CD33 antibodies and methods of treating cancer using the same

Abstract

The present disclosure generally relates to immunoglobulin-related compositions (eg, antibodies or antigen-binding fragments thereof) that can bind to CD33 protein. Antibodies of the present technology can be used in methods for detecting and treating Alzheimer's disease or CD33-related cancers in subjects in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 809,091, filed February 22, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] 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 uses 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

[0004] The following description of the technical background of the present invention is provided only to help understand the present invention, and it is not admitted that the description describes or constitutes prior art to the present invention.

[0005] Acute myeloid leukemia (AML) accounts for 25% of pediatric leukemias, but accounts for more than half of childhood leukemia deaths. 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, AML is curable in 35%-40% of cases in those patients diagnosed before the age of 60, while only 5%-15% of those patients who present later in life can be cured (Dohner et al., N Engl J Med 373:1136-52 (2015)). Summary of the Invention

[0006] In one aspect, the present disclosure provides a method comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) of an antibody or 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) the 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.

[0007] In one aspect, the present disclosure provides a method comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) of an antibody or antigen-binding fragment thereof, wherein: (a) said V H comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 133; and / or (b) V L Comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0008] 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 comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen-binding fragment is 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.

[0009] 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 variants 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 variants thereof having one or more conservative amino acid substitutions. In certain embodiments, the antibody comprises a HC amino acid sequence and a LC amino acid sequence, respectively, selected from the group consisting of: 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-hHIM34xCD3 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 xmC825); SEQ ID NO: 20 and SEQ ID NO: 27 (VL3VH5 x hC825); SEQ ID NO: 22 and SEQ ID NO: 27 (VL3VH5 x hC825); 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).

[0010] 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 a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 11, 12 or 13; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7 or 133.

[0011] 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 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.

[0012] In any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. In certain embodiments, the antibodies of the present technology comprise an IgG4 constant region comprising an S228P mutation. In any of the above embodiments, the antibody binds to the IgC2 domain of CD33. Additionally or alternatively, in some embodiments, the antibodies of the present technology lack α-1,6-fucose modification.

[0013] 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 NOs. 30-113 or 142-153. In certain embodiments, the bispecific antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs. 30-113 or 142-153.

[0014] 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 that is 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 that is 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 and decomposing (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. NO:133; and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0015] 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 that is 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 that is 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 and decomposing (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. NO: 133; and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises a tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 30-113 or 142-153.

[0016] 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 complementary heavy chain variable domain of the second immunoglobulin. The first immunoglobulin comprises a light chain variable domain of the second immunoglobulin connected to a complementary light chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin connected 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 connected together to form a single-chain variable fragment via a flexible peptide linker comprising the amino acid sequence (GGGGS) 6; 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 the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or a small molecule DOTA hapten.

[0017] 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 the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 135, and 137.

[0018] In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0019] 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 an isotope, a dye, a chromagen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0020] 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 a 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.

[0021] In another aspect, the present disclosure provides a method of treating CD33-related cancer or Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the antibody comprises a HC amino acid sequence and a LC amino acid sequence, respectively, selected from the group consisting of: 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 xmC825); SEQ ID NO: 20 and SEQ ID NO: 27 (VL3VH5 x hC825); SEQ ID NO: 22 and SEQ ID NO: 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.

[0022] In some embodiments, the CD33-associated cancer is AML, biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

[0023] 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, anti-estrogen drugs, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutics.

[0024] 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 tumor expressing CD33 and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody or antigen-binding fragment that is higher than a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody or antigen-binding fragment can be detected using positron emission tomography or single photon emission computed tomography.

[0025] 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-emitting isotope, a beta-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta-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 methods, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (eg, via an N297A mutation in the Fc region, which results in deglycosylation).

[0026] Also disclosed herein is 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) and instructions for use. In certain embodiments, the immunoglobulin-related composition is coupled 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.

[0027] Additionally or alternatively, in some embodiments, the kit further comprises a second antibody that specifically binds to an anti-CD33 immunoglobulin-related composition described herein. In some embodiments, the second antibody is conjugated to at least one detectable label selected from a radiolabel, a fluorescent label, or a chromogenic label.

[0028] 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 a 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.

[0029] In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy 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 or antigen-binding fragment of the present technology that recognizes and binds to 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 or antigen-binding fragment of the complex.

[0030] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, 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 a 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.

[0031] In any of the above embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments of the methods disclosed herein, the subject is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten 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 contains an alpha-emitting isotope, a beta-emitting isotope, or an Auger emitter.

[0032] In one aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiation therapy 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 a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In another aspect, the present disclosure provides a method 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 a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In some embodiments, the methods of the present technology further comprise administering to the subject an effective amount of a clearing agent prior to administering the radiolabeled DOTA hapten.

[0033] 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 comprising an alpha particle emitting isotope, a beta particle emitting isotope, 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

[0034] Figure 1 Schematic structures of CD33 isoforms are shown. The full-length CD33 protein is shown on the left, and the short isoform (a splice variant lacking the outer IgV domain) is shown on the right.

[0035] Figure 2 Shown is 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 short isoforms of CD33.

[0036] Figure 3 Shown is a schematic diagram of a modular tetravalent IgG-scFv format comprising an IgG molecule with two binding sites covalently linked to two scFvs, providing two additional binding domains.

[0037] Figure 4 Shown are FACS data demonstrating binding of the chimeric HIM34 x CD3 bispecific antibody (chHIM34 BsAb) to the CD33(+) AML cell lines U937, THP1, SET2, C1498-CD33, and M-07e, while sparing the CD33(-) leukemia cell line CMLT1.

[0038] Figure 5 (A) shows a schematic diagram of the full-length CD33 protein and the short isoform (a splice variant of CD33). Figure 5 (B) shows FACS data demonstrating that the chHIM34 BsAb binds to both the full-length and short isoforms of CD33, whereas the humanized M195-based BsAb (BC133) does not bind to the short isoform.

[0039] Figure 6(A) and 6(B) Shown are T cell-dependent cytotoxicity assays using chHIM34 BsAb on CD33(+) AML cell lines C1498-CD33 and THP1, respectively.

[0040] Fig. 7 (A) shows bioluminescent images showing the growth of MOLM13 AML tumors in different groups. Fig. 7 (B) shows the signal quantification of mice from different groups in Fig. 7 (A).

[0041] Figure 8 The amino acid sequences of the murine and humanized HIM34 heavy chain variable domains are shown (SEQ ID NOs: 1-7 and 133). HIM34_VH-1, HIM34_VH-2, HIM34_VH-3, HIM34_VH-4, HIM34_VH-5, HIM34-VH-6, and HIM34-VH-7 are seven forms of the humanized HIM34 heavy chain variable domains. H -CDR1, V H -CDR2 and V H - CDR3 sequence is shown in bold.

[0042] Figure 9 The amino acid sequences of the murine and humanized HIM34 light chain variable domains are shown (SEQ ID NOs: 8-13). HIM34_VL-1, HIM34_VL-2, HIM34_VL-3, HIM34_VL-4, and HIM34_VL-5 are five forms of the humanized HIM34 light chain variable domains. L -CDR1, V L -CDR2 and V L - CDR3 sequence is shown in bold.

[0043] Figures 10(A) and 10(B) show the amino acid sequence and nucleotide sequence of the light chain and heavy chain of chHIM34 BsAb, respectively (SEQ ID NOs: 14-17). The signal peptide is underlined, the variable domain of the chimeric anti-CD33 antibody is indicated in bold font, and the linker sequence is italicized and underlined. The V domain of chHIM34 BsAb is also disclosed. H and V L The amino acid sequence of the SEQ ID NO: 114-115 domain.

[0044] Figures 11(A) and 11(B) show the amino acid sequence and nucleotide sequence of the light and heavy chains of the humanized HIM34 x CD3 BsAb (BC249), respectively (SEQ ID NOs: 18-21). Figures 11(C) and 11(D) show the amino acid sequence and nucleotide sequence of the light and heavy chains of the humanized HIM34 x CD3 BsAb (BC275), respectively (SEQ ID NOs: 134-137). The signal peptide is underlined, the variable domain of the humanized anti-CD33 antibody is indicated in bold, and the linker sequence is italicized and underlined.

[0045] Figures 12(A) and 12(B) show the amino acid sequence and nucleotide sequence of the light chain and heavy chain of humanized HIM34 x CD3 BsAb (BC267), respectively (SEQ ID NOs: 18, 19, 22, and 23). The signal peptide is underlined, the variable domain of the humanized anti-CD33 antibody is indicated in bold, and the linker sequence is italicized and underlined.

[0046] Figures 13(A) and 13(B) show the amino acid sequence and nucleotide sequence of the light chain and heavy chain of humanized HIM34 x CD3 BsAb (BC268), respectively (SEQ ID NOs: 24, 25, 22, and 23). The signal peptide is underlined, the variable domain of the humanized anti-CD33 antibody is indicated in bold, and the linker sequence is italicized and underlined.

[0047] Figures 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 format are shown. The light chain amino acid sequences are shown in SEQ ID NOs: 26-29, and the heavy chain amino acid sequences are shown in SEQ ID NOs: 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 sequence is italicized and underlined.

[0048] Figures 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 format are shown. The light chain amino acid sequences are shown in SEQ ID NOs: 138 and 140, and the heavy chain amino acid sequences are shown in SEQ ID NOs: 139 and 141. The signal peptide is underlined, the variable domains of the mouse anti-CD33 antibody are indicated in bold font, and the linker sequence is italicized and underlined.

[0049] Figure 15(A) to Figure 15(BB) The amino acid sequences of the humanized HIM34 x C825 (anti-DOTA) BsAbs in the form of a single-chain bispecific tandem fragment variable (scBsTaFv) are shown (SEQ ID NOs: 30-113). The signal peptide is underlined, the variable domains of the humanized anti-CD33 antibody are indicated in bold font, and the linker sequence is italicized and underlined.

[0050] Figure 15(CC) to Figure 15(FF)The amino acid sequence of the mouse HIM34 x C825 (anti-DOTA) BsAb in the form of a single-chain bispecific tandem fragment variable (scBsTaFv) is shown (SEQ ID NOs: 142-153). The signal peptide is underlined, the variable domain of the mouse-based anti-CD33 antibody is indicated in bold font, and the linker sequence is italicized and underlined.

[0051] Figures 16(A) and 16(B) show a summary of the characteristics of chimeric and humanized HIM34 BsAbs, including binding kinetics.

[0052] Figure 17 Shown are FACS analyses of MOLM13 CD33(+) cells contacted with different concentrations of humanized CD33 BsAbs of the present technology.

[0053] Figure 18(A) to Figure 18(B) The cytotoxicity of humanized BsAbs of the present technology on MOLM13 CD33(+) cells in a T cell-dependent cytotoxicity assay (TDCC) is shown.

[0054] Figure 19 The stability of humanized BsAbs of the present technology at 40°C is shown.

[0055] Figure 20 (A) shows T cell-engaging bispecific antibodies (BsAbs) that recruit T cells to AML cells. Figure 20 (B) shows that the BC275 BsAb disclosed herein 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 properties of BC275, BC133, and BC269 BsAbs to various CD33 (+) and CD33 (-) human cancer cell lines.

[0056] Figure 21(A) to Figure 21(C) Figure 21(D) shows the correlation between the level of CD33 expression on cancer cells and the potency of the tested BsAbs in the TDCC assay. The combined data for all three CD3×CD33 BsAbs are shown.

[0057] Figure 22(A) to Figure 22(B)Shown are tumor sizes in immunodeficient NSG mice that were intravenously inoculated with CD33(+)MOLM13-luciferase human AML xenografts (10 6 cells) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). 6 A single injection of activated T cells and 0.025 μg BsAb (10 μg / Kg / dose) was performed. The timing of T cell and antibody injections is shown in FIG22(B).

[0058] Figure 23(A) to Figure 23(B) Shown are tumor sizes in immunodeficient NSG mice that were intravenously inoculated with human CD33-transduced NALM6-luciferase leukemia xenografts (0.5×10 6 cells) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). Mice received two injections of activated T cells (2.7×10 6 and 5×10 6 , one week 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 three CD3×CD33 BsAbs. The potency of the CD3×CD33 BsAbs was ranked as follows: BC275 > BC269 = BC133.

[0059] Figure 24(A) to Figure 24(D) Figure 2 shows tumor size in immunodeficient DKO mice that were subcutaneously inoculated with THP1 human leukemia xenografts (having the CC genotype of the rs12459419 SNP) and subsequently treated with BC275, BC133, and BC269 BsAbs (CD3×CD33 BsAbs) or control BC119 BsAb (CD3×GD2 BsAb). Mice received two injections of activated T cells (10 7 The T cells were injected with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose, respectively). The timing of T cell and antibody injections is shown in Figure 24(A). The potency of the CD3×CD33 BsAbs was ranked as follows: BC275 > BC269 > BC133.

[0060] Figure 25(A) to Figure 25(D)Figure 2 shows tumor size in immunodeficient DKO mice that were 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 control BC119 BsAb (CD3×GD2 BsAb). Mice received three injections of activated T cells (10 7 The T cells were injected with 0.05 μg or 0.5 μg of BsAb (≈2 μg / kg / dose or 20 μg / kg / dose, respectively). The timing of T cell and antibody injections is shown in Figure 25(A). The potency of the CD3×CD33 BsAbs was ranked as follows: BC275 > BC269 > BC133. DETAILED DESCRIPTION

[0061] It should be understood that certain aspects, modes, embodiments, variations and features of the present methods are described below in varying levels of detail to provide a substantial understanding of the present technology.

[0062] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can specifically bind to CD33 polypeptides. The immunoglobulin-related compositions of the present technology can be used in methods for detecting or treating CD33-related cancers or Alzheimer's disease in subjects in need thereof. Therefore, 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 present technology can be used alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related compositions are humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0063] 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.; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; 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 ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. 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 Biology. 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 levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods, such as antibody detection and quantification techniques. (See also Strachan and Read, Human Molecular Genetics, 2nd ed. (John Wiley and Sons, Inc., New York, 1999)).

[0064] definition

[0065] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present 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 used in the art.

[0066] Unless the context indicates otherwise or it is otherwise apparent, as used herein, the term "about" with respect to a number is generally considered to include numbers that fall within 1%, 5%, or 10% in either direction (greater than or less than) of the stated number (except where such a number is less than 0% or more than 100% of the possible value).

[0067] 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 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.

[0068] "Adjuvant" refers to one or more substances that cause immune system stimulation. In this case, the adjuvant is used to enhance the immune response to one or more vaccine antigens or antibodies. The adjuvant can be administered to the subject before, in combination with, or after the administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory 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.

[0069] 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, combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals (e.g., humans, goats, rabbits, and mice), as well as non-mammalian species (e.g., shark immunoglobulins). 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) to the substantial exclusion of binding to other molecules (e.g., a binding constant for the molecule of interest that is at least 10 greater than a binding constant for other molecules in a biological sample). 3 M -1 , at least 10 4 M -1 or at least 10 5 M -1 The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0070] More specifically, an antibody refers to a polypeptide ligand that specifically recognizes and binds to an antigen epitope and contains at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region. Antibodies are composed of heavy chains and light chains, each of which has a variable region, called a heavy chain variable region (V H ) region and light chain variable (V L ) area. H Area and V LThe regions are jointly responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, namely λ (lambda) and κ (kappa). 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 chain and light chain contains a constant region and a variable region (the regions are also called "domains"). Combined, the heavy chain variable region and the light chain variable region specifically bind to the antigen. The light chain variable region and the heavy chain variable region contain a "framework" region interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs"). The scope of the framework region and CDR has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US 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 species. The framework regions of antibodies (i.e., the combined framework regions of the constituent light and heavy chains) primarily adopt a β-sheet conformation, and the CDRs form loops connecting the β-sheet structure, and in some cases, the loops form part of the β-sheet structure. Therefore, the framework region serves to form a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions.

