level between the tumor and the 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.

[0283] In some embodiments, the subject is diagnosed with or suspected of having cancer. The radioactive level emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0284] 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 isotope that emits alpha particles, an isotope that emits beta particles, an Auger emitter, or any combination thereof. Examples of isotopes that emit beta particles include 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu. Examples of isotopes that emit alpha particles include 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At and 255Fm. 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., via an N297A mutation in the Fc region, which results in deglycosylation). The therapeutic efficacy of such an immunoconjugate can be determined by calculating the tumor: normal tissue ratio of the area under the curve (AUC). In some embodiments, the tumor: normal tissue ratio of the AUC of the immunoconjugate 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.

[0285] PRIT. In one aspect, the present disclosure provides a method for detecting a tumor in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a CD33 antigen, wherein the complex is configured to localize to a solid tumor expressing the CD33 antigen recognized by the bispecific antibody of the complex; and (b) detecting the presence of a solid tumor in the subject by detecting a radioactive level emitted by the complex that is above a reference value. In some embodiments, the subject is a human.

[0286] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the CD33 antigen recognized by the bispecific antibody of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pretargeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value. In some embodiments, the subject is a human.

[0287] Examples of DOTA haptens include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2;(xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2 and (xx) DOTA. The radioactive label can be an isotope that emits alpha particles, an isotope that emits beta particles, or an Auger emitter. Examples of radioactive labels 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.

[0288] In some embodiments of the methods disclosed herein, positron emission tomography or single photon emission computed tomography is used to detect the level of radioactivity emitted by the complex. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with, or suspected of having, Alzheimer's disease or a CD33-related cancer, such as AML, biphenotypic leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

[0289] Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intraventricularly, orally, intratumorally, or intranasally. In certain embodiments, the complex is administered into the cerebrospinal fluid or blood of a subject.

[0290] In some embodiments of the methods disclosed herein, the radioactive level emitted by the complex is detected between 2 and 120 hours after administration of the complex. In certain embodiments of the methods disclosed herein, the radioactive level emitted by the complex is expressed as percentage of injected dose per gram of tissue (%ID / g). The reference value can be calculated by measuring the radioactive level present in non-tumor (normal) tissue and calculating the mean radioactive level ± standard deviation present in non-tumor (normal) tissue. In some embodiments, the reference value is the standardized uptake value (SUV). See Thie JA, J Nucl Med. 45(9):1431-4(2004). In some embodiments, the ratio of the radioactive level 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.

[0291] In another aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with a CD33-related cancer, the method comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing the CD33 antigen target; and (b) administering to the subject an effective amount of a radioactively labeled DOTA hapten, wherein the radioactively labeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human.

[0292] The anti-DOTA bispecific antibody is administered under certain conditions and for a certain period of time (e.g., according to a dosing regimen) 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 radioactively labeled DOTA hapten is administered after a period of time sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0293] The radiolabeled DOTA hapten can be administered at any time between 1 minute and 4 days or more after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, 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, the radiolabeled DOTA hapten is administered. Alternatively, the radiolabeled DOTA hapten can be administered at any time after 4 days or more after administration of the anti-DOTA bispecific antibody.

[0294] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of a scavenger prior to administration of the radiolabeled DOTA hapten. The scavenger can be any molecule (dextran or dendrimer or polymer) capable of conjugating to the C825 hapten. In some embodiments, the scavenger is no more than 2000 kD, 1500 kD, 1000 kD, 900 kD, 800 kD, 700 kD, 600 kD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 10 kD or 5 kD. In some embodiments, the scavenger is 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.).

[0295] In some embodiments, the scavenger and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody of the technology. For example, in some embodiments, the anti-DOTA bispecific antibody of the technology is administered according to a regimen comprising at least one of the following cycles: (i) administering the anti-DOTA bispecific antibody of the technology (optionally, such that relevant tumor cells are saturated); (ii) administering the radiolabeled DOTA hapten and an optional scavenger; (iii) optionally additionally administering the radiolabeled DOTA hapten and / or the scavenger without additionally administering the anti-DOTA bispecific antibody. In some embodiments, the method may comprise multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0296] Additionally or alternatively, in some embodiments of the method, the anti-DOTA bispecific antibody and / or the radiolabeled DOTA hapten are administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intraventricularly, intratumorally, orally or intranasally.

[0297] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with a CD33-related cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the technology that recognizes and binds the radiolabeled DOTA hapten and a CD33 antigen target, wherein the complex is configured to localize to a tumor expressing the CD33 antigen target recognized by the bispecific antibody of the complex. The complex may be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intraventricularly, orally, intratumorally or intranasally. In some embodiments, the subject is a human.

[0298] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing the 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. The anti-DOTA bispecific antibody is administered under conditions and for a period of time (e.g., according to a dosing regimen) 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 of time sufficient to allow clearance of unbound anti-DOTA bispecific antibody. In some embodiments, the subject is a human.

[0299] Thus, in some embodiments, the method further comprises administering to the subject an effective amount of a scavenger prior to administering the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered at any time between 1 minute and 4 days or more 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 at any time after 4 days or more after administration of the anti-DOTA bispecific antibody.

[0300] The scavenger can be a 500 kD aminoglucan-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 scavenger and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one of the following cycles: (i) administering the anti-DOTA bispecific antibody of the present technology (optionally, such that the relevant tumor cells are saturated); (ii) administering the radiolabeled DOTA hapten and an optional scavenger; (iii) optionally additionally administering the radiolabeled DOTA hapten and / or the scavenger without additionally administering the anti-DOTA bispecific antibody. In some embodiments, the method may include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0301] Also provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds the radiolabeled DOTA hapten and the CD33 antigen target, wherein the complex is configured to localize to tumors expressing the CD33 antigen target 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. In some embodiments, the AUC tumor:AUC normal tissue ratio of the complex 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.

