Anti-CD33 antibodies, anti-CD33 / anti-CD3 bispecific antibodies and uses thereof

Abstract

The present invention describes anti-CD33 antibodies and antigen-binding fragments thereof and anti-CD33 / anti-CD3 bispecific antibodies or antigen-binding fragments thereof. The present invention also describes nucleic acids encoding the antibodies, compositions comprising the antibodies, methods for producing the antibodies, and methods for using the antibodies to treat or prevent diseases such as cancer.

Description

Technical Field

[0001] The present invention relates to monoclonal anti-CD33 antibodies, bispecific anti-CD33 anti-CD3 antibodies, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions containing the antibodies. Also provided are methods for preparing the antibodies, and methods for using the antibodies to treat diseases including cancer. Background Art

[0002] Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of leukemic cells. Despite the increasing understanding of the underlying disease biology in AML, standard treatment with cytotoxic chemotherapy has remained largely unchanged over the past few decades, and the overall five-year survival rate remains poor, less than 30% (Burnett, Wetzler, & Lowenberg, 2011; Cancer Genome Atlas Research et al., 2013). Therefore, new therapies with increased efficacy and reduced toxicity, ideally targeting AML stem cells, are urgently needed, as these cells are believed to be crucial in the pathogenesis of AML, and inadequate eradication by standard therapies is believed to be a high-incidence factor leading to relapse (Hope, Jin, & Dick, 2004; Ishikawa et al., 2007). Although therapeutic antibodies directed against cell surface molecules have been shown to be effective for treating malignancies such as lymphoma and acute lymphoblastic leukemia as well as solid tumors (Hoelzer, 2013; Jackson and Chester, 2015), only one antibody-based therapy is currently approved for AML (Godwin, Gale, and Walter, 2017).

[0003] CD33 is a 67kD single-pass transmembrane glycoprotein and a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family. Although its exact biological function is unclear, in normal individuals, it is primarily considered a myeloid differentiation antigen, with low expression in myeloid progenitor cells, neutrophils and macrophages, and high expression in circulating monocytes and dendritic cells. Importantly, CD33 has been detected in blasts and leukemic stem cells in 85% to 90% of patients with AML. Interestingly, CD33 expression is restricted to hematopoietic cells (Paul, Taylor, Stansbury, & McVicar, 2000; Ulyanova, Blasioli, Woodford-Thomas, & Thomas, 1999), but is absent on normal hematopoietic stem cells (Andrews, Torok-Storb, & Bernstein, 1983; Griffin, Linch, Sabbath, Larcom, & Schlossman, 1984; Jilani et al., 2002). These findings suggest that CD33 is a suitable target for antibody-based therapy in AML.

[0004] The structure of CD33 consists of an amino-terminal V-set Ig-like domain (encoded by exon 2 of CD33) that mediates sialic acid binding and a C2-set Ig-like domain (encoded by exons 3 and 4) in its extracellular portion (Laszlo et al., 2016). Alternative splicing of CD33 RNA can result in a shorter isoform expressed on the cell surface that lacks the V-set but retains the C2-set Ig-like domain (Laszlo, Estey, & Walter, 2014; Laszlo et al., 2016). The biological relevance of this splicing process was largely unknown until recent studies showed that the single nucleotide polymorphism (SNP) rs12459419 is present in approximately 50% of the AML population and results in a skipped deletion of exon 2 of CD33, resulting in the loss of the V domain of CD33 (Lamba et al., 2017). Interestingly, several CD33 antibody-based therapies (including Mylotarg, the only approved antibody for AML) bind to and recognize the V domain of CD33. The aforementioned study in fact showed that Mylotarg was not effective in patients expressing the SNP and was therefore only effective in about 50% of the AML population (Lamba et al., 2017). Given the data for Mylotarg, it is reasonable to assume that other V-binding CD33 antibodies will also be effective only in a limited pool of AML patients (specifically those patients who do not have the SNP rs12459419 mutation).