[0071] The CDRs are primarily responsible for binding to the antigenic epitope. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are often also identified by the chain in which a particular CDR is located. 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 Area and V L Region sequence, thus having a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs are different between different antibodies, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. These positions in the CDR are called specificity determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) and antibody fragments. Antibodies or their antigen-binding fragments specifically bind to antigens.

[0072] As used herein, the term "antibody-related polypeptide" means an antigen-binding antibody fragment including a single-chain antibody, which may comprise one or more variable regions alone or in combination with all or part of the following polypeptide elements: the hinge region, CH1, CH2, and CH3 domains of an antibody molecule. The technology also includes any combination of one or more variable regions and hinge regions, CH1, CH2, and CH3 domains. Antibody-related molecules that can be used 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 V L or V H Examples include: (i) Fab fragments, i.e., fragments of V L 、V H 、C L and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments connected by a disulfide bridge in the hinge region; (iii) H and CH1 domains; (iv) a V fragment consisting of a single-arm antibody L and V H Fv fragments composed of V domains; (v) dAb fragments (Ward et al., Nature 341: 544-546, 1989), which are composed of V H domain composition; 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 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.

[0073] As used herein, "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 includes a V H and / or V L region; in some such embodiments, V H and / or V L The V domains are those found in specific monoclonal antibodies. In some embodiments, a bispecific antibody contains two antigen-binding portions, each comprising a V domain from a different monoclonal antibody. H and / or V LIn some embodiments, the bispecific antibody contains two antigen-binding moieties, wherein one of the two antigen-binding moieties comprises a region having a V H and / or V L region of the immunoglobulin molecule, the V H and / or V L The region contains the CDRs from the first monoclonal antibody; and the other antigen binding portion includes a V H and / or V L The antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.) of the V H and / or V L The region contains the CDRs from the second monoclonal antibody.

[0074] As used herein, a "clearing agent" is an agent that binds to excess bispecific antibody present in the blood compartment of a subject to promote rapid clearance via the kidneys. The use of clearing agents prior to hapten administration (e.g., DOTA) helps achieve a better tumor-to-background ratio in pretargeted radioimmunotherapy (PRIT) systems. Examples of clearing agents include 500 kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6):1365-1372 (2012)), 500 kD aminodextran-DOTA conjugates, antibodies against pretargeting antibodies, and the like.

[0075] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipole-dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic ring stacking.

[0076] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites contained within the same polypeptide chain in conjunction with a light chain variable domain (V L ) linked to the heavy chain variable domain (V H )(V H V L By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0077] As used herein, the term "single-chain antibody" or "single-chain Fv (scFv)" refers to the two domains V and V of an Fv fragment. L and V H Single-chain antibody molecules may include polymers having multiple individual molecules, such as dimers, trimers, or other polymers. In addition, although F v The two domains of the fragment V L and V H They are encoded by separate genes, but they can be joined by synthetic linkers using recombinant methods to enable them to become a single protein chain, in which V L and V H The domains pair to form monovalent molecules (called 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.

[0078] Fragments of any of the foregoing antibodies are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as intact antibodies.

[0079] As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., a CD33 polypeptide). Antigens can also be administered to an animal to generate an immune response in the animal.

[0080] The term "antigen-binding fragment" refers to a fragment of an intact immunoglobulin structure having a polypeptide portion responsible for antigen binding. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0081] "Binding affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) represents. Affinity can be measured by standard methods known in the art (including those described herein). Low-affinity complexes contain antibodies that generally tend to dissociate easily from the antigen, while high-affinity complexes contain antibodies that generally tend to remain bound to the antigen for a longer period of time.

[0082] As used herein, term " biological sample " means the sample material derived from living cells. Biological sample can include the tissue separated from the subject, cell, protein or membrane extract of the cell and biological fluid (for example, ascites or cerebrospinal fluid (CSF)), and tissue, cell and fluid present in the subject. The biological sample of this technology includes but is not limited to taking from following sample: mammary tissue, kidney tissue, cervix uteri, endometrium, head or neck, gall bladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, cheek, skin, serum, plasma, CSF, sperm, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological sample can also be obtained from the biopsy of internal organs or from cancer. Biological samples can be obtained from subjects for diagnosis or research; or can be obtained from individuals without the disease as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0083] As used herein, the term "CDR-grafted antibody" refers to 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.

[0084] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse 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 technology. 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.

[0085] As used herein, the term "consensus FR" refers to the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0086] As used herein, a "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, where the purpose of an experiment is to determine the relevance of a therapeutic agent's efficacy in treating a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive the treatment or receives a placebo) are typically used.

[0087] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or preventive effect, such as an amount that prevents or reduces a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the case of therapeutic or preventive applications, the amount of the composition administered to the subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics (such as general health, age, sex, weight, and drug tolerance). A skilled person will be able to determine the appropriate dosage based on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition can be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are improved or eliminated. The therapeutically effective amount can be administered in one or more administrations.

[0088] As used herein, the term "effector cell" means an immune cell participating in the effector phase of an immune response, which is opposite to the cognitive phase and activation phase of an immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), 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 can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils 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 are combined with cells presenting antigens.

[0089] As used herein, the term "epitope" means a protein determinant capable of specific binding to an antibody. An epitope is typically composed of chemically active surface groups of a molecule (such as amino acids or sugar side chains) and typically has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that binding to a conformational epitope, but not a non-conformational epitope, is lost in the presence of a denaturing solvent. In some embodiments, an "epitope" of a CD33 protein is a region of the protein that specifically binds to an anti-CD33 antibody of the present technology. In some embodiments, an epitope is a conformational epitope or a non-conformational epitope. To screen for anti-CD33 antibodies that bind to an epitope, a conventional cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow and David Lane (1988), can be performed. 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, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different approach, peptides corresponding to different regions of the CD33 protein can be used in competition assays with multiple test antibodies, or with a test antibody and an antibody with a characterized or known epitope.

[0090] As used herein, "expression" includes one or more of the following: transcription of the gene into pre-mRNA; splicing and other processing of the pre-mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product (if required for proper expression and function).

[0091] As used herein, the term "gene" means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0092] "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 comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences 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" to another sequence, meaning that, when aligned, the bases (or amino acids) of that percentage are the same in the two sequences being compared. This alignment and the homology or sequence identity percentage can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, which uses default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting = HIGH SCORE; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percent homology and encode polypeptides with the same or similar biological activity. Two sequences are considered "unrelated" or "nonhomologous" if they share less than 40% identity or less than 25% identity with each other.

[0093] As used herein, " humanized " form of non-human (for example, mouse) antibody is chimeric antibody, and it contains the minimum sequence that is derived from non-human immunoglobulin.For most, humanized antibody is human immunoglobulin, and wherein the hypervariable region residues of acceptor are replaced by the hypervariable region residues (donor antibody) with required specificity, affinity and ability from non-human species (such as mouse, rat, rabbit or non-human primate).In some embodiments, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues.In addition, humanized antibody can be included in the residue that is not found in acceptor antibody or donor antibody.Carry out these modifications to further improve antibody performance such as binding affinity. Typically, a humanized antibody will comprise substantially all of at least one, typically 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 a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence, 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 typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a 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, eg, Ahmed and Cheung, FEBS Letters 588(2):288-297 (2014).

[0094] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., V L before and after residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), as well as V H 31-35B (H1), 50-65 (H2), and 95-102 (H3) in (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from the "hypervariable loops" (e.g., V LResidues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in V H 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0095] As used herein, when used in the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "identity" percentage refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison window or specific region. Such sequences are referred to as "substantially identical." This term also refers to or can be applied to the complement of a test sequence. The term also includes sequences with deletions and / or additions, as well as those with substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0096] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain consists 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 consists of a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The regions can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, arranged in the following order from amino terminus to 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 region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0097] As used herein, the term "individual," "patient," or "subject" can be a single organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0098] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for the possible abiogenous mutation that may exist in a small amount, the individual antibodies constituting the population are identical. For example, a monoclonal antibody can be an antibody derived from a single clone (including any eukaryotic, prokaryotic or phage clone) rather than the method for producing it. Monoclonal antibody compositions demonstrate single binding specificity and affinity to a specific epitope. Monoclonal antibodies have a high degree of specificity for a single antigenic site. In addition, compared with conventional (polyclonal) antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population, and should not be construed as needing to produce the antibody by any ad hoc method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, for example, but not limited to, hybridoma, recombinant and phage display technology. For example, the monoclonal antibody to be used according to the method of the present invention can be prepared by the hybridoma method originally described by Kohler et al., Nature 256:495 (1975), or can be prepared 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 technology described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0099] 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, etc., that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, A. Gennaro, 2000, Lippincott, ed., Williams & Wilkins, Philadelphia, Pennsylvania).

[0100] As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies containing antibodies that specifically bind to different epitopes or regions of an antigen.

[0101] As used herein, the term "polynucleotide" or "nucleic acid" means any RNA or DNA, which can 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 comprising DNA and RNA, which can be single-stranded or more generally double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA whose backbone has been modified for stability or other reasons.

[0102] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer comprising two or more amino acids linked 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 gene-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 fully described in basic textbooks and more detailed monographs as well as lengthy research literature.

[0103] As used herein, "PRIT" or "pretargeted radioimmunotherapy" refers to a multi-step process that addresses the slow blood clearance of tumor-targeting antibodies, which leads to undesirable toxicity to normal tissues such as the bone marrow. In pretargeting, a radionuclide or other diagnostic or therapeutic agent is attached to a small hapten. A pretargeting bispecific antibody with binding sites for the hapten as well as the target antigen is first administered. Unbound antibody is then allowed to clear from the circulation, and the hapten is then administered.

[0104] As used herein, the term "recombinant" when used with respect 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 a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0105] As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

[0106] 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 refers to the entire administration of one active ingredient before the start of administration of the other active ingredient or ingredients. Thus, one active ingredient can be administered minutes, hours, or days before the administration of the other active ingredient or ingredients. In this case, there is no simultaneous treatment.

[0107] As used herein, "specific binding" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize and bind to other molecules. As used herein, the terms "specifically bind to" a particular molecule (e.g., a polypeptide or an epitope on a polypeptide), "specifically bind to" or have "specificity" for a particular molecule can be defined, for example, by the K of a molecule for the molecule to which it binds. d About 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 - 8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12The term "specific binding" may also refer to binding in which a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a specific polypeptide (e.g., a CD33 polypeptide) or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0108] As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or substantially the same time.

[0109] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0110] As used herein, "treating" or "treatment" encompasses treating a disease or disorder described herein in a subject, such as a human, 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 the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in a state of remission.

[0111] It is also understood that the various treatment modalities for disorders as 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 continuous, extended treatment for chronic conditions, or single or several administrations for acute conditions.

[0112] 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 antibodies may be 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 may be performed to obtain the desired antibody, provided that the resulting antibody possesses the desired properties. Modifications also include changes in the glycosylation pattern of the protein. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. "Conservative substitutions" are shown in the table below.

[0113]

[0114] One type of substitution variant involves replacing one or more hypervariable region residues of the parent antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. In particular, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus produced are displayed as fusions of the gene III product of the M13 packaged in each particle in a monovalent manner from filamentous phage particles. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity), as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. According to the technology described in detail herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are produced, this group of variants is screened as described herein, and antibodies with similar or better properties in one or more related assays can be selected for further development.

[0115] CD33

[0116] CD33 (also known as Siglec-3, SIGLEC3, gp67, p67) is a 67kDa plasma membrane protein that is bound to sialic acid and is a member of the sialic acid-binding Ig-associated lectin (SIGLEC) protein family. Siglec proteins are believed 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 (an IgV and an IgC2 domain) ( Figure 1 The intracellular portion of CD33 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). In immune responses, CD33 can act as an inhibitory receptor by recruiting one or more cytoplasmic phosphatases upon ligand-induced tyrosine phosphorylation, which block signal transduction by dephosphorylating signaling molecules.

[0117] It is known that CD33 is 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 are currently no effective CD33-targeted therapeutics on the market. Existing anti-CD33 agents have, in particular, 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 ).

[0118] 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 almost 50% of AML patients, do not benefit from current anti-CD33-IgV domain antibody-based therapies (e.g., gemtuzumab ozogamicin, lintuzumab, AMV564).

[0119] Immunoglobulin-related compositions of the present technology

[0120] The present technology describes methods and compositions for producing and using anti-CD33 immunoglobulin-related compositions (e.g., anti-CD33 antibodies or antigen-binding fragments thereof). The anti-CD33 immunoglobulin-related compositions of the present disclosure can be used to diagnose or treat CD33-related cancers or Alzheimer's disease. Anti-CD33 immunoglobulin-related compositions within the scope of the present technology include, for example, but not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, 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 fragment is selected from Fab, F(ab)'2, Fab', scF v and F v The present technology discloses anti-CD33 bispecific antibody formats that, in addition to binding to both the full-length CD33 protein and the short isoform of CD33, also address the existing problems of poor tumor antigen binding affinity and short in vivo half-life. Importantly, the short isoform of CD33 is expressed in half of AML patients. In one aspect, the present technology provides chimeric and humanized variants of HIM34, including multispecific immunoglobulin-related compositions (e.g., bispecific antibody agents).

[0121] In one aspect, the present disclosure provides a method comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (VL ) of an antibody or 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) the 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.

[0122] In one aspect, the present technology provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) of an antibody or antigen-binding fragment thereof, wherein (a) said V H comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 133; and / or (b) V L Comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0123] In any of the above embodiments, the antibody further comprises an Fc domain of any 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:

[0124] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 116)

[0125] APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0126] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO:117)

[0127] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0128] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO:118)

[0129] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0130] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO:119)

[0131] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0132] Human IgM constant region, Uniprot: P01871 (SEQ ID NO:120)

[0133] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0134] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO:121)

[0135] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0136] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO:122)

[0137] ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0138] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO:123)

[0139] ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0140] Human Igκ constant region, Uniprot: P01834 (SEQ ID NO:124)

[0141] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs: 116-123. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 124. In some embodiments, the immunoglobulin-related compositions of the present technology bind to the IgC2 domain of CD33. In some embodiments, the epitope is a conformational epitope.

[0143] In another aspect, the present disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or an antigen-binding fragment thereof) comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 136, SEQ ID NO: 139, SEQ ID NO: 141, or a variant thereof having one or more conservative amino acid substitutions.

[0144] Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:140, or variants thereof having one or more conservative amino acid substitutions.

[0145] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a HC amino acid sequence and a LC amino acid sequence, respectively, selected from the group consisting of: 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: 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 xhC825), 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).

[0146] In any of the above embodiments of the immunoglobulin-related compositions, 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.

[0147] 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.

[0148] 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 present in an amount of 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 ) 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.

[0149] In certain embodiments, the immunoglobulin-related compositions include one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 11, 12 or 13; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7 or 133. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution can be a "conservative substitution" as defined herein.

[0150] 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 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.

[0151] 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.

[0152] 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 that is 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 that is 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 and decomposing (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. NO:133; and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0153] 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 that is 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 that is 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 and decomposing (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. NO:133; and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0154] In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises a tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs. 30-113 or 142-153.

[0155] 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 complementary heavy chain variable domain of the second immunoglobulin. The first immunoglobulin comprises a light chain variable domain of the second immunoglobulin connected to a complementary light chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin connected 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 connected together to form a single-chain variable fragment via a flexible peptide linker comprising the amino acid sequence (GGGGS) 6; 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 the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or a small molecule DOTA hapten.