[0302] Toxicity. Optimally, an effective amount (e.g., dose) of the anti-CD33 antibody described herein will provide a therapeutic benefit without causing substantial toxicity to the subject. The toxicity of the anti-CD33 antibody described herein can be determined in cell culture or experimental animals by standard pharmaceutical procedures, such as by determining the LD 50 (dose lethal to 50% of the population) or LD 100Determined by the dose that is lethal to 100% of the population. The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic to humans. The doses of the anti-CD33 antibodies described herein are within the range of circulating concentrations that include effective doses with little or no toxicity. The doses can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dose can be chosen by an individual physician based on the condition of the subject. See, e.g., Fingl et al., The Pharmacological Basis of Therapeutics, Chapter 1 (1975).

[0303] Formulation of pharmaceutical compositions. According to the methods of the present technology, the anti-CD33 antibodies can be incorporated into a pharmaceutical composition suitable for administration. Pharmaceutical compositions generally comprise a recombinant or substantially purified antibody and a pharmaceutically acceptable carrier, in a form suitable for administration to a subject. The pharmaceutically acceptable carrier depends in part on the particular composition being administered and in part on the particular method used to administer the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th ed., 1990). Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the United States Food and Drug Administration.

[0304] The terms "pharmaceutically acceptable", "physiologically tolerable" and their grammatical variants, when they relate to compositions, carriers, diluents and reagents, are used interchangeably and denote that the materials are capable of being administered to a subject or on a subject without producing an undesirable physiological effect to an extent that would prohibit the administration of the composition. For example, "pharmaceutically acceptable excipient" means an excipient that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic and desirable, and includes excipients acceptable for veterinary as well as human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or in the case of aerosol compositions, gaseous. "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include those that can be formed when an acidic proton present in the composition is capable of reacting with an inorganic or organic base. 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 amine bases, for example ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc. Such salts also include acid addition salts formed with inorganic acids (such as hydrochloric acid and hydrobromic acid) and organic acids (such as acetic acid, citric acid, maleic acid and alkane sulfonic acids and aromatic sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxyl, sulfonyloxy and phosphonyloxy groups present in the anti-CD33 antibody, such as C 1-6 alkyl esters. When there are two acidic groups, the pharmaceutically acceptable salt or ester can be a monoacid monosalt or ester or a disalt or diester; and similarly, when there are more than two acidic groups, some or all of such groups can be salified or esterified. The anti-CD33 antibody named in this technology can exist in an unsalted or unesterified form, or in a salified and / or esterified form, and this naming of the anti-CD33 antibody is intended to include the initial (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Additionally, certain embodiments of this technology can exist in more than one stereoisomeric form, and this naming of the anti-CD33 antibody is intended to include all individual stereoisomers and all mixtures of such stereoisomers (whether racemic or otherwise). It is not difficult for a person of ordinary skill in the art to determine the appropriate timing, sequence and dosage for administering the specific drugs and compositions of this technology.

[0305] Examples of such carriers or diluents include but are not limited to water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and compounds for pharmaceutical active substances is well known in the art. Their use in the composition is contemplated unless any conventional medium or compound is incompatible with the anti-CD33 antibody. Supplementary active compounds can also be incorporated into the composition.

[0306] The pharmaceutical compositions of the present technology are formulated to be compatible with the intended route of administration. The anti-CD33 antibody compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; or intramuscular routes, or as an inhalant. The anti-CD33 antibody can be optionally administered in combination with other agents that are at least partially effective in treating various CD33-related cancers or Alzheimer's disease.

[0307] Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, 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 can be adjusted with an acid or a base (such as hydrochloric acid or sodium hydroxide). Parenteral preparations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0308] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it is easy to inject. Under the conditions of manufacture and storage, the composition must be stable and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium that contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. The action of microorganisms can be prevented 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, polyols (such as mannitol, sorbitol), sodium chloride. Prolonged absorption of injectable compositions can be achieved by including a compound that delays absorption, such as aluminum monostearate and gelatin.

[0309] A sterile injectable solution can be prepared by incorporating the anti-CD33 antibody of the present technology in a required amount into a suitable solvent which optionally has one or a combination of the ingredients listed above, followed by filtration sterilization. Generally, a dispersion is prepared by incorporating the anti-CD33 antibody into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional required ingredients from a previously sterile filtered solution. The antibodies of the present technology can be administered in the form of depot injections or implant formulations, which can be formulated in a manner that allows for sustained or pulsed release of the active ingredient.

[0310] Oral compositions generally include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the anti-CD33 antibody can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier as a mouthwash, wherein the compound in the fluid carrier is orally administered and swished and spat out or swallowed. Pharmaceutically compatible binding compounds and / or auxiliary materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose, disintegrating compounds such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening compounds such as sucrose or saccharin; or flavoring compounds such as peppermint, methyl salicylate, or orange flavor.

[0311] For administration by inhalation, the anti-CD33 antibody is delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, such as a gas (e.g., carbon dioxide), or a nebulizer.

[0312] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by using a nasal spray or a suppository. For transdermal administration, the anti-CD33 antibody is formulated into an ointment, an ointment, a gel, or a cream as is commonly known in the art.

[0313] The anti-CD33 antibody can also be prepared as a pharmaceutical composition in the form of a suppository (e.g., together with conventional suppository bases such as cocoa butter and other glycerides) or a retention enema for rectal delivery.

[0314] In one embodiment, the anti-CD33 antibody is prepared with a carrier that prevents the rapid clearance of the anti-CD33 antibody from the body, such as a controlled release formulation, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. Such materials are also commercially available from Alza Corporation and NovaPharmaceuticals, Inc. Liposome suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as the method described in U.S. Patent No. 4,522,811. C. Kit

[0315] The present technology provides a kit for detecting and / or treating CD33-related cancers or Alzheimer's disease, the kit comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant). Optionally, the above components of the kit of the present technology are packaged in a suitable container and labeled for the diagnosis and / or treatment of CD33-related cancers or Alzheimer's disease. The above components can be stored in unit containers or multi-dose containers (e.g., sealed ampoules, vials, bottles, syringes, and test tubes) in the form of an aqueous solution (preferably a sterile solution) or a lyophilized (preferably sterile) formulation for reconstitution. The kit can also include a second container that contains a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and saline solutions. In addition, the kit can include instructions for diluting the pharmaceutical composition and / or instructions for administering the diluted or undiluted pharmaceutical composition. The container can be made of a variety of materials (such as glass or plastic) and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous injection needle). The kit can also include additional containers that contain pharmaceutically acceptable buffers, such as phosphate buffered saline, Ringer's solution, and dextrose solution. The kit can also include other materials that are desirable from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, media for one or more suitable hosts. The kit can optionally include instructions typically included in the commercial packaging of a therapeutic or diagnostic product, the instructions containing information about, for example, indications, usage, dosage, manufacture, administration, contraindications, and / or warnings regarding the use of such a therapeutic or diagnostic product.