[0005] Indeed, when studying the CD33 clinical field, additional anti-CD33 antibodies include Amgen's AMG330 and AMG673, Amphivena's AMV564, Immunogen's IMGN779, Boehringer Ingelheim's BI836858, Actinium Pharma's Actinimab, and Seattle Genetic's SGN33A. Amgen's AMG330 is a CD33×CD3 BiTE, and it is reported that it "recognizes a linear epitope with a core sequence IPYYDKN located in the V-set domain of CD33" (Friedrich et al., 2014). Given that AMG673 is a half-life extended version of the CD33 BiTE, it is believed that it binds to the same epitope as the BiTE. Amphivena's AMV564 is a tetravalent bispecific CD33 / CD3 antibody, and according to US9803029, the antibody binds to the V domain of CD33. Immunogen's IMGN779 is a CD33 antibody (My9-6) conjugated to a DNA alkylating agent, and according to FIG. 1 in US9359442, 125 I-labeled My9-6 antibody and My9 antibody compete for binding to CD33-positive U-937 cells. My9 antibody binds to the V domain of CD33 (Perez-Oliva et al., 2011). Together, the evidence suggests that IMGN779 binds to the V domain of CD33. BI 836858 from Boehringer Ingelheim is an Fc-engineered anti-CD33 antibody that mediates NK cell-mediated ADCC and binds to the V domain of CD33 (Vasu et al., 2016). Additionally, Vasu et al., as well as Malik et al. and Perez-Oliva et al., show evidence for mapping rituzumab (HuM195) to the V domain of CD33 (Malik et al., 2015; Perez-Oliva et al., 2011). The HuM195 antibody is currently in clinical trials and is conjugated to actinium by Actinium pharma to make Actimab. The HuM195 antibody has also been conjugated to a DNA binder by Seattle Genetics to produce SGN33A; however, this drug is currently on hold due to toxicity issues. Thus, anti-CD33 antibodies known in the art bind to the V domain of CD33.

[0006] In light of these data, there is a significant unmet medical need when it comes to CD33-based antibody therapy in AML, and antibodies with a C2 domain that bind CD33 are needed for the treatment of cancers that express CD33. Summary of the invention

[0007] In one general aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof that binds to CD33. In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof binds to the C2 domain of the CD33. In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof binds to the V domain of the CD33.

[0008] In another general aspect, the invention relates to an isolated bispecific antibody or antigen binding fragment thereof that binds to CD33 and CD3. In certain embodiments, the bispecific antibody or antigen binding fragment thereof binds to the C2 domain of the CD33. In certain embodiments, the bispecific antibody or antigen binding fragment thereof binds to the V region of the CD33.

[0009] The present invention provides an isolated monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to the C2 domain of CD33. In certain embodiments, the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0010] a. SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively;

[0011] b. SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively;

[0012] c. SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively;

[0013] d. SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively;

[0014] e. SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively;

[0015] f. SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively;

[0016] g. SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively;

[0017] h. SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively;

[0018] i. SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively;

[0019] j. SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively;

[0020] k. SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively;

[0021] l. SEQ ID NO: 402, 403, 404, 522, 523 and 524, respectively;

[0022] m. SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively;

[0023] n. SEQ ID NO: 423, 424, 425, 543, 544 and 545, respectively; or

[0024] o. SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively.

[0025] The antibody or antigen-binding fragment thereof can, for example, specifically bind to CD33, preferably human CD33.

[0026] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 292, 291, 261, 269, 280, 259, 263, 264, 265, 266, 272, 277, 279, 284 or 285, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 332, 331, 302, 310, 320, 300, 304, 305, 306, 307, 317, 319, 324 or 325.

[0027] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0028] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332;

[0029] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331;

[0030] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302;

[0031] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310;

[0032] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322;

[0033] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300;

[0034] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304;

[0035] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305;

[0036] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306;

[0037] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307;

[0038] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307;

[0039] 1. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317;

[0040] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319;

[0041] n. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; or

[0042] o. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 325.

[0043] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof has an in vitro EC of less than about 2 nM. 50 Inducing antibody-dependent cellular cytotoxicity (ADCC). The antibody or antigen-binding fragment thereof may, for example, comprise an IgG1 low-fucose backbone.

[0044] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is present in an amount of less than about 5×10 -9 The dissociation constant (KD) of M binds to CD33.