[0156] In certain embodiments, the immunoglobulin-related composition comprises an IgG1 constant region comprising 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 comprising an S228P mutation.

[0157] 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 can 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.

[0158] In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein is a recombinant nucleic acid sequence encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from SEQ ID NO: 15, 17, 19, 21, 23, 25, 135, and 137.

[0159] In another aspect, the present technology provides a host cell that expresses any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0160] The immunoglobulin-related compositions (e.g., anti-CD33 antibodies) of the present technology 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 (e.g., a heterologous polypeptide or solid support material). See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 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 compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized.

[0161] The immunoglobulin-related compositions of the present technology can be further recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules and effector molecules (such as heterologous polypeptides, drugs or toxins) that can be used as markers in detection assays. See, for example, WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Patent No. 5,314,995; and EP 0 396 387.

[0162] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen-binding fragment can be optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. For chemical or physical binding, the functional groups on the immunoglobulin-related composition are typically associated with the functional groups on the agent. Alternatively, the functional groups on the agent are associated with the functional groups on the immunoglobulin-related composition.

[0163] The functional groups on the agent and the functional groups on the immunoglobulin-related composition can be directly associated. For example, the functional groups on the agent (e.g., sulfhydryl) can be associated with the functional groups on the immunoglobulin-related composition (e.g., sulfhydryl) to form a disulfide bond. Alternatively, the functional groups can be associated through a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent 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 party. 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.

[0164] In another embodiment, the conjugate includes a kind of immunoglobulin related composition associated with a kind of medicament.In one embodiment, the conjugate includes at least one medicament chemically bonded (such as conjugated) to at least one immunoglobulin related composition.The medicament and immunoglobulin related composition can be chemically bonded by any method known to those skilled in the art.For example, the functional group on the medicament can be directly attached to the functional group on the immunoglobulin related composition.Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate and hydroxyl.

[0165] The agent can also be chemically bonded to the immunoglobulin-related composition using a cross-linking agent such as a dialdehyde, carbodiimide, dimaleimide, or the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc. of Rockford, Illinois. The Pierce Biotechnology, Inc. website can provide assistance. Additional cross-linking agents include the platinum cross-linkers described in U.S. Patent Nos. 5,580,990, 5,985,566, and 6,133,038 to Kreatech Biotechnology, BV of Amsterdam, The Netherlands.

[0166] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homobifunctional crosslinkers are generally used to crosslink the same functional groups. Examples of homobifunctional crosslinkers include EGS (ie, ethylene glycol bis[succinimidyl succinate]), DSS (ie, disuccinimidyl suberate), DMA (ie, dimethyl adipimidate.2HCl), DTSSP (ie, 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (ie, 1,4-bis-[3'-(2'-pyridyldithio)-propionamido] butane) and BMH (ie, bismaleimidohexane). Such homobifunctional crosslinkers can also be obtained from Pierce Biotechnology, Inc.

[0167] In other cases, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The aforementioned Pierce Biotechnology, Inc. website can also assist those skilled in the art in selecting suitable cross-linking agents that can be cleaved, for example, by enzymes in cells. This allows the agent to 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., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (e.g., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0168] In another embodiment, the conjugate comprises at least one agent physically associated with at least one immunoglobulin-related composition. The agent can be physically associated 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 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 stirred, for example, by shaking the container, to mix the immunoglobulin-related composition and the agent.

[0169] The immunoglobulin-related compositions can be modified by any method known to those skilled in the art. For example, as described above, the immunoglobulin-related compositions can be modified by cross-linking agents or functional groups.

[0170] A. Methods for preparing anti-CD33 antibodies of the present technology

[0171] Overview. First, a target polypeptide is selected, and antibodies of the present technology can be generated against the target polypeptide. For example, antibodies can be generated against the full-length CD33 protein, the CD33 protein lacking the external IgV domain, or against a portion of the extracellular domain of the CD33 protein (e.g., the IgC2 domain). Techniques for generating antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, xenogeneic or human mice, hybridomas, and the like. 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.

[0172] It will be appreciated that recombinantly engineered antibodies and antibody fragments (eg, antibody-related polypeptides) directed against CD33 protein and fragments thereof are suitable for use in accordance with the present disclosure.

[0173] Anti-CD33 antibodies that can be subjected to the techniques described herein include monoclonal 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 producing high yields 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.

[0174] Typically, antibodies are obtained from a species of origin. More specifically, nucleic acid or amino acid sequences representing the variable portion of the light chain, heavy chain, or both of an antibody from the species of origin that is specific for the target polypeptide antigen are obtained. The species of origin can be any species that can be used to generate antibodies or antibody libraries for the present technology, such as rat, mouse, rabbit, chicken, monkey, or human.

[0175] Phage or phagemid display technology is a technology that can be used to derive antibodies of the present technology. The technology for producing and cloning monoclonal antibodies is well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology can be carried out in Escherichia coli.

[0176] 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 comprising all or part of the nucleic acid sequences encoding the above-described polypeptides, wherein the nucleic acid sequences are altered by substitution of different codons encoding functionally equivalent amino acid residues within the coding sequence, thereby producing a silent change. It should be understood that the nucleotide sequences of immunoglobulins according to the present technology tolerate up to 25% sequence homology variation as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form effective antibodies that recognize CD33 protein. For example, one or more amino acid residues within a polypeptide sequence can be substituted with another amino acid of similar polarity that acts as a functional equivalent, thereby resulting in a silent change. Substitutes for amino acids within the sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar 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 this technology are proteins or fragments or derivatives thereof that are differentially modified during or after translation, such as by glycosylation, proteolytic cleavage, connection to antibody molecules or other cellular ligands, etc. In addition, the nucleic acid sequence encoding the immunoglobulin can be mutated in vitro or in vivo to generate 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 pre-existing sites to facilitate further in vitro modification. Any mutagenesis technique known in the art can be used, including but not limited to in vitro site-directed mutagenesis (J. Biol. Chem. 253:6551), use of Tab linkers (Pharmacia), and the like.

[0177] Preparation of polyclonal antisera and immunogens. Methods for producing antibodies or antibody fragments of the present technology generally include immunizing a subject (typically a non-human subject, such as a mouse or rabbit) with a purified CD33 protein or fragment thereof or with cells expressing a CD33 protein or fragment thereof. Suitable immunogenic preparations may contain, for example, recombinantly expressed CD33 protein or chemically synthesized CD33 peptides. Using standard techniques for polyclonal and monoclonal antibody preparation, the extracellular domain of the CD33 protein or a portion or fragment thereof (e.g., an IgC2 domain) may be used as an immunogen to produce anti-CD33 antibodies that bind to the CD33 protein or a portion or fragment thereof.

[0178] The full-length CD33 protein or a fragment thereof can be used as an immunogen. In some embodiments, the CD33 fragment comprises the IgC2 domain of CD33, such that antibodies raised against the peptide form specific immune complexes with CD33 proteins, including short isoforms of CD33 (CD33 splice variants lacking the IgV domain).

[0179] The IgC2 domain of CD33 is 84 amino acids in length. In some embodiments, the antigenic CD33 peptide comprises at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, or 80 amino acid residues. Depending on the intended use and according to methods well known to those skilled in the art, longer antigenic peptides may sometimes be desirable rather than shorter ones. Multimers of a given epitope may sometimes be more effective than monomers.

[0180] If desired, the immunogenicity of the CD33 protein (or 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 conventional adjuvants, such as Freund's complete or incomplete adjuvant, to enhance the subject's immune response to the polypeptide. Various adjuvants used to enhance the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants (e.g., Bacille Calmette-Guerin and Corynebacterium parvum), or similar immunostimulatory compounds. These techniques are standard in the art.

[0181] When describing the present technology, the immune response may be described as a "primary" or "secondary" immune response. A primary immune response, also referred to as a "protective" immune response, refers to an immune response generated in an individual as a result of initial exposure (e.g., initial "immunization") to a specific antigen (e.g., CD33 protein). In some embodiments, immunization can be performed by vaccinating an individual with a vaccine containing the antigen. For example, the vaccine can be a CD33 vaccine comprising one or more antigens derived from CD33 proteins. Over time, the primary immune response may weaken or diminish, and may even disappear or at least become so diminished that it cannot be detected. Therefore, 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.

[0182] Thus, a secondary immune response can be elicited, for example, to boost an existing immune response that has weakened or diminished, or to regenerate 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 following stimulation of memory B cells generated upon initial presentation of an antigen. A delayed-type hypersensitivity (DTH) reaction is a CD4 + A type of cellular secondary or memory immune response mediated by T cells. The first exposure to an antigen primes the immune system, and one or more additional exposures result in DTH.

[0183] After appropriate immunization, anti-CD33 antibodies can be prepared from the subject's serum. 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 an IgG fraction.

[0184] 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 mouse anti-CD33 monoclonal antibody. To prepare monoclonal antibodies against CD33 protein or its derivatives, fragments, analogs or homologs, any technique for producing antibody molecules by continuous cell line culture can be used. Such techniques include, but are not limited to, hybridoma technology (see, for example, Kohler and Milstein, 1975. Nature 256: 495-497); trioma technology; human B cell hybridoma technology (see, for example, Kozbor et al., 1983. Immunol. Today 4: 72) and EBV hybridoma technology to produce human monoclonal antibodies (see, for example, 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 this technology, and can be produced by using human hybridomas (see, for example, Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr (EpsteinBarr) virus (see, for example, Cole et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, nucleic acid groups encoding antibody regions can be isolated. PCR using primers derived from sequences encoding antibody conserved regions is used to amplify the sequence encoding the antibody portion from the colony, and then the DNA encoding the antibody or its fragment (such as variable domains) is reconstructed from the amplified sequence. Such amplified sequences can also be fused with DNA encoding other proteins, such as phage coats or bacterial cell surface proteins, to express and display 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 antibodies or fragments thereof for an antigen or epitope present on the CD33 protein. Alternatively, hybridomas expressing anti-CD33 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using conventional methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73:3-46). Screening of hybridomas using standard methods will produce monoclonal antibodies with different specificities (i.e., directed against different epitopes) and affinities.Selected monoclonal antibodies with desired properties (e.g., CD33 binding) can be used as expressed by hybridomas, can be conjugated to molecules (e.g., 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 substitutions or deletions of specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of antibodies for the CD33 protein.

[0185] Hybridoma technology. In some embodiments, the antibody of the present technology is an anti-CD33 monoclonal antibody produced by a hybridoma, wherein the hybridoma includes a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused with immortalized cells. Hybridoma technology includes those known in the art and taught in the following literature: 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 producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0186] Phage display technology. As noted above, antibodies of the present technology can be produced by applying recombinant DNA technology and phage display technology. For example, anti-CD33 antibodies can be prepared using various phage display methods known in the art. In the phage display method, 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 (e.g., human or mouse) by direct selection with an antigen (usually an antigen bound to or captured on a solid surface or beads). The phage used in these methods are typically filamentous phages that include fd and M13 with Fab, Fv, or disulfide-stabilized Fv antibody domains that are recombinantly fused to phage gene III or gene VIII proteins. Furthermore, the method is suitable for the construction of Fab expression libraries (see, eg, Huse et al., Science 246: 1275-1281, 1989) to rapidly and efficiently identify monoclonal Fab fragments with the desired specificity for a CD33 polypeptide, eg, a polypeptide or derivative, fragment, analog, or homolog thereof.Other examples of phage display methods that can be used to make antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci USA, 85:5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87:1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182:41-50, 1995; Ames et al., J. Immunol. Methods 184:177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24:952-958, 1994; Persic et al., Gene 187:9-18, 1997; Burton et al., Advances in Immunology 57:191-280, 1994; PCT / GB91 / 01134; WO90 / 02809; 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); WO 92 / 01047 (CAT / MRC); WO 91 / 17271 (Affyma x) 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. U.S. Patent No. 6,753,136 to Lohning has described a method for displaying polypeptides on the surface of phage particles by attaching the polypeptides via disulfide bonds. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce complete 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 recombinantly producing Fab, Fab′ and F(ab′)2 fragments can also be utilized using methods known in the art, such as those disclosed in 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.

[0187] Typically, hybrid antibodies or hybrid antibody fragments cloned into display vectors can be selected for the appropriate antigen to identify variants that retain good binding activity because the antibodies or antibody fragments will be present on the surface of the phage or phagemid particles. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). However, other vector formats can be used for this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or Lambda Zap systems) for selection and / or screening.

[0188] Expression of recombinant anti-CD33 antibodies. As described above, the antibodies of the present technology can be produced by applying recombinant DNA technology. The recombinant polynucleotide construct encoding the anti-CD33 antibodies of the present technology generally includes an expression control sequence operably linked to the coding sequence of the anti-CD33 antibody chain, and the expression control sequence includes a naturally associated or heterologous promoter region. Therefore, another aspect of the present technology includes a vector containing one or more nucleic acid sequences encoding the anti-CD33 antibodies of the present technology. For the 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 vector or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA technology well known in the art and described in detail 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.

[0189] In general, expression vectors that can be used in recombinant DNA technology are usually in the form of plasmids. In this disclosure, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector form. However, the present technology is intended to include other forms of expression vectors that are not technically plasmids and that 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 the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-CD33 antibody and for collecting and purifying the anti-CD33 antibody (e.g., cross-reactive anti-CD33 antibody). See US2002 / 0199213 in general. These expression vectors can generally be replicated in the host organism as an episome or as an integral part of the host chromosomal DNA. Typically, expression vectors contain a selection marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectin lyase, that can be used to direct secretion of the extracellular antibody fragment. See U.S. Patent No. 5,576,195.

[0190] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having CD33 binding properties, and the recombinant expression vector is in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences selected according to the host cell for expression, and the one or more regulatory sequences are operably linked to the nucleic acid sequence to be expressed. In the recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory sequences in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired polypeptide, and the like. Typical regulatory sequences that can be used as promoters for expression of recombinant polypeptides (e.g., anti-CD33 antibodies) include, for example, but are not limited to, promoters for 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters for alcohol dehydrogenase, isocytochrome C, and 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 the host cell. See U.S. Patent No. 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., anti-CD33 antibody, etc.).

[0191] 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 discussed in the following literature: Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods that can be used to prepare and screen for polypeptides (e.g., 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.

[0192] The expression of polypeptides in prokaryotes is most often carried out in Escherichia coli using a vector containing a constitutive or inducible promoter that guides the expression of fusion or non-fusion polypeptides. A fusion vector adds a number of amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors generally have three purposes: (i) increasing the expression of the recombinant polypeptide; (ii) increasing the solubility of the recombinant polypeptide; and (iii) assisting in 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 moiety and the recombinant polypeptide so that the recombinant polypeptide can be separated from the fusion moiety after purification of the fusion polypeptide. Such enzymes and their homologous 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, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0193] 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 INENZYMOLOGY 185, Academic Press, San Diego, CA (1990) 60-89). Pack et al., U.S. Patent Nos. 6,294,353; 6,692,935, have described methods for targeted assembly of different active peptides or protein domains via polypeptide fusion to produce multifunctional polypeptides. One strategy for maximizing expression of recombinant polypeptides (e.g., anti-CD33 antibodies) in E. coli is to express the polypeptide in a host bacterium that is impaired in its ability to proteolytically cleave the recombinant polypeptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990) 119-128. Another strategy is to change the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are those preferentially used in the expression host (e.g., E. coli) (see, for example, Wada et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such changes in the nucleic acid sequence of the present technology can be performed by standard DNA synthesis techniques.