[0316] The kit can be used to detect the presence of immunoreactive CD33 protein in a biological sample, which can be any body fluid, including but not limited to, for example, serum, plasma, lymph fluid, cyst fluid, urine, feces, cerebrospinal fluid, ascites, or blood, and includes biopsy samples of body tissues. For example, the kit can comprise: one or more humanized, chimeric, or bispecific anti-CD33 antibodies (or antigen-binding fragments thereof) of the present technology that are capable of binding to CD33 protein in the biological sample; means for determining the amount of CD33 protein in the sample; and means for comparing the amount of immunoreactive CD33 protein in the sample with a standard. One or more of the anti-CD33 antibodies can be labeled. The kit components (such as reagents) can be packaged in suitable containers. The kit can also contain instructions for using the kit to detect immunoreactive CD33 protein.

[0317] For an antibody-based kit, the kit can comprise, for example, 1) a first antibody attached to a solid support, such as a humanized, chimeric, or bispecific CD33 antibody (or antigen-binding fragment thereof) of the present technology, which binds to CD33 protein; and, optionally; 2) a different second antibody that binds to CD33 protein or the first antibody and is conjugated to a detectable label.

[0318] The kit can also comprise, for example, buffers, preservatives, or protein stabilizers. The kit can also contain other components required to detect the detectable label, such as enzymes or substrates. The kit can also contain a control sample or a series of control samples that can be assayed and compared with the test sample. Each component of the kit can be encapsulated in a separate container, and all the different containers can be placed together in a single package along with instructions for interpreting the results of the assay using the kit. The kit of the present technology can contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, such as for detecting CD33 protein in vitro or in vivo, or for treating CD33-related cancers in a subject in need. In certain embodiments, the reagents can be used according to the methods of the present technology. Examples

[0319] 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 illustrative anti-CD33 antibodies of the present technology. The following examples demonstrate the production of chimeric, humanized, and bispecific antibodies of the present technology, and the characterization of their binding specificities and in vitro and in vivo biological activities. Example 1: Structure and binding affinity of chimeric and humanized anti-CD33 antibodies of the present disclosure

[0320] Humanization of murine HIM34. The CDRs of the heavy and light chains of HIM34 were transplanted onto a human IgG1 framework based on their homology to the human framework IGHV1-2*02 IGHJ4*01 (for VH), IGKV1-33*01-IGKJ2*01 (for VL), respectively. Thirty forms of huHIM34 were gene synthesized based on six heavy chains and five light chains and expressed in DG44 cells. The amino acid sequences of murine, chimeric, and humanized HIM34 variable heavy chains are shown in SEQ ID NOs 2-7 and 114, and Figure 8 and FIG. 10(B). The amino acid sequences of murine, chimeric, and humanized HIM34 variable light chains are shown in SEQ ID NOs. 9-13 and 115, and Figure 9 and FIG. 10(A).

[0321] Generation of CD33-BsAb bispecific antibody. The CD33-BsAb was designed in the IgG-scFv format ( Figure 3 ). The CD33-BsAb was constructed by fusing the humanized OKT3 scFv to the C-terminus of the light chain of a chimeric or humanized anti-CD33 antibody via a (G4S)3 linker, as previously described in: Xu H et al., Cancer Immunology Research 3:266-277 (2015) and Lopez-Albaitero A et al., OncoImmunology 6:e1267891 (2017). N297A and K322A mutations were introduced into the Fc region of the antibody to abrogate FcR and complement binding activities, 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 construct was then transfected into CHO-S cells, and stable clones were selected for high antibody production levels. For larger-scale antibody purification, the selected stable clones were amplified in shake flasks. The bispecific antibody was purified from the supernatant using one-step protein A affinity chromatography.

[0322] FIG. 10 shows the amino acid sequences of the light and heavy chains of the chimeric CD33-BsAb (chHIM34-BsAb), which correspond to SEQ ID NO:14 and SEQ ID NO:16, respectively. Figure 11(A) to Figure 11(B) 、 Figure 11(C) to Figure 11(D) 、 Figure 12(A) to Figure 12(B) and Figure 13(A) to Figure 13(B)The light and heavy chains of the following four humanized CD33-BsAbs (hHIM34-BsAbs) are shown: BC249, BC275, BC267, and BC268, respectively. Figure 11(A) to Figure 11(B) The amino acid sequences of the light and heavy chains of BC249 are shown, which correspond to SEQ ID NO:18 and SEQ ID NO:20, respectively, and are based on the humanized HIM34 variable light chain VL-3 and variable heavy chain VH-5. Figure 11(C) to Figure 11(D) The amino acid sequences of the light and heavy chains of BC275 are shown, which correspond to SEQ ID NO:134 and SEQ ID NO:136, respectively, and are based on the humanized HIM34 variable light chain VL-4 and variable heavy chain VH-5.

[0323] Figure 12(A) to Figure 12(B) The amino acid sequences of the light and heavy chains of BC267 are shown, which correspond to SEQ ID NO:18 and SEQ ID NO:22, respectively, and are based on the humanized HIM34 variable light chain VL-3 and variable heavy chain VH-6. Figure 13(A) to Figure 13(B) The amino acid sequences of the light and heavy chains of BC268 are shown, which correspond to SEQ ID NO:24 and SEQ ID NO:22, respectively, and are based on the humanized HIM34 variable light chain VL-4 and variable heavy chain VH-6.