[0045] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to CD33 with an EC of less than about 2 nM. 50 Induce internalization.

[0046] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent.

[0047] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric, partially humanized, or fully humanized.

[0048] Also provided herein is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, comprising an anti-CD33 antibody or antigen-binding fragment thereof and an anti-CD3 antibody or antigen-binding fragment thereof, wherein the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0049] a. SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively;

[0050] b. SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively;

[0051] c. SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively;

[0052] d. SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively;

[0053] e. SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively;

[0054] f. SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively;

[0055] g. SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively;

[0056] h. SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively;

[0057] i. SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively;

[0058] j. SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively;

[0059] k. SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively;

[0060] l. SEQ ID NO: 402, 403, 404, 522, 523 and 524, respectively;

[0061] m. SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively;

[0062] n. SEQ ID NO: 423, 424, 425, 543, 544 and 545, respectively; or

[0063] o. SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively;

[0064] And the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0065] 1) SEQ ID NO: 342, 343, 344, 465, 466 and 467, respectively; or

[0066] 2) are SEQ ID NOs: 345, 346, 347, 468, 469 and 470, respectively.

[0067] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 292, 291, 261, 269, 280, 259, 263, 264, 265, 266, 272, 277, 279, 284 or 285, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 332, 331, 302, 310, 320, 300, 304, 305, 306, 307, 317, 319, 324 or 325; and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 257 or 258, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 298 or 299.

[0068] In certain embodiments, the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof comprises:

[0069] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0070] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0071] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0072] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0073] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0074] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0075] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0076] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0077] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0078] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0079] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0080] 1. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0081] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0082] n. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0083] o. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0084] p. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0085] q. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0086] r. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0087] s. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0088] t. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0089] u. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0090] v. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0091] w. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0092] x. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0093] y. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0094] z. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0095] aa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0096] bb. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0097] cc. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299; or

[0098] dd. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299.

[0099] In certain embodiments, the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof is expressed in cells expressing CD33 in vitro at an EC of less than about 1 nM. 50 Values ​​induce T cell-dependent cytotoxicity.

[0100] In certain embodiments, the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof is chimeric, partially humanized, or fully humanized.

[0101] Also provided are isolated nucleic acids encoding the monoclonal and / or bispecific antibodies or antigen-binding fragments thereof of the invention.

[0102] Also provided are vectors comprising the isolated nucleic acid encoding the monoclonal and / or bispecific antibodies or antigen-binding fragments thereof of the invention.

[0103] Also provided is a host cell comprising a vector comprising the isolated nucleic acid encoding the monoclonal and / or bispecific antibody or antigen-binding fragment thereof of the invention.

[0104] In certain embodiments, a pharmaceutical composition is provided, comprising the isolated monoclonal antibody or its antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition is provided, comprising the isolated bispecific antibody or its antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier.

[0105] Also provided is a method for treating cancer in a subject in need thereof, the method comprising administering the pharmaceutical composition of the present invention to the subject. In certain embodiments, the cancer is a hematological cancer. The hematological cancer may be, for example, selected from, but not limited to, leukemia, lymphoma, or multiple myeloma. In certain embodiments, the hematological cancer may be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0106] Also provided is a method for preparing the monoclonal or bispecific antibody or antigen-binding fragment thereof of the present invention. The method comprises culturing a cell comprising a nucleic acid encoding the monoclonal or bispecific antibody or antigen-binding fragment under conditions for preparing the monoclonal or bispecific antibody or antigen-binding fragment, and recovering the monoclonal or bispecific antibody or antigen-binding fragment from the cell or culture.

[0107] Also provided is a method for preparing a pharmaceutical composition comprising the monoclonal and / or bispecific antibody or antigen-binding fragment of the present invention. The method comprises combining the monoclonal and / or bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] The foregoing summary and the following detailed description of preferred embodiments of the present patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the precise embodiments shown in the drawings.