[0194] 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, California) and picZ (Invitrogen Corp, San Diego, California). Alternatively, baculovirus expression vectors can be used to express anti-CD33 antibodies in insect cells. Baculovirus vectors that can be used to express polypeptides (eg, anti-CD33 antibodies) in cultured insect cells (eg, 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).

[0195] 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 are not limited to, pCDM8 (Seed, Nature 329:840, 1987) and pMT2PC (Kaufman et al., EMBO J. 6:187-195, 1987). When used in mammalian cells, the control functions of the expression vector are typically provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, 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 antibodies of the present technology, see, for example, 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, Chapters 16 and 17.

[0196] In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of the 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), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also encompassed, such as the mouse hox promoter (Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989).

[0197] Another aspect of the inventive method relates to a host cell into which a recombinant expression vector of the present 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 specific subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may not in fact be different from the parental cell, but are still included within the scope of the term as used herein.

[0198] 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 such 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 replication origins, promoters, enhancers, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89:49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0199] In some embodiments, the present invention relates to a method for the conversion of mammalian cells. The method of ...

[0200] 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 can integrate the exogenous DNA into their genome. In order to identify and select these integrants, a gene encoding a selectable marker (e.g., resistance to antibiotics) 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 stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells incorporating the selectable marker gene will survive, while other cells die).

[0201] Host cells (e.g., prokaryotic or eukaryotic host cells in culture) containing the anti-CD33 antibodies of the present technology can be used to produce (i.e., express) recombinant anti-CD33 antibodies. In one embodiment, the method comprises culturing host cells (into which a recombinant expression vector encoding the anti-CD33 antibody has been introduced) in a suitable culture medium, thereby producing the anti-CD33 antibody. In another embodiment, the method further comprises the step of isolating the anti-CD33 antibody from the culture medium or host cells. Once expressed, the anti-CD33 antibody, such as a collection of anti-CD33 antibodies or anti-CD33 antibody-related polypeptides, is purified from the culture medium and host cells. 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. Typically, the anti-CD33 antibody chain is expressed together with a signal sequence and thereby released into the culture 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, and the like (see generally Scopes, Protein Purification (Springer-Verlag, New York, 1982)).

[0202] Polynucleotides encoding anti-CD33 antibodies, such as the coding sequence of an anti-CD33 antibody, can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequently expressed in the milk of the transgenic animal. See, for example, U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes, such as casein or beta-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 transferred into enucleated oocytes.

[0203] 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 CD33 protein (see, for example, U.S. Patent No. 4,946,778). Examples of technologies 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.

[0204] Chimeric and humanized antibodies. In one embodiment, the anti-CD33 antibodies of the present technology are chimeric anti-CD33 antibodies. In one embodiment, the anti-CD33 antibodies of the present technology are humanized anti-CD33 antibodies. In one embodiment of the present technology, the donor antibody and the recipient antibody are monoclonal antibodies from different species. For example, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

[0205] Recombinant anti-CD33 antibodies comprising human and non-human portions (e.g., 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 the anti-CD33 antibodies of the present technology in humans and use of these agents in in vitro detection assays, 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 are not limited to, those described in 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 Nos. 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, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP 460 167), 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. Pat. No. 5,565,332). In one embodiment, the cDNA encoding the murine anti-CD33 monoclonal antibody is digested with specifically selected restriction endonucleases to remove sequences encoding the Fc constant region and substitute the 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 No. 6,180,370; U.S. Patent Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0206] In one embodiment, the present technology provides for the construction of humanized anti-CD33 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response while still possessing effective antibody effector function. As used herein, the terms "human" and "humanized" with respect to antibodies refer to any antibody that is expected to elicit a weak immunogenic response in a human subject that is therapeutically tolerable. In one embodiment, the present technology provides for humanized anti-CD33 antibodies, heavy chain and light chain immunoglobulins.

[0207] CDR antibodies. In some embodiments, the anti-CD33 antibodies of the present technology are anti-CD33 CDR antibodies. Typically, the donor and recipient antibodies used to generate anti-CD33 CDR antibodies are monoclonal antibodies from different species; typically, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. The graft may have a single V H or V L A single CDR (or even a portion of a single CDR) within a V H and V L In general, all three CDRs in all variable domains of the recipient antibody will be replaced by the corresponding donor CDRs, but only the required number of replacements is required so that the resulting CDR-grafted antibody fully binds to the CD33 protein. Methods for producing CDR-grafted and humanized antibodies are taught in the following literature: U.S. Pat. No. 5,585,089 to Queen et al.; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and U.S. Pat. No. 5,225,539 to Winter; and EP 0682040. It can be used to prepare V H and V L Polypeptide methods are taught in Winter et al., US Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP 0451216; and EP 0120694.

[0208] After selecting the suitable framework region candidate from the same family and / or same family member, one or both of the heavy chain and light chain variable regions are produced by transplanting the CDR from the species of origin into the hybrid framework region. Conventional methods well known to those skilled in the art can be used to complete the assembling of the hybrid antibody or hybrid antibody fragment with hybrid variable chain region about any of the above aspects. For example, the DNA sequence encoding the hybrid variable domains as described herein (that is, the framework based on the target species and the CDR from the species of origin) can be produced by oligonucleotide synthesis and / or PCR. Suitable restriction enzymes can also be used to separate the nucleic acid encoding the CDR region from the species of origin antibody, and be connected to the target species framework by being connected with a suitable ligase. Alternatively, the framework region of the variable chain of the species of origin antibody can be changed by site-directed mutagenesis.

[0209] Since hybrids are constructed from a selection 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 whose members have different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0210] This process typically does not alter the FRs of the recipient antibody flanking the transplanted CDRs. However, one skilled in the art can sometimes improve the antigen-binding affinity of the resulting anti-CD33 CDR-grafted antibody by replacing certain residues in a given FR to make the FR more similar to the corresponding FRs of the donor antibody. Suitable substitution positions include amino acid residues adjacent to the CDRs or amino acid residues that can interact with the CDRs (see, for example, US Pat. No. 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with the donor FRs and modify them to make them more similar to the recipient FRs or human consensus FRs. Techniques for making these modifications are known in the art. In particular, if the resulting FRs conform to the human consensus FRs at that position or are at least 90% or more identical to such consensus FRs, doing so may not significantly increase the antigenicity of the resulting modified anti-CD33 CDR-grafted antibody compared to the same antibody with fully human FRs.

[0211] Bispecific antibodies (BsAb). Bispecific antibodies are antibodies 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). BsAb can be prepared, for example, by combining heavy chains and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, through molecular function, a bispecific binding agent binds to one antigen (or epitope) on one of its two binding arms (a VH / VL pair) and binds to a different antigen (or epitope) on its second arm (a different VH / VL pair). According to this definition, a bispecific binding agent has two different antigen binding arms (both specificity and CDR sequence are different) and is monovalent for each antigen it binds.

[0212] The bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) 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, a T cell, a myeloid cell, a plasma cell, or a 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, BsAb is capable of binding to tumor cells that express the CD33 antigen on the cell surface. In some embodiments, BsAb has been engineered to promote killing of tumor cells by guiding (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen binding site specific for CD33 and a second antigen binding site specific for a small molecule hapten (e.g., DTP A, 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).

[0213] Use molecular engineering to produce a variety of bispecific fusion proteins.For example, the BsAb utilizing complete immunoglobulin framework (for example IgG), single-chain variable fragment (scFv) or its combination has been constructed.In some embodiments, bispecific fusion protein is divalent, and it comprises for example the scFv with a single binding site for a kind of antigen and the Fab fragment with a single binding site for a second antigen.In some embodiments, bispecific fusion protein is divalent, and it comprises for example the scFv with a single binding site for a kind of antigen and another scFv fragment with a single binding site for a second antigen.In other embodiments, bispecific fusion protein is tetravalent, and it comprises for example the immunoglobulin (for example IgG) with two binding sites for a kind of antigen and two identical scFv for a second antigen.The BsAb that has been shown to be composed of two series-connected scFv units is a clinically successful bispecific antibody form. In some embodiments, the BsAb comprises two single-chain variable fragments (scFv) in series, which are designed so that the scFv that binds to a tumor antigen (e.g., CD33) is connected to the scFv that engages T cells (e.g., by binding to CD3). In this way, T cells are recruited to the tumor site so that they can mediate cytotoxic killing of tumor cells. See, for example, Dreier et al., J. Immunol. 170: 4397-4402 (2003); Bargou et al., Science 321: 974-977 (2008)). In some embodiments, the BsAb of the present technology comprises two single-chain variable fragments (scFv) in series, which are designed so that the scFv that binds to a tumor antigen (e.g., CD33) is connected to the scFv that engages the small molecule DOTA hapten.

[0214] The latest methods for producing BsAbs include engineered recombinant monoclonal antibodies that have additional cysteine ​​residues so that they are more firmly cross-linked than more common immunoglobulin isotypes. See, for example, FitzGerald et al., Protein Eng. 10 (10): 1221-1225 (1997). Another method is engineered recombinant fusion proteins that connect two or more different single-chain antibodies or antibody fragment segments with the desired dual specificity. See, for example, Coloma et al., Nature Biotech. 15: 159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0215] Bispecific fusion proteins connecting 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 technology comprises an immunoglobulin containing a heavy chain and a light chain and an scFv. In some specific embodiments, the scFv is connected to the C-terminus of the heavy chain of any CD33 immunoglobulin disclosed herein. In some specific embodiments, the scFv is connected to the C-terminus of the light chain of any CD33 immunoglobulin disclosed herein. In various embodiments, the scFv is connected to the heavy chain or light chain via a linker sequence. By PCR reaction, the appropriate linker sequence necessary for in-frame connection of the heavy chain Fd to the scFv is introduced into the V L and V κ The DNA fragment encoding the scFv was then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct was excised and ligated to a V vector containing a DNA sequence encoding the CD33 antibody. H The resulting vector can be used to transfect appropriate host cells, such as mammalian cells, to express the bispecific fusion protein.

[0216] In some embodiments, the length of 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 some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but to provide flexibility (e.g., a first and / or second antigen binding site) for the polypeptide. In some embodiments, based on the specific characteristics of the BsAb, such as increased stability, a linker is used in the BsAb described herein. In some embodiments, the BsAb of the present technology comprises a G4S linker. In some specific embodiments, the BsAb of the present technology comprises (G4S) n A linker wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0217] Self-assembly decomposition (SADA) conjugate. In some embodiments, the anti-CD33 antibodies of the present technology include one or more SADA domains. SADA domains can be designed and / or customized to achieve environmentally dependent multimerization with beneficial kinetics, thermodynamics and / or pharmacological properties. For example, it has been recognized that a SADA domain can be a part of a conjugate that allows effective 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 may include a SADA domain connected to one or more binding domains. In some embodiments, such conjugates are characterized in that they multimerize to form a complex of desired size under relevant conditions (e.g., in a solution present when the conjugate is 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 a payload), and decompose into smaller forms under other conditions (e.g., in the absence of a relevant environmental multimerization trigger).

[0218] Compared with the conjugate without SADA domain, SADA conjugate can have improved characteristics. In some embodiments, the improved characteristics of polymer conjugate include: the increased affinity / combination to target, the increased specificity to target cell or tissue and / or the initial serum half-life of extension. In some embodiments, the improved characteristics include by dissociating into smaller states (for example, dimers or monomers), and SADA conjugates show reduced non-specific binding, reduced toxicity and / or improved renal clearance. In some embodiments, SADA conjugates include the SADA polypeptides with the amino acid sequence of the amino acid sequence of the human homologous multimeric polypeptide showing at least 75% homogeneity, and are determined by one or more polymer dissociation constants (K D ) representation.

[0219] In some embodiments, the SADA conjugate is constructed and arranged so 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 70kDa. In some embodiments, the first multimerization state is a non-multimerization state (e.g., a 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 multimerization state (e.g., a trimer or tetramer). In some embodiments, the high order multimerization state is a homotetramer or higher order homomultimer having a size greater than 150kDa. In some embodiments, when the high order homomultimerization conjugate is formed at a size greater than the SADA polypeptide K D In some embodiments, when the concentration of the SADA conjugate is lower than the concentration of the SADA polypeptide K DWhen the conjugate is activated, it switches from one or more higher-order multimerization states to a first multimerization state under physiological conditions.

[0220] In some embodiments, the SADA polypeptide is covalently linked to the binding domain via a joint. Any suitable joint known in the art can be used. In some embodiments, the SADA polypeptide is connected 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, wherein 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.

[0221] 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 that can associate in a parallel or antiparallel orientation. In some embodiments, the SADA polypeptide is capable of reversible multimerization. In some embodiments, the SADA domain is a tetramerization domain, a heptamerization domain, a hexamerization domain, or an octamerization domain. In certain embodiments, the SADA domain is a tetramerization domain. In some embodiments, the SADA domain is composed of a multimerization domain, each of which is composed of a helical bundle associated 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), 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-associated protein (CBFA2T1). Examples of suitable SADA domains are described in PCT / US2018 / 031235, which is hereby incorporated by reference in its entirety. The polypeptide sequences of exemplary SADA domains are provided below.

[0222] Human p53 tetramerization domain amino acid sequence (321-359)

[0223] KPLDGEYFTLQIRGRERRFEMFRELNEALELKDAQAGKEP(SEQ ID NO:125)

[0224] Human p63 tetramerization domain amino acid sequence (396-450)

[0225] RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ(SEQ ID NO:126)

[0226] Human p73 tetramerization domain amino acid sequence (348-399)

[0227] RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (SEQ ID NO: 127).

[0228] Human HNRNPC tetramerization domain amino acid sequence (194-220)

[0229] QAIKKELTQIKQKVDSLLENLEKIEKE(SEQ ID NO:128)

[0230] Human SNAP-23 tetramerization domain amino acid sequence (23-76)

[0231] STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL(SEQ ID NO:129)

[0232] Human Stefin B tetramerization domain amino acid sequence (2-98)

[0233] MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF(SEQ ID NO:130)

[0234] KCNQ4 tetramerization domain amino acid sequence (611-640)

[0235] DEISMMGRVVKVEKQVQSIEHKLDLLLGFY(SEQ ID NO:131)

[0236] CBFA2T1 tetramerization domain amino acid sequence (462-521)

[0237] TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH(SEQ IDNO:132)

[0238] In some embodiments, in some embodiments, the SADA polypeptide is or comprises a tetramerization domain of p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), synaptosome-associated protein 23 N-terminal domain (SNAP-23), Stefin B (cystatin 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 sequence shown in any one of SEQ ID NOs: 125-132.

[0239] Fc modification. In some embodiments, the anti-CD33 antibodies of the present technology comprise a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or a parent Fc region) such that the affinity of the molecule for an Fc receptor (e.g., FcγR) is altered, provided that the variant Fc region is not substituted at a position that directly contacts the Fc receptor, based on crystallographic and structural analyses of Fc-Fc receptor interactions (e.g., those disclosed in Sondermann et al., Nature, 406:267-273 (2000)). Examples of positions within the Fc region that directly contact an Fc receptor (e.g., FcγR) include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop.

[0240] In some embodiments, the anti-CD33 antibodies of the present technology have altered affinities for activating and / or inhibitory receptors, wherein the variant Fc region has one or more amino acid modifications, wherein the one or more amino acid modifications are N297 to alanine or K322 to alanine.

[0241] Glycosylation modifications. In some embodiments, the anti-CD33 antibodies of the present technology have an Fc region that contains variant glycosylation compared to a parent 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.

[0242] In some embodiments, the antibodies of the present technology may have modified glycosylation sites relative to an appropriate reference antibody that binds to an antigen of interest (e.g., CD33) without altering the functionality of the antibody, such as its binding activity to the antigen. As used herein, "glycosylation site" includes any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) will be specifically and covalently attached.