[0324] Comparing the binding kinetics of chimeric and humanized forms of anti-CD33 antibodies by surface plasmon resonance. To determine whether the humanized anti-CD33 HIM34 antibody binds CD33 with an affinity equal to that of the chimeric form, surface plasmon resonance assays were performed using a Biacore T-200. The Biacore T-200 biosensor, CM5 sensor chip, and related reagents were purchased from GE Healthcare. CD33 as the active surface and a blank as the reference were immobilized onto the CM5 sensor chip at approximately 1000 RU (response units) using an amine coupling kit (GE Healthcare). The 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) and then analyzed. The samples were injected onto the sensor surface at a flow rate of 30 μL / min for 1 min. After the association phase was completed, dissociation was monitored in HBS-EP buffer at the same flow rate for 3 min, or for antibodies with slow koff, dissociation was monitored at 37 °C for 30 min. At the end of each cycle, the surface was regenerated with 10 mM NaOH at a flow rate of 50 μL / min for 30 s. The biosensor curves obtained after injecting the samples onto the active surface were subtracted from the control curves obtained from the samples injected onto the reference surface, and then kinetic analysis was performed. The data were analyzed using the Biacore T-200 evaluation software. As Figure 16(A) to Figure 16(B) shown, the humanized anti-CD33 HIM34 antibody binds with a k on and k off similar to that of the chimeric form.

[0325] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to the CD33 antigen with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect CD33 protein in a sample. Example 2: Biological activity of the T cell-engaging CD33-BsAb antibody of the present technology

[0326] The chimeric CD33-BsAb binds to AML cell lines. The binding of chHIM34 BsAb to target cells was tested by FACS immunostaining. It binds to the CD33(+) AML cell lines U937, THP1, SET2, C1498-CD33, and M-07e, without harming the CD33(-) leukemia cell line CMLT1( Figure 4 ).

[0327] The chimeric CD33-BsAb binds to both the splice variant and full-length CD33. The full-length CD33 molecule has two extracellular domains: the membrane-distal IgV and the membrane-proximal IgC2 domain( Figure 1 ). There are no FDA-approved antibodies that bind to the CD33 splice variant lacking the IgV domain( Figure 2 ). Importantly, since 50% of AML patients have the spliced CD33 protein, they cannot benefit from current CD33 antibody drugs. The chimeric CD33-BsAb of the present disclosure, namely chHIM34 BsAb, binds to both the full-length and splice variant of CD33, while the BsAb based on humanized M195 (BC133) does not bind to the splice variant CD33( Figure 5(A) to Figure 5(B) ). Figure 19 The stability of the humanized BsAb (hHIM34 x CD3) of the present technology at 40 °C as determined by HPLC is shown. Among all the tested clones, the hHIM34L4H5 (BC275) clone showed the highest stability at 40 °C.

[0328] The chimeric CD33-BsAb redirects T cells to kill CD33(+) AML cell lines. To evaluate whether the chimeric CD33-BsAb can redirect T cells to kill leukemia cells, the cytotoxicity of T cells against CD33(+) AML cells was tested in a standard 4-hour 51Cr release assay. Significant killing of the AML cell line was observed when chHIM34 BsAb was present, with an EC50 as low as 1 fM (for C1498-CD33) to 200 fM (for THP1)( Figure 6(A) to Figure 6(B) ).

[0329] The binding kinetics of the chimeric and humanized forms of the HIM34 bispecific antibody were compared by surface plasmon resonance. The affinity of the humanized HIM34BsAb for CD33 was tested using surface plasmon resonance technology. The results are shown below: ka (1 / Ms) kd (1 / s) KD (M) chH34 914000 0.000117 1.28E-10 L3H4 1840000 0.00175 9.55E-10 L4H1 4950000 0.00597 1.21E-09 L4H4 4500000 0.00743 1.65E-09 L4H5 692000 0.000154 2.22E-10 L4H7 861000 0.00017 1.98E-10

[0330] The mean fluorescence intensity (MFI) is a reliable criterion for predicting the in vitro potency of the CD33 antibodies of the present technology. To test the binding of the humanized HIM34 BsAb, MOLM13 CD33(+) cells were contacted with different concentrations of the humanized anti-CD33 BsAb disclosed herein. The flow cytometry results are shown in Figure 17 . The cytotoxicity of the humanized BsAb against MOLM13 cells was tested in a T cell-dependent cytotoxicity assay (TDCC). As shown in Figure 18(A) to Figure 18(B)As shown, clones with excellent binding to CD33 (higher MFI) are more effective (lower EC50). These results demonstrate that MFI is a reliable criterion for predicting the in vitro potency of CD33 antibodies of the present technology.

[0331] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically target CD33-positive cancer cells. Accordingly, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in a subject in need thereof. Example 3: In vivo therapeutic study using CD33-BsAb

[0332] Patients with mutations in the FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) (FLT3 / ITD) generally have a poor prognosis. In pediatric AML, the negative consequences of these mutations are more prominent (Levis and Small, Leukemia 17:1738-52 (2003)). To evaluate whether chimeric CD33-BsAb can redirect T cells to AML with FLT3 / ITD mutations, in vivo experiments were performed using NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ (NSGS) mice. The mice were randomized into 4 groups and all received 1 million MOLM13 cells containing the firefly luciferase gene: Group 1: Activated T cells only (ATC); Group 2: ATC plus chHIM34 BsAb (0.1 μg / dose); Group 3: ATC plus control BsAb (0.1 μg / dose); and Group 4: ATC plus chHIM34 BsAb (0.01 μg / dose). Treatment began on day 7 when leukemia was established. Within three weeks, the mice received a weak injection of 10 million ATC mixed with BsAb. The BsAb was administered twice / week, once mixed with ATC and once alone. A total of five doses of BsAb were injected. To support in vivo T cell growth, 1000 IU of interleukin 2 was administered subcutaneously twice / week. Bioluminescence imaging was performed weekly to evaluate the leukemia burden. As Figure 7(A) to Figure 7(B) shown, administration of ATC in the presence of 100 or 10 ng / dose chHIM34 BsAb treated the leukemic mice. However, 100 ng / dose of control BsAb plus ATC had no anti-tumor effect, which was similar to the ATC-only group.

[0333] Accordingly, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in a subject in need thereof. Example 4: Activity comparison of the anti-CD33 BsAb of the present technology with existing CD33 antibodies

[0334] The BC275 (CD3×CD33) BsAb disclosed herein binds to the membrane-proximal domain (IgC) of the extracellular region of CD33. See Figure 20(A) to Figure 20(B) . For other examples of existing CD3×CD33 BsAbs, BC133 based on the M195 clone (lintuzumab) and BC269 based on My96 (gemtuzumab) 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 their binding to CD33. A secondary antibody was used to detect the binding of the BsAb to the human cancer cell lines. As shown in Figure 20(C), all three BsAbs bound to CD33(+) cells but did not react with CD33(−) cells. Activated human T cells were mixed with various CD33(+) or CD33(−) (NALM6) cancer cells to evaluate the T cell-dependent cell-mediated cytotoxicity (TDCC) of the three CD3×CD33 BsAbs. As Figure 21(A) to Figure 21(C) shown, all three BsAbs effectively lysed leukemia cells presenting CD33(+) but did not harm CD33(−) NALM6 cells. These results demonstrate that CD3×CD33 BsAbs selectively target CD33(+) cancers. In addition, there was a positive correlation between the level of CD33 expression on cancer cells and the potency of the BsAb in the TDCC assay (pooled data for all three CD3×CD33 BsAbs are shown in Figure 21(D)).

[0335] Several animal experiments were conducted to evaluate and compare the in vivo potency of the three BsAbs. In the first experiment, immunodeficient NSG mice were intravenously inoculated with CD33(+) MOLM13-luciferase (which has the CC genotype of rs12459419 SNP) human AML xenografts (10 6 cells). Treatment was started 11 days later ( Figure 22(A) to Figure 22(B) ). The mice were given a single injection of 5×10 6 activated T cells and 0.025 μg of the test BsAb (10 μg / Kg / dose). The timing of T cell and antibody injections is shown in Figure 22(B). In the control group, the mice were given T cells plus BC119 (CD3×GD2 BsAb). The injection of the individual BsAb was continued for 4 more doses. The growth of leukemia was monitored using an IVIS bioluminescence imager. All three CD3×CD33 BsAbs redirected T cells to treat mice inoculated with AML cells, while mice receiving the control BsAb and T cells showed leukemia progression ( Figure 22(A) to Figure 22(B) ).

[0336] Next, immunodeficient NSG mice were intravenously inoculated with human CD33-transduced NALM6-luciferase leukemia xenografts (0.5×10 6 cells). Treatment was initiated 3 days later (Figure 23(A)). Mice were given two injections of activated T cells (2.7×10 6 and 5×10 6 , with a one-week interval between doses) and 10 ng BsAb / 10 6 T cells. In the control group, mice were given T cells plus 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 imager. To support T cell engraftment, all mice received two subcutaneous injections of interleukin 2 (1000 IU) during the first week of treatment. Figure 23(C) shows the survival curves of mice treated with three CD3×CD33 BsAbs. The most effective BsAb was BC275 (the CD33 antibody of the present technology), which promoted a higher degree of survival compared to the conventional BC269 and BC133 BsAbs (which showed equivalent effects).

[0337] The next experiment was conducted on immunodeficient DKO mice subcutaneously inoculated with THP1 human leukemia xenografts (which have the CC genotype of rs12459419 SNP). Treatment was initiated 14 days later ( Figure 24(A) to Figure 24(D) ). Mice were given two injections of activated T cells (10 7 , with a one-week interval between doses) and 0.05 μg or 0.5 μg BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). In the control group, mice were given T cells plus 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 received two subcutaneous injections of interleukin 2 (1000 IU) twice a week for the first three weeks after T cell injection. The most effective BsAb was BC275 (the CD33 antibody of the present technology), which showed the highest degree of tumor reduction compared to the conventional BC269 and BC133 BsAbs (i.e., BC275>BC269>BC133).

[0338] Animal experiments were conducted on immunodeficient DKO mice subcutaneously inoculated with K562 human leukemia xenografts (which have the TT genotype of rs12459419 SNP). Treatment was initiated 7 days later ( Figure 25(A) to Figure 25(D) ). Mice were given three injections of activated T cells (10 7, at intervals of one week between doses) and 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose). In the control group, mice were given T cells plus BC119 (CD3×GD2 BsAb). The timing of T cell and antibody injections is shown in Fig. 25(A). Leukemia progression was monitored by measuring tumor size. To support T cell engraftment, all mice received 2 subcutaneous injections of interleukin 2 (1000 IU) per week for the three weeks prior to T cell injection. The most effective BsAb was BC275 (the CD33 antibody of the present technique), and BC275 showed the highest degree of tumor reduction compared to the conventional BC269 and BC133 BsAbs (i.e., BC275 > BC269 > BC133).

[0339] Thus, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in subjects in need thereof. Example 5: Use of the anti-CD33 BsAb of the present technology in PRIT

[0340] IgG-based CD33-C825 BsAb. CD33(+) leukemia cells were injected into animals subcutaneously, intraperitoneally, intravenously, or via other routes. After tumor establishment (depending on the tumor type and injection route), treatment will begin. Treatment consists of one or more cycles. Each cycle includes administering the test BsAb (250 μg intravenously), followed 24 to 48 hours later by injection of a clearing agent (DOTA dextran or DOTA dendrimer; the dose is 5%-15% of the BsAb dose, see Cheal SM et al., Mol Cancer Ther 13:1803-12, 2014). Four hours later, DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi) will be injected intravenously. Generally, DOTA- 225 Ac is more effective than DOTA- 177 Lu and may require fewer cycles to eradicate the tumor.