[0243] Oligosaccharide side chains are typically linked to the antibody backbone via N-linked or O-linked. N-linked glycosylation refers to the attachment of the oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of the oligosaccharide moiety to a hydroxyamino 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 specialized CHO cells and exhibits enhanced ADCC effector function.

[0244] 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 present technology, see, for example, U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US2002 / 0028486; International Patent Application Publication 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 all 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 portions of the antibody. In some specific embodiments, the present technology includes deleting a glycosylation site in the Fc region of an antibody by modifying the asparagine at position 297 to an alanine.

[0245] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms can be produced 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-acetylglucosamine transferase III (GnTIII)), by expressing molecules comprising an Fc region in various organisms or cell lines from various organisms, or by modifying one or more carbohydrates after a molecule comprising 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 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 Nos. 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 documents is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication No. WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0246] Fusion protein. In one embodiment, the anti-CD33 antibody of the present technology is a fusion protein. When fused with 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 polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion is not necessarily direct, but can be performed through a linker sequence. Moreover, the fusion protein of the present technology can also be engineered to improve the characteristics of the anti-CD33 antibody. For example, a region of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the anti-CD33 antibody to improve stability and durability during purification from host cells or subsequent processing and storage. In addition, a peptide portion can be added to the anti-CD33 antibody to facilitate purification. Such regions can be removed before the final preparation of the anti-CD33 antibody. Adding a peptide portion to facilitate the processing of the polypeptide is a conventional technique well known in the art. The anti-CD33 antibody of the present technology can be fused with a marker sequence (such as a peptide that promotes the purification of the fusion polypeptide). In selected embodiments, the marker amino acid sequence is a hexahistidine peptide, particularly as provided in the pQE vector (QIAGEN, Inc., Chatsworth, CA), many of which are commercially available. For example, hexahistidine allows for convenient purification of the fusion protein, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Another peptide tag that can be used for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984.

[0247] Thus, any of these aforementioned fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology.Additionally, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0248] Fusion proteins with a disulfide-linked dimeric structure (due to IgG) can bind and neutralize other molecules more efficiently than individual monomeric secreted proteins or protein fragments. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

[0249] Similarly, EP-AO 464 533 (Canadian counterpart 2 045 869) discloses fusion proteins comprising portions of the constant region of an immunoglobulin molecule and another human protein or fragment thereof. In many cases, the Fc portion of the fusion protein is beneficial in therapy and diagnosis, thus resulting in, for example, improved pharmacokinetic properties. See EP-A 0 232 262. Alternatively, it may be necessary 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 hinder 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.

[0250] Labeled anti-CD33 antibodies. In one embodiment, the anti-CD33 antibodies of the present technology are coupled to a labeling moiety, i.e., a detectable group. The specific label or detectable group conjugated to the anti-CD33 antibody is not a key aspect of the present technology, as 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 with 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. Therefore, 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.), radioactive labels (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 (e.g., 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.).

[0251] 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 sensitivity required, ease of conjugation to the compound, stability requirements, available instrumentation, and disposal regulations.

[0252] Non-radioactive labels are typically attached by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to a molecule. The ligand is then bound to an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Many ligands and anti-ligands can be used. Where a ligand (e.g., biotin, thyroxine, and cortisol) has a natural anti-ligand, the ligand can be used in conjunction with the naturally occurring anti-ligand of the label. Alternatively, any hapten or antigenic compound can be used in combination with an antibody such as an anti-CD33 antibody.

[0253] The molecule can also be directly conjugated to the compound that produces the signal, for example, by being conjugated to an enzyme or a fluorophore. The target enzyme as the label will mainly be a hydrolase, particularly a phosphatase, an esterase and a glycosidase, or an oxidoreductase, particularly a peroxidase. Fluorescent compounds that can be used as labeling 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 labeling moieties include, but are not limited to, for example, fluorescein and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labels or signal generating systems that can be used, see U.S. Patent No. 4,391,904.

[0254] The means of detecting labeling are well known to those skilled in the art. Therefore, for example, when labeling is radioactive labeling, detection means include scintillation counter or film, as in radioautography. When labeling is fluorescent labeling, it can be detected by exciting fluorescent dye with the light of appropriate wavelength and detecting the fluorescence produced. Fluorescence can be detected in visual form, by film, by using electronic detectors such as charge coupled device (CCD) or photomultiplier tube etc. Similarly, enzyme labeling can be detected by providing the appropriate substrate of enzyme and detecting the resulting reaction product. Finally, simple colorimetric labeling can be simply detected by observing the color relevant to labeling. Therefore, in various dip-dye rod assays, conjugated gold often appears pink, and the beads of various conjugations present the color of beads.

[0255] 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.

[0256] B. Identification and Characterization of Anti-CD33 Antibodies of the Present Technology

[0257] Methods for identifying and / or screening anti-CD33 antibodies of the present technology. Methods for identifying and screening antibodies against CD33 polypeptides for antibodies with desired specificity for CD33 protein (e.g., those that bind to the IgC2 domain of CD33) include any immune-mediated techniques known in the art. Components of the 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 radiolabeled target cells, and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2:373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen, and the presentation of the antigen on MHC can be detected by T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with an agent (which cross-links their Fc-ε receptors) and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); (5) enzyme-linked immunosorbent assay (ELISA).

[0258] Similarly, the products of the immune response in model organisms (e.g., mice) or human subjects can also be detected by various 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 scratch site (Peters et al., Blood, 72:1310-5, 1988); (3) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (4) 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 scratch site (Peters et al., Blood, 72:1310-5, 1988); (5) the production of antibodies in response to vaccination can be easily detected by ELISA; (6) the production of antibodies in response to vaccination can be easily detected by ELISA; (7) the production of antibodies in response to vaccination can be easily detected by ELISA; (8) the production of antibodies in response to vaccination can be easily detected by ELISA; (9) the production of antibodies in response to vaccination can be easily detected by ELISA; (10) the production of antibodies in response to vaccination can be easily detected by ELISA; (11) the production of antibodies in response to vaccination can be easily detected by ELISA; (12) the production of antibodies in response to vaccination can be easily detected by ELISA; (13) the production of antibodies in response to vaccination can be easily detected by ELISA; (14) the production of antibodies in response to vaccination can be easily detected by ELISA; (15) the production of antibodies in response to vaccination can be easily detected by ELISA; (16) the production of antibodies in response to vaccination can be easily detected by ELISA; (17) the production of antibodies in response to vaccination can be easily detected by ELISA; (18) the 3 H-thymidine measures the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) the differentiation of cells of the immune system can be measured by labeling PBMCs with antibodies to CD molecules (such as CD4 and CD8) and measuring the fraction of PBMCs that express these markers.

[0259] In one embodiment, the anti-CD33 antibodies of the present technology are selected by displaying CD33 peptides on the surface of replicable genetic packages. See, e.g., 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 605 522; EP 616 640; EP 1 024 191; EP 589 877; EP 774 511; EP 844 306. Methods for producing / selecting filamentous phage particles containing a phagemid genome encoding a binding molecule with a desired specificity have been described. See, for example, EP 774 511; US ​​5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0260] In some embodiments, anti-CD33 antibodies of the present technology are selected using display of CD33 peptides on the surface of yeast host cells. Methods that can be used to isolate scFv polypeptides by display on the surface of yeast have been described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0261] In some embodiments, anti-CD33 antibodies of the present technology are selected using ribosome display. Methods that can be used to identify ligands in peptide libraries using ribosome display 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.

[0262] In certain embodiments, tRNA display of CD33 peptides is used to select anti-CD33 antibodies of the present technology. Methods that can be used for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. Biol., 9:741-46, 2002.

[0263] In one embodiment, anti-CD33 antibodies of the present technology are selected using RNA display. Methods that can be used to select 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 that can be used to select peptides and proteins using non-natural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13:506-12, 2003.

[0264] In some embodiments, an anti-CD33 antibody of the present technology is expressed in the periplasm of Gram-negative bacteria and mixed with a labeled CD33 protein. See WO 02 / 34886. In clones expressing a recombinant polypeptide with affinity for the CD33 protein, the concentration of the labeled CD33 protein bound to the anti-CD33 antibody is increased, 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.

[0265] After the desired anti-CD33 antibody is selected, it is expected that the antibody can be produced in large quantities by any technique known to those skilled in the art (e.g., prokaryotic or eukaryotic 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 an antibody heavy chain using conventional techniques, in which a minimal portion of the CDRs and (if necessary) the variable region framework required to retain the binding specificity of the originating species antibody (engineered according to the techniques described herein) is derived from the originating species antibody, and the remainder of the antibody is derived from a target species immunoglobulin that can be manipulated as described herein, thereby generating a vector for expressing the hybrid antibody heavy chain.

[0266] Measurement of CD33 binding. In some embodiments, a CD33 binding assay refers to an assay format in which a 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 the CD33 protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassays, proximity scintillation assays, fluorescence energy transfer assays, liquid chromatography, membrane filtration assays, and the like. Biophysical assays for directly measuring CD33 protein bound to anti-CD33 antibodies include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips), and the like. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, and the like. If the specific binding of the candidate anti-CD33 antibody is at least 1% greater than the binding observed in the absence of the candidate anti-CD33 antibody, the candidate anti-CD33 antibody can be used as an anti-CD33 antibody against the present technology. In some embodiments, the CD33 protein is a short isoform of CD33 (a splice variant lacking the IgV domain).

[0267] Uses of anti-CD33 antibodies of this technology

[0268] 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 proteins (e.g., for measuring the level of CD33 protein in appropriate physiological samples, for diagnostic methods, for polypeptide imaging, etc.). The antibodies of the present technology can be used to separate CD33 proteins by standard techniques such as affinity chromatography or immunoprecipitation. The anti-CD33 antibodies of the present technology can facilitate the purification of naturally immunoreactive CD33 proteins from biological samples such as mammalian serum or cells, as well as the purification of recombinantly produced immunoreactive CD33 proteins expressed in host systems. In addition, the anti-CD33 antibodies can be used to detect immunoreactive CD33 proteins (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 proteins in tissues as part of a clinical testing procedure, for example, to determine the efficacy of a given treatment regimen. As described above, detection can be facilitated by coupling (i.e., physically linking) the anti-CD33 antibodies of the present technology to a detectable substance.

[0269] Detection of CD33 protein. An exemplary method for detecting the presence of an immunoreactive CD33 protein in a biological sample involves 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 an immunoreactive CD33 protein, thereby detecting the presence of the immunoreactive CD33 protein in the biological sample. Detection can be accomplished by attaching a detectable label to the antibody.

[0270] The term "labeled" with respect to anti-CD33 antibodies is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound (e.g., a secondary antibody) that is directly labeled. Examples of indirect labeling include detection of the primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected using fluorescently labeled streptavidin.

[0271] In some embodiments, the anti-CD33 antibodies disclosed herein are conjugated to 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.

[0272] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0273] 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 et al. 111 In is an exemplary isotope when used for in vivo imaging because it avoids 125 I or 131 The problem of CD33 binding antibodies labeled with I being dehalogenated by the liver is solved. 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, a monoclonal antibody conjugated with 1-(P-isothiocyanatobenzyl)-DPTA is 111 In exhibits minimal uptake in non-tumor tissues (particularly the liver) and enhances tumor localization specificity (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe.

[0274] 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 toxin and cholera toxin.

[0275] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0276] The detection methods of the present technology can be used to detect immunoreactive CD33 proteins in biological samples in vitro and in vivo. In vitro techniques for detecting immunoreactive CD33 proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, and immunofluorescence. In addition, in vivo techniques for detecting immunoreactive CD33 proteins include introducing labeled anti-CD33 antibodies into a subject. For example, the anti-CD33 antibodies can be labeled with a radioactive marker, the presence and location of which in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains CD33 protein molecules from the test subject.

[0277] 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 (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096, 1987. Other antibody-based methods that can be used to detect protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). 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), as well as biotin.

[0278] In addition to measuring the level of immunoreactive CD33 protein 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 X-ray radiography, NMR or ESR. For X-ray radiography, suitable labels include radioisotopes, such as barium or cesium, which emit detectable radiation but are not significantly harmful to the subject. Labels suitable for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the anti-CD33 antibody by labeling nutrients used for the relevant scFv clone.

[0279] The cells have been treated with an appropriate detectable imaging moiety such as a radioisotope (e.g. 131 I. 112 In, 99 An anti-CD33 antibody labeled with a radiopaque substance (e.g., mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance) is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into a subject. As will be understood in the art, the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for human subjects, the amount of radioactivity injected is typically between about 5 and 20 millicuries. 99 The labeled anti-CD33 antibodies will then accumulate in cells containing the specific target polypeptide. For example, the labeled anti-CD33 antibodies of the present technology will accumulate in cells and tissues in the subject where the CD33 protein has been localized.

[0280] Thus, the present technology provides methods for diagnosing a medical condition, which involve: (a) determining the expression of an immunoreactive CD33 protein by measuring the binding of an anti-CD33 antibody of the present technology in cells or body fluids of an individual; and (b) comparing the amount of immunoreactive CD33 protein present in the sample with a standard reference, wherein an increase or decrease in the level of the immunoreactive CD33 protein compared to the standard is indicative of a medical condition.

[0281] Affinity purification. The anti-CD33 antibodies of the present technology can be used to purify immunoreactive CD33 proteins from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics (such as polycarbonates), complex carbohydrates (such as agarose and sepharose), acrylic resins, and beads such as polyacrylamide and latex. Techniques for coupling antibodies 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)).

[0282] The simplest method of combining antigen with antibody-support matrix is ​​to collect beads in a column, and make the antigen solution pass through the column downwards. The efficiency of this method depends on the contact time between immobilized antibody and the antigen, and can extend the contact time by using a low flow rate. Immobilized antibody captures the antigen when the antigen flows through. Alternatively, antigen can be contacted with the antibody support matrix in the following manner: antigen solution is mixed with a support (for example, beads) and rotated or shaken slurry, thereby realizing maximum contact between the antigen and the immobilized antibody. After binding reaction is completed, slurry is passed in a column to collect beads. Use suitable washing buffer to wash beads, then elute pure or substantially pure antigen.

[0283] The antibody or polypeptide of interest can be conjugated to a solid support (e.g., 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 of the 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, wherein the first and second solid supports can be the same or different.

[0284] Suitable linkers (which may be cross-linking 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 cross-linking agents include homobifunctional agents and particularly heterobifunctional agents. Useful bifunctional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. The cross-linking agent can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, a photolabile cross-linking agent such as 3-amino-(2-nitrophenyl) propionic acid can be used as a means of 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. Pat. No. 5,643,722). Other cross-linking agents are well known in the art. (See, eg, Wong (1991), supra; and Hermanson (1996), supra).

[0285] Antibodies or polypeptides can be immobilized on solid supports (such as beads) by covalent amide bonds formed between carboxyl-functionalized beads and the amino terminus of the polypeptide, or conversely by covalent amide bonds formed between amino-functionalized beads and the carboxyl terminus of the polypeptide. In addition, bifunctional trityl linkers can be attached to the support via amino resins through amino or carboxyl groups on the resin, such as 4-nitrophenyl active esters attached to resins (such as Wang resins). When using the bifunctional trityl method, the solid support may need to be treated with volatile acids (such as formic acid or trifluoroacetic acid) to ensure that the polypeptide is cut and can be removed. In this case, the polypeptide can be deposited on the bottom of the hole of the solid support or on the flat surface of the solid support as a plaque without beads. After adding the matrix solution, the polypeptide can be desorbed into the MS.