[0341] Tetramerized BsAb. CD33(+) leukemia cells were injected into animals subcutaneously, intraperitoneally, intravenously, or via other routes, and after tumor establishment (depending on the type and injection route of the tumor), treatment will begin. Treatment consists of one or more cycles. Each cycle consists of: administering BsAb (250 μg intravenously), followed 24 - 48 hours later by intravenous injection of DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi). Generally, DOTA- 225 Ac is more effective than DOTA-177 Lu is more effective and may require fewer cycles to eradicate the tumor.

[0342] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit the progression of tumor growth and / or metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in subjects in need thereof. Example 6: Use of the anti-CD33 BsAb of the present technology in the treatment of Alzheimer's disease

[0343] A variety of rodent models for Alzheimer's disease (AD) have been developed (Nazem et al., J Neuroinflammation 17; 12:74 (2015)). Since the HIM34 antibody binds to human CD33 and does not cross-react with mouse CD33, a knock-in mouse model that produces human CD33 (hCD33) will be generated. 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 study the therapeutic effects of the CD33 antibodies or antigen-binding fragments of the present technology in vivo.

[0344] In one experiment, the anti-CD33 BsAb comprising the C825 anti-DOTA binding moiety disclosed herein is intracranially injected or injected into the cerebrospinal fluid, followed by intravenous injection of a radio-labeled metal-DOTA conjugate, such as 177 Lu-DOTA-Bn. In addition to behavioral analysis of the mice, histopathological evaluation of the brain tissue over time is used as an outcome measure. It is expected that animals receiving the anti-CD33 BsAb of the present technology will show improvement in one or more symptoms of Alzheimer's disease (AD) compared to those observed in untreated controls.

[0345] Accordingly, the immunoglobulin-related compositions disclosed herein can be used to treat Alzheimer's disease in subjects in need thereof. Equivalent content

[0346] The technology of the present invention is not limited to the specific embodiments described in this application, which are intended to be illustrative of the individual aspects of the technology of the present invention. As will be apparent to those skilled in the art, many modifications and changes can be made to the technology of the present invention without departing from the spirit and scope of the technology of the present invention. As will be clear to those skilled in the art from the foregoing description, there are functionally equivalent methods and devices within the scope of the technology of the present invention in addition to the methods and devices enumerated herein. Such modifications and changes are intended to fall within the scope of the technology of the present invention. It is to be understood that the technology of the present invention is not limited to the specific methods, reagents, compounds, compositions or biological systems, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0347] In addition, where features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is thus also described in terms of any individual member or subgroup of members of the Markush group.

[0348] 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 also cover any and all possible subranges and combinations of subranges thereof. Any recited range can be readily recognized as fully describing the same range and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into lower third, middle third, and upper third, etc. Also as will be understood by those skilled in the art, all such words as "up to", "at least", "greater than", "less than", etc. include the recited numbers and refer to ranges that can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so on.

[0349] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent that they are not inconsistent with the explicit teachings of this specification.

[0350] More specifically, the present application provides the following: 1. An antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ), wherein (a) said V H comprises a V H -CDR1 sequence of GYSFTDYN (SEQ ID NO:154), a V H -CDR2 sequence of IDPYKGGT (SEQ ID NO:155), and a V (SEQ ID NO:156) of V H -CDR3 sequence; and (b) said V L comprises a V L -CDR1 sequence of QDINKY (SEQ ID NO:157), a V L-CDR2 sequence and V of LQYDNLLT (SEQ ID NO:159) L -CDR3 sequence. 2. An antibody or antigen-binding fragment thereof comprising a heavy-chain immunoglobulin variable domain (V H ) and a light-chain immunoglobulin variable domain (V L ), wherein: (a) The V H comprises an amino acid sequence selected from: 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) The V L comprises an amino acid sequence selected from: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. 3. The antibody or antigen-binding fragment according to item 1 or 2, further comprising an Fc domain of an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. 4. The antibody according to item 3, comprising an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A. 5. The antibody according to item 3, comprising an IgG4 constant region containing an S228P mutation. 6. The antigen-binding fragment according to item 1 or 2, wherein the antigen-binding fragment is selected from Fab, F(ab’)2, Fab’, scF v and F v . 7. The antibody or antigen-binding fragment according to any one of items 1-6, wherein the antibody or antigen-binding fragment binds to the IgC2 domain of CD33. 8. The antibody according to any one of items 1-5 or 7, wherein the antibody is a monoclonal antibody, chimeric antibody, humanized antibody or bispecific antibody. 9. An antibody comprising SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, The heavy chain (HC) amino acid sequence of 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 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, The light chain (LC) amino acid sequence of SEQ ID NO: 140 or a variant thereof having one or more conservative amino acid substitutions. 10. The antibody according to item 9, which comprises an HC amino acid sequence and an LC amino sequence selected from the following respectively: SEQ ID NO: 16 and SEQ ID NO: 14 (chHIM34 x CD3 BsAb); SEQ ID NO: 20 and SEQ ID NO: 18 (BC249-hHIM34 x CD3 BsAb); SEQ ID NO: 136 and SEQ ID NO: 134 (BC275-hHIM34 x CD3 BsAb); SEQ ID NO: 22 and SEQ ID NO: 18 (BC267-hHIM34 x CD3 BsAb); SEQ ID NO: 22 and SEQ ID NO: 24 (BC268-hHIM34 x CD3 BsAb); SEQ ID NO: 20 and SEQ ID NO: 26 (VL3VH5 x mC825); SEQ ID NO: 20 and SEQ ID NO: 27 (VL3VH5 x hC825); SEQ ID NO: 22 and SEQ ID NO: 26 (VL3VH6 x mC825); SEQ ID NO: 22 and SEQ ID NO: 27 (VL3VH6 x hC825); SEQ ID NO: 22 and SEQ ID NO: 28 (VL4VH6 x mC825); SEQ ID NO: 22 and SEQ ID NO: 29 (VL4VH6 x hC825); 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). 11. An antibody comprising (a) a light - chain immunoglobulin variable domain sequence that is at least 95% identical to the light - chain immunoglobulin variable domain sequence of any one of SEQ ID NO:9, 10, 11, 12, or 13; and / or (b) a heavy - chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy - chain immunoglobulin variable domain sequence of any one of SEQ ID NO:2, 3, 4, 5, 6, 7, or 133. protein variable domain sequence. 12. An antibody comprising: (a) an LC sequence that is at least 95% identical to the LC sequence present in any one of SEQ ID NO:14, 18, 24, 26, 27, 28, 29, 134, 138, or 140; and / or (b) an HC sequence that is at least 95% identical to the HC sequence present in any one of SEQ ID NO:16, 20, 22, 136, 139, or 141. 13. The antibody according to any one of items 9 - 12, wherein the antibody is a chimeric antibody, a humanized antibody, or a bispecific antibody. 14. The antibody according to any one of items 9 - 13, wherein the antibody binds to the IgC2 domain of CD33. 15. The antibody according to any one of items 9 - 14, wherein the antibody comprises an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A. 16. The antibody according to any one of items 9 - 14, wherein the antibody comprises an IgG4 constant region containing the 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 NO.30 - 113 or 142 - 153. 18. The antibody or antigen - binding fragment according to item 17, wherein the antibody or antigen - binding fragment comprises an amino acid sequence selected from any one of SEQ ID NO.30 - 113 or 142 - 153. 19. A recombinant nucleic acid sequence encoding an antibody or antigen-binding fragment according to any one of items 1-18. 20. A recombinant nucleic acid sequence selected from: SEQ ID NO:15, 17, 19, 21, 23, 25, 135, and 137. 21. A host cell or vector comprising the recombinant nucleic acid sequence according to item 19 or item 20. 22. A composition comprising an antibody or antigen-binding fragment according to any one of items 1-8 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from: isotopes, dyes, chromogens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. 23. A composition comprising an antibody according to any one of items 9-18 and a pharmaceutically acceptable carrier, wherein the antibody is optionally conjugated to an agent selected from: isotopes, dyes, chromogens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. 24. The antibody according to any one of items 1-5, 7, or 8, wherein the antibody lacks α-1,6-fucose modification. 25. The antibody according to any one of items 9-16, wherein the antibody lacks α-1,6-fucose modification. 26. The bispecific antibody according to item 8 or 13, wherein the bispecific antibody binds to T cells, B cells, myeloid cells, plasma cells, or mast cells. 27. The bispecific antibody or antigen-binding fragment according to item 8, 13, 17, or 18, wherein 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, TCRγ / δ, NKp46, KIR, or a small molecule DOTA hapten. 28. A method for treating a CD33-related cancer or Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody comprising an HC amino acid sequence and an LC amino acid sequence, the HC amino acid sequence and the LC amino acid sequence each independently comprising a sequence selected from: SEQ ID NO:16 and SEQ ID NO:14 (chHIM34 x CD3 BsAb); SEQ ID NO: 20 and SEQ ID NO: 18 (BC249-hHIM34 x CD3 BsAb); SEQ ID NO: 136 and SEQ ID NO: 134 (BC275-hHIM34 x CD3 BsAb); SEQ ID NO: 22 and SEQ ID NO: 18 (BC267-hHIM34 x CD3 BsAb); SEQ ID NO: 22 and SEQ ID NO: 24 (BC268-hHIM34 x CD3 BsAb); SEQ ID NO: 20 and SEQ ID NO: 26 (VL3VH5 x mC825); SEQ ID NO: 20 and SEQ ID NO: 27 (VL3VH5 x hC825); SEQ ID NO: 22 and SEQ ID NO: 26 (VL3VH6 x mC825); SEQ ID NO: 22 and SEQ ID NO: 27 (VL3VH6 x hC825); SEQ ID NO: 22 and SEQ ID NO: 28 (VL4VH6 x mC825); SEQ ID NO: 22 and SEQ ID NO: 29 (VL4VH6 x hC825); 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), wherein said antibody specifically binds to CD33. 29. A method for treating a CD33-related cancer or Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a bispecific antibody or antigen-binding fragment, the bispecific antibody or antigen-binding fragment comprising an amino acid sequence selected from any one of SEQ ID NO. 30-113 or 142-153. 30. The method according to item 28 or 29, wherein the CD33-related cancer is acute myeloid leukemia (AML), biphenotypic leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia. 31. The method according to any one of items 28 - 30, wherein the antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent. 32. The method according to item 31, wherein the additional therapeutic agent is one or more of the following: alkylating agent, platinum agent, taxane, vinca agent, antiestrogen drug, aromatase inhibitor, ovarian inhibitor, VEGF / VEGFR inhibitor, EGF / EGFR inhibitor, PARP inhibitor, alkaloid that inhibits cells, cytotoxic antibiotic, antimetabolite, endocrine / hormone agent, bisphosphonate therapeutic agent. 33. A method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of the antibody or antigen-binding fragment according to any one of items 1 - 18, wherein the antibody is configured to localize to a tumor expressing CD33 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactive level emitted by the antibody or antigen-binding fragment that is higher than a reference value. 34. The method according to item 33, wherein the subject is diagnosed with or suspected of having cancer or Alzheimer's disease. 35. The method according to item 33 or 34, wherein the radioactive level emitted by the antibody or antigen-binding fragment is detected using positron emission tomography or single photon emission computed tomography. 36. The method according to any one of items 33 - 35, further comprising administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen-binding fragment according to any one of items 1 - 18 conjugated to a radionuclide. 37. The method according to item 36, wherein the radionuclide is an isotope that emits alpha particles, an isotope that emits beta particles, an Auger emitter, or any combination thereof. 38. The method according to item 37, wherein the isotope that emits beta particles is selected from 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and67 Cu. 39. A kit, comprising the antibody or antigen-binding fragment according to any one of items 1-18 and an instruction manual. 40. The kit according to item 39, wherein the antibody or antigen-binding fragment according to any one of items 1-18 is conjugated to at least one detectable label selected from a radiolabel, a fluorescent label, and a chromogenic label. 41. The kit according to item 39 or 40, further comprising a secondary antibody that specifically binds to the antibody according to any one of items 1-18. 42. The bispecific antibody or antigen-binding fragment according to item 8, 13, 17, or 18, wherein the bispecific antibody binds to a radiolabeled DOTA hapten and a CD33 antigen. 43. A method for selecting a subject for pretargeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment according to item 42, wherein the complex is 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 when the level of radioactivity emitted by the complex is higher than a reference value. 44. A method for increasing the sensitivity of tumors to radiotherapy in a subject diagnosed with CD33-related cancer, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment according to item 42, wherein the complex is configured to localize to tumors expressing CD33. 45. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment according to item 42, wherein the complex is configured to localize to tumors expressing CD33. 46. A method for increasing the sensitivity of tumors to radiotherapy in a subject diagnosed with CD33-related cancer, the method comprising (a) administering an effective amount of the bispecific antibody or antigen-binding fragment according to item 42, wherein the bispecific antibody or antigen-binding fragment is configured to localize to tumors expressing CD33; 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 bispecific antibody or antigen-binding fragment. 47. A method for treating cancer in a subject in need thereof, the method comprising (a) Administering an effective amount of the bispecific antibody or antigen-binding fragment according to item 42, wherein the bispecific antibody or antigen-binding fragment is configured to localize to tumors expressing CD33; 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 bispecific antibody or antigen-binding fragment. 48. The method according to item 46 or 47, further comprising administering to the subject an effective amount of a scavenger before administering the radiolabeled DOTA hapten. 49. The method according to any one of items 43-48, wherein the subject is a human. 50. The method according to any one of items 43-45, wherein the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intraventricularly, orally, intratumorally, or intranasally. 51. The method according to any one of items 43-50, wherein the radiolabeled DOTA hapten comprises an isotope emitting alpha particles, an isotope emitting beta particles, or an Auger emitter. 52. The method according to any one of items 43-51, wherein the radiolabeled DOTA hapten comprises 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. 53. A bispecific antigen-binding fragment comprising a first polypeptide chain, wherein: The first polypeptide chain comprises, in the N-terminal to C-terminal direction: i. The heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. A flexible peptide linker comprising the amino acid sequence (GGGGS)6; iii. The light chain variable domain of the first immunoglobulin; iv. A flexible peptide linker comprising the amino acid sequence (GGGGS)4; v. The heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; vi. A flexible peptide linker comprising the amino acid sequence (GGGGS)6; vii. The light chain variable domain of the second immunoglobulin; viii. A flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and ix. A self-assembling degrading (SADA) polypeptide; wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. 54. A bispecific antigen-binding fragment comprising a first polypeptide chain, wherein: The first polypeptide chain comprises, in the N-terminal to C-terminal direction: i. The light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. A flexible peptide linker comprising the amino acid sequence (GGGGS)6; iii. The heavy chain variable domain of the first immunoglobulin; iv. A flexible peptide linker comprising the amino acid sequence (GGGGS)4; v. The heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; vi. A flexible peptide linker comprising the amino acid sequence (GGGGS)6; vii. The light chain variable domain of the second immunoglobulin; viii. A flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and ix. A self - assembling degrading (SADA) polypeptide; wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. 55. The antigen - binding fragment according to item 53 or 54, wherein the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. 56. The antigen - binding fragment according to item 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. The antigen - binding fragment according to any one of items 53 - 56, wherein the antigen - binding fragment comprises an amino acid sequence selected from SEQ ID NO.30 - 113 or 142 - 153. 58. A bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other, and wherein: a. Each of the first polypeptide chain and the fourth polypeptide chain comprises, in the N - terminal to C - terminal direction: i. The light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. The light chain constant domain of the first immunoglobulin; iii. A flexible peptide linker comprising the amino acid sequence (GGGGS)3; and iv. The light chain variable domain of the second immunoglobulin linked to the complementary heavy chain variable domain of the second immunoglobulin, or the heavy chain variable domain of the second immunoglobulin linked to the complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and b. Each of the second polypeptide chain and the third polypeptide chain comprises, in the N-terminal to C-terminal direction: i. The heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; ii. The heavy chain constant domain of the first immunoglobulin; and wherein the heavy chain variable domain of the first immunoglobulin is selected from: 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: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. 59. The bispecific antibody according to 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, TCRγ / δ, NKp46, KIR or a small molecule DOTA hapten.