[0286] Hydrophobic trityl linkers can also be used as acid-labile linkers by cleaving the amino-linked trityl group from the polypeptide using a volatile acid or an appropriate matrix solution (e.g., a matrix solution containing 3-HPA). Acid instability can also be altered. For example, the trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl group can be replaced with 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 or by cleaving the trityl ether bond or the tritylamine bond under acidic conditions, including (if necessary) under typical MS conditions, where a matrix such as 3-HPA is used as the acid.

[0287] Orthogonal cleavable linkers can also be used to bind a first solid support (e.g., beads) to a second solid support, or can be used to bind a polypeptide of interest to a solid support. Using such linkers, a first solid support (e.g., beads) can be selectively cleaved from a 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 bind beads to a second solid support, and an acid-cleavable bifunctional trityl group can be used to fix the polypeptide to the support. As needed, the connection between the polypeptide and the solid support can be cleaved first, for example, to keep the connection between the first and second supports intact. Trityl linkers can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions.

[0288] For example, beads can be bound to a second support via a linking group, and a linking group having a length and chemical properties can be selected that promotes high-density binding of beads to a solid support or high-density binding of polypeptides to beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing multiple functional groups for each attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythrole, and trihydroxyaminomethane.

[0289] Non-covalent binding association. Through non-covalent interactions, antibodies or polypeptides can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of ferromagnetic materials that can be magnetized can be attracted to a magnetic solid support and can be 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 the polypeptide (e.g., a polypeptide containing an attached trityl group) or with a second solid support having hydrophobic characteristics, respectively.

[0290] The solid support may also have members of a specific binding pair and thus may be conjugated to a polypeptide containing a complementary binding moiety or to a second solid support. For example, beads coated with avidin or with streptavidin may be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or a biotin derivative such as iminobiotin.

[0291] It should be appreciated 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 solid support, and conversely, avidin or other biotin-binding moiety can be incorporated into a support or polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and their specifically interacting antigens, and other such pairs known to those skilled in the art.

[0292] A. Diagnostic Uses of Anti-CD33 Antibodies of the Present Technology

[0293] Overview. The anti-CD33 antibodies of the present technology can be used in diagnostic methods. Thus, the present technology provides methods for diagnosing CD33 activity in a subject using the antibodies. The anti-CD33 antibodies of the present technology can be selected such that they have any level of epitope binding specificity and very high binding affinity for the CD33 protein. Generally speaking, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed in the immunoassay to remove non-specifically bound material without removing the target polypeptide. Thus, the anti-CD33 antibodies of the present technology used in diagnostic assays typically have a specificity of about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1 Furthermore, it is desirable that anti-CD33 antibodies for use as diagnostic reagents have sufficient kinetic association rates to reach equilibrium under standard conditions within at least 12 h, at least five (5) h, or at least one (1) hour.

[0294] Anti-CD33 antibodies can be used to detect immunoreactive CD33 protein in a variety of standard assay formats, including immunoprecipitation, Western blot, ELISA, radioimmunoassay, and immunoassay. 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 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.

[0295] Immunometric assays or sandwich assays are a form of 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 anti-CD33 antibody population) fixed to a solid phase and another anti-CD33 antibody or anti-CD33 antibody population in solution. Typically, the solution anti-CD33 antibody or anti-CD33 antibody population is labeled. If an antibody population is used, the population can contain antibodies that bind specifically to different epitopes within the target polypeptide. Therefore, the same population can be used for both solid phase and solution antibodies. If anti-CD33 monoclonal antibodies are used, first and second CD33 monoclonal antibodies with different binding specificities are used for the solid phase and solution phase. The solid phase (also referred to as "capture") and solution (also referred to as "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 referred to as a simultaneous assay. After contacting the CD33 protein with the anti-CD33 antibody, the sample is incubated for a period of time, typically ranging from about 10 minutes to about 24 hours, and typically about 1 hour. A washing step is then performed to remove components of the sample that are not specifically bound to the anti-CD33 antibody used as a diagnostic reagent. When the solid phase antibody and the solution antibody are combined in separate steps, washing can be performed after either or both of the binding steps. After washing, binding is quantified, typically by detecting the label attached to the solid phase via binding of the labeled solution antibody. Typically, for a given antibody pair or antibody population and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of the target antigen. The concentration of immunoreactive CD33 protein in the sample being tested is then read by interpolation from the calibration curve (i.e., standard curve). The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurement of the bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of this curve is a measure of the CD33 protein concentration in the sample.

[0296] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, and also particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX TM (Amersham Pharmacia Biotech, Piscataway, NJ) 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) for attachment to a surface-bound linker (such as avidin).

[0297] In some embodiments, the present disclosure provides anti-CD33 antibodies of the present technology conjugated to a diagnostic agent. The diagnostic agent can comprise a radioactive or non-radioactive label, a contrast agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label can be a gamma, beta, alpha, Auger electron, or positron emitting isotope. The diagnostic agent is a molecule that is administered conjugated to an antibody portion, i.e., an antibody or antibody fragment or subfragment, and can be used to diagnose or detect disease by localizing cells containing the antigen.

[0298] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules and the enhancers (such as paramagnetic ions) of magnetic resonance imaging (MRI). U.S. Patent number 6,331,175 describes MRI technology and the preparation of antibodies conjugated with MRI enhancers, and is incorporated by reference in its entirety. In some embodiments, diagnostic agents are selected from radioisotopes, enhancers for magnetic resonance imaging and fluorescent compounds. In order to make the antibody component load radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail, the long tail being attached with a variety of chelating groups for binding ions. This tail can be a polymer, such as polylysine, polysaccharides or other derivatization or derivatizable chains, the polymer having a side group that can be combined with a chelating group, the chelating group being, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes and similar groups known to be useful for this purpose. Chelates can be coupled to the antibodies of the present technology using standard chemical methods. Chelates are typically linked to antibodies via a group that is capable of forming a bond with the molecule with minimal loss of immunoresponsiveness and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used for radioimaging with diagnostic isotopes. When complexed with non-radioactive metals such as manganese, iron, and gadolinium, the same chelates can be used for MRI when used with the CD33 antibodies of the present technology.

[0299] Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), respectively, have been used with a variety of metals and radioactive metals such as radionuclides of gallium, yttrium, and copper. Such metal-chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Other examples of DOTA chelates include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-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(H SG)-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-L ys(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-C ys)-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(H SG)-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(D TPA)-D-Lys(Tscg-Cys)-NH2; and (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2. ;

[0300] Also considered are the stable binding nuclides (e.g., for RAIT) 223 Other cyclic chelates of interest include macrocyclic polyethers.

[0301] B. Therapeutic Uses of Anti-CD33 Antibodies of the Present Technology

[0302] 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 for 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 CD33-related cancer or Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to, AML, biphenotypic leukemia, bilineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

[0303] The compositions of the present technology can be used in combination with other therapeutic agents that can be used to treat 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, anti-estrogen drugs, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutics, and targeted biological therapeutics (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, a Nastrazole, exemestane, letrozole, leuprolide, 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 poison, annonaceous lactone, or a combination thereof.

[0304] The compositions of the present technology can optionally be administered to a subject in need thereof as a single bolus injection. Alternatively, the dosing regimen can include multiple administrations at different times after the tumor appears.

[0305] The method for treating Alzheimer's disease may further comprise administering to the subject sequentially, separately or simultaneously at least one additional therapy selected from donepezil, galantamine, memantine, rivastigmine, memantine extended release and donepezil, aducanumab, solanezumab, insulin, verubecestat, AADvacl, CSP-1103 and intepirdine.

[0306] 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 is also understood that the various treatment modalities for medical conditions as 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.

[0307] In some embodiments, the antibodies of the present technology comprise pharmaceutical formulations that 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).

[0308] Typically, the effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect is within the range of about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is 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 about 0.0001 to 100 mg / kg of subject body weight per week, every two weeks, or every three weeks, and more typically 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 per week, every two weeks, or every three weeks, or within the range of 1-10 mg / kg per week, every two weeks, or every three weeks. In one embodiment, the single dose range of the antibody is 0.1-10,000 micrograms per kilogram of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter of delivery. Exemplary treatment regimens require administration once every two weeks, once a month, or once every 3 to 6 months. Anti-CD33 antibodies can be administered on multiple occasions. The intervals between individual doses can be hourly, daily, weekly, monthly, or annual. Intervals can also be irregular, as indicated by measuring blood levels of the antibody in the subject. In some methods, the dose is adjusted to achieve the following serum antibody concentration 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 dose and frequency vary depending on the half-life of the antibody in the subject. The dose and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, it is sometimes necessary to administer a relatively high dose at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

[0309] 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 thereof) of the present technology, wherein the antibody is configured to localize to a tumor expressing CD33 and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (% ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and calculating the mean radioactivity level ± standard deviation present in the non-tumor (normal) tissue. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0310] In some embodiments, the subject is diagnosed with or suspected of having cancer.The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0311] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha-emitting isotope, a beta-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta-emitting isotopes include 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re、 188 Re、 177 Lu and 67 Cu. Examples of alpha-emitting isotopes include 213 Bi, 211 At 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At and 255Examples of Fm. 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 In some embodiments of the method, non-specific FcR-dependent binding in normal tissue is eliminated or reduced (e.g., via an N297A mutation in the Fc region, which results in deglycosylation). The therapeutic effectiveness of the immunoconjugate can be determined by calculating the area under the curve (AUC) tumor: AUC normal tissue ratio. In some embodiments, the immunoconjugate has an AUC tumor: AUC normal tissue ratio of about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1, or 100: 1.

[0312] 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 the solid tumor in the subject by detecting a level of radioactivity emitted by the complex that is above a reference value. In some embodiments, the subject is a human.

[0313] 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 a 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.

[0314] 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 alpha-emitting isotope, a beta-emitting isotope, 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.

[0315] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with, or is suspected of having, Alzheimer's disease or a CD33-associated cancer, such as AML, biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

[0316] Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intraventricularly, orally, intratumorally, or intranasally. In certain embodiments, the complex is administered to the cerebrospinal fluid or blood of the subject.

[0317] In some embodiments of the methods disclosed herein, the radioactivity 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 radioactivity level emitted by the complex is expressed as a percentage of the injected dose per gram of tissue (% ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissue and calculating the average radioactivity level ± standard deviation present in non-tumor (normal) tissue. In some embodiments, the reference value is a standard uptake value (SUV). See Thie JA, J Nucl Med. 45(9): 1431-4(2004). In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0318] In another aspect, the present disclosure provides a method for increasing the sensitivity of a tumor to radiation therapy 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 a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human.

[0319] The anti-DOTA bispecific antibody is administered under conditions and for a period of time (e.g., according to a dosing regimen) sufficient to allow it to saturate the tumor cells. In some embodiments, after administration of the anti-DOTA bispecific antibody, unbound anti-DOTA bispecific antibody is removed from the bloodstream. In some embodiments, a radiolabeled DOTA hapten is administered after a period of time sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0320] The radiolabeled DOTA hapten can be administered any time between 1 minute and 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range thereof, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time after 4 or more days after administration of the anti-DOTA bispecific antibody.

[0321] Additionally or alternatively, 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 scavenger can be any molecule (glucan or dendrimer or polymer) that can be conjugated 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.).

[0322] In some embodiments, the scavenger and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody of the present technology. For example, in some embodiments, the anti-DOTA bispecific antibody of the present technology is administered according to a regimen comprising at least one of the following cycles: (i) administration of the anti-DOTA bispecific antibody of the present technology (optionally, to saturation of relevant tumor cells); (ii) administration of a radiolabeled DOTA hapten and an optional scavenger; (iii) optional additional administration of the radiolabeled DOTA hapten and / or the scavenger without additional administration of the anti-DOTA bispecific antibody. In some embodiments, the method can include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cycles).

[0323] Additionally or alternatively, in some embodiments of the methods, the anti-DOTA bispecific antibody and / or the radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, intratumorally, orally, or intranasally.

[0324] 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 present technology that recognizes and binds to 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 can be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments, the subject is a human.

[0325] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a CD33 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. The anti-DOTA bispecific antibody is administered under conditions and for a time (e.g., according to a dosing regimen) sufficient to allow it to saturate tumor cells. In some embodiments, after administration of the anti-DOTA bispecific antibody, unbound anti-DOTA bispecific antibody is removed from the bloodstream. In some embodiments, the radiolabeled DOTA hapten is administered after a time period sufficient to allow clearance of unbound anti-DOTA bispecific antibody. In some embodiments, the subject is human.

[0326] Thus, in some embodiments, the method further comprises administering to the subject an effective amount of a clearing agent prior to administering the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered any time between 1 minute and 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range thereof, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time after 4 or more days after administration of the anti-DOTA bispecific antibody.

[0327] The clearing agent can be a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In), etc.). In some embodiments, the clearing agent 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) administration of an anti-DOTA bispecific antibody of the present technology (optionally, to saturation of relevant tumor cells); (ii) administration of a radiolabeled DOTA hapten and, optionally, a clearing agent; (iii) optionally, additional administration of the radiolabeled DOTA hapten and / or the clearing agent without additional administration of the anti-DOTA bispecific antibody. In some embodiments, the method can include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cycles).

[0328] Also provided herein are methods for treating cancer in a subject in need thereof, 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 to 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 therapeutic effectiveness of such a complex can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the complex has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0329] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-CD33 antibody described herein will provide therapeutic benefit without causing substantial toxicity to the subject. The toxicity of the anti-CD33 antibodies described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by measuring the LD 50 (dose lethal to 50% of the population) or LD 100The therapeutic index is determined by the dose ratio between toxicity and therapeutic effect (the dose that is lethal to 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dose range that is non-toxic to humans. The dose of the anti-CD33 antibodies described herein is within a range of circulating concentrations that includes an effective dose with little or no toxicity. The dose can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the subject's condition. See, for example, Fingl et al., The Pharmacological Basis of Therapeutics, Chapter 1 (1975).

[0330] Formulation of pharmaceutical compositions. According to the methods of the present technology, the anti-CD33 antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions typically comprise recombinant or substantially purified antibodies and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier depends, in part, on the specific composition being administered, as well as on the specific method for administering the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th edition, 1990). Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0331] The terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, when they relate to compositions, carriers, diluents, and reagents, are used interchangeably and indicate that the material is capable of being administered to or onto a subject without producing undesirable physiological effects to the extent that would prohibit administration of the composition. For example, a "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 that are acceptable for veterinary as well as human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, a gas. "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. 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 alkanesulfonic and arenesulfonic acids such as methanesulfonic acid and benzenesulfonic acid. Pharmaceutically acceptable esters include esters formed with carboxyl, sulfonyloxy, and phosphonoxy groups present in the anti-CD33 antibody, for example, C 1-6 Alkyl ester. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid monosalt or ester or a disalt or ester; and similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The anti-CD33 antibodies named in this technology may exist in an unsalted or unesterified form, or in a salified and / or esterified form, and the naming of such anti-CD33 antibodies is intended to include the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. In addition, certain embodiments of the present technology may exist in more than one stereoisomer form, and the naming of such anti-CD33 antibodies is intended to include all single stereoisomers and all mixtures of such stereoisomers (whether racemic or otherwise). It is not difficult for one of ordinary skill in the art to determine the appropriate timing, sequence, and dosage for administering a particular drug and composition of the present technology.

[0332] 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, may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional media or compounds are incompatible with the anti-CD33 antibody, their use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0333] The pharmaceutical compositions of the present technology are formulated to be compatible with their intended route of administration. The anti-CD33 antibody compositions of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally, or intramuscularly, or as an inhalant. The anti-CD33 antibodies can optionally be administered in combination with other agents that are at least partially effective in treating various CD33-related cancers or Alzheimer's disease.