Claims

1. An antibody or an antigen-binding fragment thereof comprising a heavy-chain immunoglobulin variable domain (V H ) and a light-chain immunoglobulin variable domain (V L ), wherein: The V H comprises the amino acid sequence of SEQ ID NO:6, and the V L comprises the amino acid sequence of SEQ ID NO:11; or The V H comprises the amino acid sequence of SEQ ID NO:6, and the V L comprises the amino acid sequence of SEQ ID NO:12; or The V H comprises the amino acid sequence of SEQ ID NO:6, and the V L comprises the amino acid sequence of SEQ ID NO:

13.

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

3. The antibody according to claim 2, comprising an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A.

4. The antibody according to claim 2, comprising an IgG4 constant region containing the S228P mutation.

5. The antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from Fab, F(ab’)2, Fab’, scF v and F v .

6. The antibody according to claim 1, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

7. The antibody according to claim 1, comprising a heavy chain (HC) amino acid sequence containing SEQ ID NO:20 or SEQ ID NO:136 or a light chain (LC) amino acid sequence containing SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:

134.

8. The antibody according to claim 7, comprising an HC amino acid sequence and an LC amino acid sequence selected from the following, respectively: SEQ ID NO:20 and SEQ ID NO:18 (BC249-hHIM34 x CD3 BsAb); SEQ ID NO:136 and SEQ ID NO:134 (BC275-hHIM34 x CD3 BsAb); SEQ ID NO:20 and SEQ ID NO:26 (VL3VH5 x mC825); SEQ ID NO:20 and SEQ ID NO:27 (VL3VH5 x hC825), wherein the antibody is a bispecific antibody.

9. The antibody according to any one of claims 1-8, wherein the antibody binds to the IgC2 domain of CD33.

10. The bispecific antibody or antigen-binding fragment according to claim 6, comprising an amino acid sequence selected from any one of SEQ ID NOs. 30-41.

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