[0334] 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; an antibacterial compound such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate or phosphate, and a compound for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The parenteral formulation can be packaged in an ampoule, disposable syringe or multiple dose vial made of glass or plastic.

[0335] 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 physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Percival, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should 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 prevent the contaminating effects of microorganisms such as bacteria and fungi during storage. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., 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 desired particle size in the case of a dispersion, and by using a surfactant. Protection against the effects of microorganisms can be achieved by various antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), sodium chloride. Prolonged absorption of the injectable composition can be achieved by including compounds that delay absorption, such as aluminum monostearate and gelatin in the composition.

[0336] Sterile injectable solutions can be prepared by incorporating the anti-CD33 antibody of the present technology in the desired amount into an appropriate solvent optionally containing one or a combination of the ingredients listed above, followed by sterilization by filtration. Typically, a dispersion is prepared by incorporating the anti-CD33 antibody into a sterile vehicle containing a base dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution. The antibodies of the present technology can be administered in the form of a depot injection or implant formulation, which can be formulated in a manner that allows for sustained or pulsatile release of the active ingredient.

[0337] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, anti-CD33 antibodies 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 used as a mouthwash, wherein the compound in the fluid carrier is administered orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants 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 mint, methyl salicylate, or orange flavor.

[0338] For administration by inhalation, the anti-CD33 antibody is delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0339] Systemic administration can also be performed transmucosally or transdermally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are 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 using nasal sprays or suppositories. For transdermal administration, the anti-CD33 antibody is formulated into an ointment, salves, gel, or cream as generally known in the art.

[0340] Anti-CD33 antibodies can also be prepared as pharmaceutical compositions for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0341] In one embodiment, the anti-CD33 antibody is prepared with a carrier that prevents rapid clearance of the anti-CD33 antibody from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0342] C. Kit

[0343] The present technology 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). Optionally, the above components of the kit of the present technology are packaged in a suitable container and labeled for use in the diagnosis and / or treatment of CD33-related cancer 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) as an aqueous solution (preferably a sterile solution) or as a lyophilized (preferably sterile) formulation for reconstitution. The kit may also comprise a second container containing a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients and saline solutions for the pharmaceutical composition. In addition, the kit may comprise 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 (for example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The test kit can also include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The test kit can also include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, culture media for one or more suitable hosts. The test kit can optionally include instructions typically included in commercial packaging for therapeutic or diagnostic products, containing information about, for example, the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings for using such therapeutic or diagnostic products.

[0344] The kit can be used to detect the presence of immunoreactive CD33 protein in a biological sample, such as any body fluid, including but not limited to serum, plasma, lymph, cystic fluid, urine, feces, cerebrospinal fluid, ascites, or blood, and including a biopsy sample of body tissue. For example, the kit can include: 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 a biological sample; a device for determining the amount of CD33 protein in a sample; and a device for comparing the amount of immunoreactive CD33 protein in a sample with a standard. One or more of the anti-CD33 antibodies can be labeled. The kit components (e.g., reagents) can be packaged in suitable containers. The kit can also include instructions for using the kit to detect immunoreactive CD33 protein.

[0345] For antibody-based kits, the kit can comprise, for example, 1) a first antibody, such as a humanized, chimeric, or bispecific CD33 antibody (or antigen-binding fragment thereof) of the present technology, attached to a solid support, which first antibody binds to a CD33 protein; and, optionally; 2) a different second antibody that binds to a CD33 protein or the first antibody and is conjugated to a detectable label.

[0346] The kit may also contain, for example, a buffer, a preservative, or a protein stabilizer. The kit may also contain other components required for detection of the detectable marker, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples, which can be assayed and compared with the test sample. Each component of the kit can be enclosed in a separate container, and all the different containers can be placed in a single package together with instructions for interpreting the results of assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, for example, for detecting CD33 protein in vitro or in vivo, or for treating CD33-related cancers in subjects in need thereof. In certain embodiments, the reagents can be used according to the methods of the present technology.

[0347] Example

[0348] 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 generation of chimeric, humanized, and bispecific antibodies of the present technology, as well as the characterization of their binding specificity and in vitro and in vivo biological activities.

[0349] Example 1: Structure and Binding Affinity of Chimeric and Humanized Anti-CD33 Antibodies of the Disclosure

[0350] Humanization of murine HIM34. The CDRs of the HIM34 heavy and light chains were transplanted onto the human IgG1 framework based on their homology to the human frameworks IGHV1-2*02IGHJ4*01 (for VH) and IGKV1-33*01-IGKJ2*01 (for VL), respectively. Thirty versions of huHIM34 were gene-synthesized based on six heavy chains and five light chains and expressed in DG44 cells. The amino acid sequences of the murine, chimeric, and humanized HIM34 variable heavy chains are shown in SEQ ID NOs 2-7 and 114, and Figure 8 and Figure 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 Figure 10(A).

[0351] Generation of CD33-BsAb bispecific antibodies. Design of CD33-BsAb using IgG-scFv format ( Figure 3 CD33-BsAb was constructed by fusing a 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 abolish 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 is then transfected into CHO-S cells and stable clones are selected for high antibody production levels. For larger-scale antibody purification, the selected stable clones are amplified in shake flasks. The bispecific antibody is purified from the supernatant using one-step protein A affinity chromatography.

[0352] FIG10 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) Shown are the light and heavy chains of the following four humanized CD33-BsAbs (hHIM34-BsAbs): BC249, BC275, BC267, and BC268, respectively. Figure 11(A) to Figure 11(B) The light and heavy chain amino acid sequences of BC249 are shown, corresponding to SEQ ID NO: 18 and SEQ ID NO: 20, respectively, and are based on humanized HIM34 variable light chain VL-3 and variable heavy chain VH-5. Figure 11(C) to Figure 11(D) The light and heavy chain amino acid sequences of BC275 are shown, corresponding to SEQ ID NO: 134 and SEQ ID NO: 136, respectively, and are based on humanized HIM34 variable light chain VL-4 and variable heavy chain VH-5.

[0353] Figure 12(A) to Figure 12(B)The light and heavy chain amino acid sequences of BC267 are shown, corresponding to SEQ ID NO: 18 and SEQ ID NO: 22, respectively, and are based on humanized HIM34 variable light chain VL-3 and variable heavy chain VH-6. Figure 13(A) to Figure 13(B) The light and heavy chain amino acid sequences of BC268 are shown, corresponding to SEQ ID NO: 24 and SEQ ID NO: 22, respectively, and are based on humanized HIM34 variable light chain VL-4 and variable heavy chain VH-6.

[0354] The binding kinetics of chimeric and humanized anti-CD33 antibodies were compared by surface plasmon resonance. To determine whether the humanized anti-CD33 HIM34 antibody binds to CD33 with an affinity equal to that of the chimeric form, surface plasmon resonance assays were performed using a Biacore T-200. Biacore T-200 biosensors, CM5 sensor chips, and related reagents were purchased from GE Healthcare. CD33 as an active surface and a blank as a reference were immobilized onto a CM5 sensor chip at approximately 1000 RU (response units) using an amino coupling kit (GE Healthcare). CD33 antibodies and related control antibodies were diluted in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20) at various concentrations (31 to 500 nM) before analysis. Samples were injected onto the sensor surface at a flow rate of 30 μL / min for 1 min. After the association phase was complete, dissociation was monitored for 3 min at the same flow rate in HBS-EP buffer, or for 30 min at 37°C for antibodies with a slow koff. At the end of each cycle, the surface was regenerated using 10 mM NaOH at a flow rate of 50 μL / min for 30 seconds. The biosensor curve obtained after injecting the sample on the active surface was subtracted from the control curve obtained with the sample injected on the reference surface before kinetic analysis. Data were analyzed using Biacore T-200 evaluation software. Figure 16(A) to Figure 16(B) As shown in Figure 2, the humanized anti-CD33 HIM34 antibody has a similar k value to the chimeric form. on and k off combination.

[0355] 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.

[0356] Example 2: Biological activity of T cell-engaged CD33-BsAb antibodies of the present technology

[0357] Chimeric CD33-BsAb binds to AML cell lines. Binding of chHIM34 BsAb to target cells was tested by FACS immunostaining. It binds to CD33(+) AML cell lines U937, THP1, SET2, C1498-CD33, and M-07e, while leaving the CD33(-) leukemia cell line CMLT1 ( Figure 4 ).

[0358] The chimeric CD33-BsAb binds to both the splice variant and the full-length CD33. The full-length CD33 molecule has two extracellular domains: a membrane distal IgV domain and a membrane proximal IgC2 domain ( Figure 1 There are no FDA-approved antibodies that bind to the CD33 splice variant lacking the IgV domain ( Figure 2 Importantly, since 50% of AML patients have spliced ​​CD33 proteins, they cannot benefit from current CD33 antibody drugs. The chimeric CD33-BsAb of the present disclosure, chHIM34 BsAb, binds to both full-length and splice variants of CD33, while the humanized M195-based BsAb (BC133) does not bind to splice variant CD33 ( Figure 5(A) to Figure 5(B) ). Figure 19 Shown is the stability of a humanized BsAb of the present technology (hHIM34 x CD3) at 40° C. determined using HPLC. Of all clones tested, the hHIM34L4H5 (BC275) clone showed the highest stability at 40° C.

[0359] Chimeric CD33-BsAb redirects T cells to kill CD33(+) AML cell lines. To evaluate whether chimeric CD33-BsAb can redirect T cells to kill leukemia cells, T cell cytotoxicity against CD33(+) AML cells was tested in a standard 4-hour 51Cr release assay. In the presence of chHIM34 BsAb, significant killing of AML cell lines was observed, with EC50 as low as 1 fM (for C1498-CD33) to 200 fM (for THP1) ( Figure 6(A) to Figure 6(B) ).

[0360] The binding kinetics of the chimeric and humanized versions of the HIM34 bispecific antibody were compared by surface plasmon resonance. The affinity of the humanized HIM34 BsAb for CD33 was tested using surface plasmon resonance technology. The results are shown below:

[0361] 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

[0362] Mean fluorescence intensity (MFI) is a reliable criterion for predicting the in vitro efficacy of the CD33 antibodies of the present technology. To test the binding of humanized HIM34 BsAb, MOLM13 CD33 (+) cells were contacted with different concentrations of humanized anti-CD33 BsAb disclosed herein. Flow cytometry results are shown in Figure 17 The cytotoxicity of humanized BsAbs against MOLM13 cells was tested in a T cell-dependent cytotoxicity assay (TDCC). Figure 18(A) to Figure 18(B) As shown in , clones with superior binding (higher MFI) to CD33 were more potent (lower EC50). These results demonstrate that MFI is a reliable criterion for predicting the in vitro potency of CD33 antibodies of the present technology.

[0363] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically target CD33-positive cancer cells. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in subjects in need thereof.

[0364] Example 3: In vivo therapeutic studies using CD33-BsAb

[0365] The prognosis of patients with FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation (FLT3 / ITD) is generally poor. In pediatric AML, the negative consequences of these mutations are more prominent (Levis and Small, Leukemia 17:1738-52 (2003)). In order to evaluate whether chimeric CD33-BsAb can redirect T cells to AML with FLT3 / ITD mutation, in vivo experiments were performed with NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ (NSGS) mice. Mice were randomized into 4 groups, and all received 1 million MOLM13 cells containing the firefly luciferase gene: Group 1: activated T cells (ATC) only; 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 the 7th day, when leukemia was established. Within three weeks, mice received a weak injection of 10 million ATC mixed with BsAb. BsAb was administered twice / week, once mixed with ATC and the other time alone. A total of five doses of BsAb were injected. To support T cell growth in vivo, 1000 IU of interleukin 2 was administered subcutaneously twice a week. Bioluminescence imaging was performed weekly to evaluate leukemia burden. Figure 7(A) to Figure 7(B)As shown in , administration of ATC in the presence of 100 or 10 ng / dose of chHIM34 BsAb treated leukemic mice. However, 100 ng / dose of control BsAb plus ATC had no anti-tumor effect, similar to the ATC-only group.

[0366] Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in subjects in need thereof.

[0367] Example 4: Anti-CD33 of the present technology Comparison of the activity of BsAb and existing CD33 antibodies

[0368] The BC275 (CD3×CD33) BsAb disclosed herein binds to the membrane proximal domain (IgC) of the extracellular region of CD33. Figure 20(A) to Figure 20(B) . 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 CD33 extracellular region (Figure 20(B)). Various CD33(+) and CD33(-) human cancer cell lines were contacted with the three bispecific antibodies to evaluate their binding to CD33. Secondary antibodies were used to detect the binding of BsAbs to 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) As shown in Figure 21, all three BsAbs effectively lysed CD33(+) leukemia cells but did not harm CD33(-) NALM6 cells. These results demonstrate that CD3×CD33 BsAbs selectively target CD33(+) cancers. In addition, there is a positive correlation between the level of CD33 expression on cancer cells and the efficacy of the BsAbs in the TDCC assay (combined data for all three CD3×CD33 BsAbs are shown in Figure 21(D)).

[0369] Several animal experiments were performed to evaluate and compare the efficacy of the three BsAbs in vivo. 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 started 11 days later ( Figure 22(A) to Figure 22(B) ). Mice were given 5×10 6A single injection of activated T cells and 0.025 μg of test BsAb (10 μg / Kg / dose). The timing of T cell and antibody injections is shown in Figure 22 (B). In the control group, mice received T cells plus BC119 (CD3×GD2BsAb). The injection of BsAb alone was continued for another 4 doses. The growth of leukemia was monitored using an IVIS bioluminescent imager. All three CD3×CD33 BsAbs redirected T cells to treat mice inoculated with AML cells, while mice receiving control BsAb and T cells showed leukemia progression ( Figure 22(A) to Figure 22(B) ).

[0370] Next, immunodeficient NSG mice were intravenously inoculated with human CD33-transduced NALM6-luciferase leukemia xenografts (0.5×10 6 cells). Treatment was started 3 days later (Fig. 23(A)). Mice received two injections of activated T cells (2.7×10 6 and 5×10 6 , one week 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). The growth of leukemia was monitored using an IVIS bioluminescent imager. To support T cell transplantation, all mice were given 2 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 (a CD33 antibody of the present technology), which promoted a higher degree of survival than conventional BC269 and BC133 BsAbs (which showed equivalent effects).

[0371] The next experiment was performed on immunodeficient DKO mice inoculated subcutaneously with THP1 human leukemia xenografts with the CC genotype of the rs12459419 SNP. Treatment was initiated 14 days later ( Figure 24(A) to Figure 24(D) Mice received two injections of activated T cells (10 7, one week 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 received T cells plus BC119 (CD3×GD2 BsAb). The schedule of T cell and antibody injections is shown in Figure 24 (A). Leukemia progression was monitored by measuring tumor size. To support T cell transplantation, all mice received 2 subcutaneous injections of interleukin 2 (1000 IU) per week within the first three weeks of T cell injection. The most effective BsAb was BC275 (a CD33 antibody of the present technology), which showed the highest degree of tumor reduction compared to conventional BC269 and BC133 BsAbs (i.e., BC275>BC269>BC133).

[0372] Animal experiments were performed on immunodeficient DKO mice subcutaneously inoculated with K562 human leukemia xenografts (which have the TT genotype of the rs12459419 SNP). Treatment was started 7 days later ( Figure 25(A) to Figure 25(D) Mice received three injections of activated T cells (10 7 , one week 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 received T cells plus BC119 (CD3×GD2 BsAb). The schedule of T cell and antibody injections is shown in Figure 25 (A). Leukemia progression was monitored by measuring tumor size. To support T cell transplantation, all mice received 2 subcutaneous injections of interleukin 2 (1000 IU) per week within the first three weeks of T cell injection. The most effective BsAb was BC275 (CD33 antibody of the present technology), which showed the highest degree of tumor reduction compared to conventional BC269 and BC133 BsAbs (i.e., BC275>BC269>BC133).

[0373] Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in subjects in need thereof.

[0374] Example 5: Anti-CD33 BsAb using the present technology in PRIT

[0375] IgG-based CD33-C825 BsAb. CD33(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously or via other routes. After tumor establishment (depending on tumor type and injection route), treatment will be started. Treatment consists of one or more cycles. Each cycle includes administration of the test BsAb (250 μg intravenously), followed by injection of a scavenger (DOTA glucan or DOTA dendrimer; the dose is 5%-15% of the BsAb dose, see Cheal SM et al., Mol Cancer Ther 13:1803-12, 2014) 4 hours later, DOTA- 177 Lu (up to 1.5mCi) or DOTA- 225 Ac(1μCi). Usually, DOTA- 225 Ac than DOTA- 177 Lu is more effective and may require fewer cycles to eradicate the tumor.

[0376] Tetramerized BsAb. CD33(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or via other routes, and after tumor establishment (depending on the type of tumor and the injection route), treatment is initiated. Treatment consists of one or more cycles. Each cycle consists of the following: administration of BsAb (250 μg intravenously), followed by intravenous injection of DOTA- 177 Lu (up to 1.5mCi) or DOTA- 225 Ac(1μCi). Usually, DOTA- 225 Ac than DOTA- 177 Lu is more effective and may require fewer cycles to eradicate the tumor.

[0377] 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. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat CD33-related cancers in subjects in need thereof.

[0378] Example 6: Anti-CD33 of the present technology Use of BsAbs in the treatment of Alzheimer's disease

[0379] Various rodent models for Alzheimer's disease (AD) have been developed (Nazem et al., J Neuroinflammation 17;12:74 (2015)). Because the HIM34 antibody binds to human CD33 and does not cross-react with mouse CD33, a knock-in mouse model of human CD33 (hCD33) will be generated. 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.

[0380] In one experiment, an anti-CD33 BsAb disclosed herein comprising a C825 anti-DOTA binding moiety was injected intracranially or into the cerebrospinal fluid, followed by intravenous injection of a radiolabeled metal-DOTA conjugate, e.g. 177 Lu-DOTA-Bn. In addition to behavioral analysis of mice, histopathological assessment of brain tissue over time was used as an outcome measure. It is expected that animals receiving the anti-CD33 BsAb of this technology will show improvement in one or more symptoms of Alzheimer's disease (AD) compared to those observed in untreated controls.

[0381] Therefore, the immunoglobulin-related compositions disclosed herein can be used to treat Alzheimer's disease in a subject in need thereof.

[0382] Equivalent content

[0383] The technology of the present invention is not limited to the specific embodiments described in this application, and the embodiments are intended to be a single illustration of the separate aspects of the technology of the present invention. As will be clear 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. It will be clear to those skilled in the art based on the foregoing description that, in addition to the methods and equipment listed herein, functionally equivalent methods and equipment within the scope of the technology of the present invention. Such modifications and changes are intended to fall within the scope of the technology of the present invention. It should be understood that the technology of the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and they can certainly change. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments and are not intended to be restrictive.

[0384] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0385] As will be understood by one skilled in the art, for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily identified as fully describing the same range and enabling the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. Also, as will be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," etc., includes the stated numbers and refers to ranges that can subsequently be decomposed into subranges as described above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, etc.

[0386] 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 not inconsistent with the explicit teachings of this specification. Sequence Listing <110> Memorial Sloan Kettering Cancer Center <120> CD33 antibodies and methods of treating cancer using the same <130> 115872-0653 <140> PCT / US2020 / 019351 <141> 2020-02-21 <150> 62 / 809,091 <151> 2019-02-22 <160> 167 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 1 Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 3 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 4 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 4 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 6 <211> 119 <212> PRT <213> artificial sequence <220> <223> Artificial sequence description: synthetic Polypeptide <400> 6 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 7 <211> 119 <212> PRT <213> artificial sequence <220> <223> Artificial sequence description: synthetic Polypeptide <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Ser Val Thr Val Ser Ser 115 <210> 8 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 8 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 9 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 10 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 11 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 12 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 13 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 14 <211> 505 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 14 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile 35 40 45 Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg 50 55 60 Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn 85 90 95 Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn 100 105 110 Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys Thr Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser 245 250 255 Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Lys 260 265 270 Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg Gln 275 280 285 Ala Pro Gly Lys Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg 290 295 300 Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser 305 310 315 320 Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu Arg 325 330 335 Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp His 340 345 350 Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser 355 360 365 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 370 375 380 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 385 390 395 400 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 405 410 415 Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp 420 425 430 Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr Asp Thr 435 440 445 Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 450 455 460 Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile 465 470 475 480 Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly 485 490 495 Cys Gly Thr Lys Leu Gln Ile Thr Arg 500 505 <210> 15 <211> 1518 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic polynucleotides <400> 15 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcacagcgac 60 atcgtcatga ctcagagccc tagcagcctg agcgcatccc tgggaggaaa ggtcactatc 120 acttgtaaag catcacagga catcaacaag tacatcgcct ggtatcagca caagccaggc 180 aaaggccccc ggctgctgat ccactacacc agcacactgc agccaggcat cccttcccgg 240 ttcagcggct ccggctctgg aagagactac agcttttcca tctctaatct ggagcccgag 300 gatatcgcca cctattattg tctgcagtat gataatctgc tgacatttgg cgctggaact 360 aagctggaac tgaaacggac cgtggccgcc ccctccgtgt tcatcttccc cccctccgac 420 gagcagctga agtccggcac cgcctccgtg gtgtgcctgc tgaacaactt ctacccccgg 480 gaggccaagg tgcagtggaa ggtggacaac gccctgcagt ccggcaactc ccaggagtcc 540 gtgaccgagc aggactccaa ggactccacc tactccctgt cctccaccct gaccctgtcc 600 aaggccgact acgagaagca caaggtgtac gcctgcgagg tgacccacca gggcctgtcc 660 tcccccgtga ccaagtcctt caaccggggc gagtgcacta gtggcggcgg aggatctggc 720 ggaggtggaa gcggaggggg aggatctcag gtgcagctgg tgcagagcgg aggcggagtg 780 gtgcagcctg gcagatccct gagactgtcc tgcaaggcct ccggctacac cttcacccgg 840 tacaccatgc actgggtgcg acaggcccct ggcaagtgcc tggaatggat cggctacatc 900 aacccctccc ggggctacac caactacaac cagaagttca aggaccggtt caccatctcc 960 cgggacaact ccaagaacac cgcctttctg cagatggact ccctgcggcc tgaggatacc 1020 ggcgtgtact tctgcgcccg gtactacgac gaccactact ccctggacta ctggggccag 1080 ggaacccctg tgacagtgtc atctggtggc ggaggaagtg ggggaggcgg atcaggtggt 1140 ggtggatcag gcgggggagg ttcagggggt ggcggttctg ggggaggggg ctctgatatt 1200 cagatgactc agagcccttc cagcctgagc gcctccgtgg gagatcgcgt gacaattacc 1260 tgctctgcct cctcctccgt gtcttacatg aattggtatc agcagacccc tgggaaggct 1320 cctaagcggt ggatctacga cacctccaag ctggcctctg gcgtgcccag caggttttct 1380 ggctccggca gcggcacaga ttataccttc accatcagct ccctgcagcc agaagatatc 1440 gctacctatt attgtcagca gtggtcctcc aaccctttca ccttcggctg cggcacaaag 1500 ctgcagatca caagatag 1518 <210> 16 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 16 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr Asp Tyr Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asp Pro Tyr Lys Gly Gly Thr Ile Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Phe Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Met Ile Thr Ala Tyr Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Ser Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 17 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polynucleotide <400> 17 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcacagcgaa 60 gtgaaactgc agcagagcgg ccctgaactg gtcaaacctg gaacctccgt gaaagtgtct 120 tgtaaagcat ccgggtactc attcaccgac tacaacatgt attgggtgaa gcagagccac 180 ggcaagtccc tggagtggat cggctacatc gacccctata agggcggcac aatctacaac 240 cagaagttca agggcaaggc caccctgaca gtggacaaga gctcctctac cgcctttatg 300 cacctgaata gcctgacatc cgaggattct gccgtgtact attgcgcccg ggagatgatt 360 acagcctact actttgatta ctggggacag gggtcttccg tcaccgtctc ctctgcctcc 420 accaagggcc cctctgtgtt tcctctggcc ccctccagca agtccacctc tggtggaaca 480 gccgccctgg gctgcctcgt gaaggactac tttcccgagc ccgtgaccgt gtcctggaac 540 tctggcgctc tgacctctgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600 tactccctgt cctccgtcgt gacagtgccc tccagctctc tgggcaccca gacctacatc 660 tgcaacgtga accacaagcc ctccaatacc aaggtggaca agcgggtgga acccaagtcc 720 tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780 gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840 acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta cgcctccacc 960 taccgggtgg tgtccgtgct gacagtgctg caccaggact ggctgaacgg caaagagtac 1020 aagtgcgccg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080 aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140 aagaaccagg tgtccctgac ctgtctcgtg aaaggcttct acccctccga tatcgccgtg 1200 gaatggggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260 tccgacggct cattcttcct gtacagcaag ctgaccgtgg acaagtcccg gtggcagcag 1320 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380 tccctgtccc tgagccccgg caaatga 1407 <210> 18 <211> 505 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthetic peptides <400> 18 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile 35 40 45 Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys 50 55 60 Leu Leu Ile His Tyr Thr Ser Thr Leu Glu Thr Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn 100 105 110 Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys Thr Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gl...

Claims

1. A method comprising a heavy chain immunoglobulin variable domain (V H ) amino acid sequence and light chain immunoglobulin variable domain (V L ) amino acid sequence, or an antigen-binding fragment thereof, wherein: The V H The amino acid sequence is SEQ ID NO: 7, and the V L The amino acid sequence is SEQ ID NO: 11; or The V H The amino acid sequence is SEQ ID NO: 7, and the V L The amino acid sequence is SEQ ID NO: 12; or The V H The amino acid sequence is comprised of SEQ ID NO: 7, and the V L The amino acid sequence is SEQ ID NO:

13.

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

3. The antibody of claim 2, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

4. The antibody of claim 2, comprising an IgG4 constant region comprising an S228P mutation.

5. The antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scF v and F v . 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 of claim 1, comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:22, or a light chain (LC) amino acid sequence comprising SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:

134.

8. The antibody according to claim 7, comprising a HC amino acid sequence and a LC amino acid sequence respectively selected from the following: 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: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); and SEQ ID NO: 22 and SEQ ID NO: 29 (VL4VH6 x hC825), Wherein the antibody is a bispecific antibody.

9. The antibody of 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. 42-65.

11. A recombinant nucleic acid encoding the antibody or antigen-binding fragment according to any one of claims 1, 7-8 or 10. 12 . The recombinant nucleic acid according to claim 11 , having a sequence selected from the group consisting of: SEQ ID NO: 19, 23, 25 and 135.

13. A host cell or vector comprising the recombinant nucleic acid sequence according to claim 11 or claim 12.

14. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1, 7-8 or 10 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

15. The antibody of any one of claims 1, 7-8 or 10, wherein the antibody lacks α-1,6-fucose modification.

16. The bispecific antibody of claim 6, wherein the bispecific antibody binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

17. The bispecific antibody or antigen-binding fragment of claim 6, 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.

18. Use of the antibody according to claim 8 in the preparation of a medicament for treating a CD33-related cancer in a subject in need thereof, wherein the antibody specifically binds to CD33, and The CD33-associated cancer is acute myeloid leukemia (AML), biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

19. Use of the bispecific antibody or antigen-binding fragment of claim 10 in the preparation of a medicament for treating a CD33-related cancer in a subject in need thereof, 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.

20. The use according to any one of claims 18-19, wherein the antibody or antigen-binding fragment is formulated for separate, sequential or simultaneous administration with an additional therapeutic agent.

21. The use according to claim 20, wherein the additional therapeutic agent is one or more of the following: an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian inhibitor, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, a bisphosphonate therapeutic agent.

22. Use of the antibody or antigen-binding fragment of any one of claims 1, 7-8 or 10 in the preparation of a kit for in vivo detection of a tumor in a subject, wherein the antibody is configured to localize to a tumor expressing CD33 and is labeled with a radioactive isotope.

23. The use according to claim 22, wherein the subject is diagnosed with or suspected of having cancer.

24. The use according to claim 22 or 23, wherein the level of radioactivity emitted by the antibody or antigen-binding fragment is detected using positron emission tomography or single photon emission computed tomography.

25. The use according to any one of claims 22-23, further comprising administering to a subject an immunoconjugate comprising the antibody or antigen-binding fragment according to any one of claims 1, 7-8 or 10 conjugated to a radionuclide.

26. The use according to claim 25, wherein the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter or any combination thereof.

27. The use according to claim 26, wherein the isotope emitting beta particles is selected from 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu.

28. A kit comprising the antibody or antigen-binding fragment according to any one of claims 1, 7-8 or 10 and instructions for use.

29. The kit of claim 28, wherein the antibody or antigen-binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

30. The kit according to claim 28 or 29, further comprising a secondary antibody that specifically binds to the antibody.

31. The bispecific antibody or antigen-binding fragment of claim 6 or 10, wherein the bispecific antibody binds to a radiolabeled DOTA hapten and a CD33 antigen.

32. Use of a complex comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment according to claim 31 in the preparation of a kit for selecting a subject for pretargeted radioimmunotherapy, wherein the complex is configured to localize to a tumor expressing CD33.

33. Use of a complex comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment of claim 31 in the preparation of a medicament for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with a CD33-associated cancer, wherein the complex is configured to localize to a tumor expressing CD33, wherein the CD33-associated cancer is acute myeloid leukemia (AML), biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

34. Use of a complex comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment of claim 31 in the preparation of a medicament for treating a CD33-associated cancer in a subject in need thereof, wherein the complex is configured to localize to a tumor expressing CD33, wherein the CD33-associated cancer is acute myeloid leukemia (AML), biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

35. Use of the bispecific antibody or antigen-binding fragment of claim 31 and a radiolabeled DOTA hapten in the preparation of a medicament for increasing the sensitivity of a tumor to radiation therapy in a subject diagnosed with a CD33-related cancer, wherein the bispecific antibody or antigen-binding fragment is configured to localize to a tumor expressing CD33; and wherein the radiolabeled DOTA hapten is configured to bind to the bispecific antibody or antigen-binding fragment, and wherein the CD33-associated cancer is acute myeloid leukemia (AML), biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

36. Use of the bispecific antibody or antigen-binding fragment of claim 31 and a radiolabeled DOTA hapten in the preparation of a medicament for treating a CD33-related cancer in a subject in need thereof, wherein the bispecific antibody or antigen-binding fragment is configured to localize to a tumor expressing CD33; and wherein the radiolabeled DOTA hapten is configured to bind to the bispecific antibody or antigen-binding fragment, and The CD33-associated cancer is acute myeloid leukemia (AML), biphenotypic leukemia, bi-lineage leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, or acute lymphoblastic leukemia.

37. The use according to claim 34 or 36, further comprising administering a clearing agent to the subject prior to administering the radiolabeled DOTA hapten.

38. The use according to any one of claims 32-36, wherein the subject is a human.

39. The use according to any one of claims 32-34, wherein the complex is formulated for intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral, intratumoral or intranasal administration.

40. The use of any one of claims 32-36, wherein the radiolabeled DOTA hapten comprises an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter.

41. The use according to any one of claims 32-36, 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.

Images