[0109] Figure 1A to Figure 1B A CD33×CD3 T-cell mediated cytotoxicity assay is shown. The CD33×CD3 bispecific antibody using the anti-CD3 arm CD3B219 was incubated with human pan T cells and CD33 +AML cell lines were incubated together. After incubation for 48 hours at 37°C, 5% CO2, total tumor cell cytotoxicity was measured by flow cytometry. Figure 1A Antibody candidates identified by OMNIRat are shown, and Figure 1B Antibody candidates identified by OMNIMouse are shown.

[0110] FIG. 2A to FIG. 2B Shown is an in vitro evaluation of cytotoxicity and T cell activation of blasts in fresh whole blood of AML patients using a CD33×CD3 bispecific antibody with anti-CD3 arms CD3B219 and CD3B376. Figure 2A Shown are the percentages of total cytotoxicity of AML cells using CD33 bispecific antibodies or CD3×Vehicle control. Figure 2B T cell activation induced by CD33 bispecific antibody or CD3×Vabove control is shown. No Fc blocker was added.

[0111] FIG. 3A to FIG. 3C CD33×CD3 T-cell mediated cytotoxicity assay is shown. CD33×CD3 bispecific antibodies using anti-CD3 arms CD3B219 and anti-CD3B376 were co-cultured with human pan T cells and wild-type for the CD33 SNPrs12459419 mutation (KG1, Figure 3A ), heterozygous (SH2, Figure 3B ) or homozygous (OCIAML3, Figure 3C After incubation for 48 hours at 37° C., 5% CO 2 , total tumor cell cytotoxicity was measured by flow cytometry.

[0112] FIG. 4A to FIG. 4B In vitro evaluation of the cytotoxicity of C33B904 antibody paired with CD3B219 or CD3B376 on MOLM-13 cells exogenously added to normal healthy human whole blood (N=6 donors) is shown: MOLM-13 cells ( Figure 4A ) and CD33 + CD14 + Monocytes ( Figure 4B ) percentage of cytotoxicity.

[0113] FIG. 5A to FIG. 5B Shown is an in vitro evaluation of cytotoxicity and T cell activation of monocytes in fresh whole blood from six normal cynomolgus macaque donors using a CD33×CD3 bispecific antibody with anti-CD3 arms CD3B219 and CD3B376. Figure 5AThe expression of CD33 using CD33 bispecific antibody or its CD3× blank control is shown. + CD14 + Percent total cytotoxicity in cynomolgus macaque monocytes. Figure 5B T cell activation induced by CD33 bispecific antibody or its CD3× blank control is shown. No Fc blocker was added.

[0114] Figure 6 The anti-tumor efficacy of C3CB189 in MOLM-13 human AML xenografts in T cell humanized NSG mice is shown. Bioluminescence (BLI) imaging of diffuse MOLM-13 tumors was performed twice per week, and the results were expressed as mean brightness (p / s / cm 2 / sr)±SEM (n=8-10 / group). *p≤0.0001 between treatment and control, calculated by two-way ANOVA and Bonferroni test.

[0115] Figure 7 The survival rate of animals treated with C3CB189 in MOLM-13 human AML xenografts in T cell humanized NSG mice is shown. The survival rate of mice carrying MOLM-13 is graphically represented using Kaplan-Meier curves and evaluated by log-rank (Mantel-Cox) test. * p≤0.0001 for treatment and control group.

[0116] Figure 8 The anti-tumor efficacy of C3CB88 in MOLM-13 human AML xenografts in T cell humanized NSG mice is shown. Bioluminescence (BLI) imaging of diffuse MOLM-13 tumors was performed twice per week, and the results were expressed as mean brightness (p / s / cm 2 / sr)±SEM (n=8-10 / group). *p≤0.0001 between treatment and control, calculated by two-way ANOVA and Bonferroni test.

[0117] Fig. 9 The survival rate of animals treated with C3CB88 in MOLM-13 human AML xenografts of T cell humanized NSG mice is shown. The survival rate of mice carrying MOLM-13 is graphically represented using Kaplan-Meier curves and evaluated by log-rank (Mantel-Cox) test. * p≤0.05 for treatment and control group.

[0118] Fig.10Shown is an in vitro protein A drug conjugate cell viability assay for detecting the internalization of five anti-CD33 antibodies in MOLM13 cells. All five anti-CD33 antibodies showed cytotoxicity in a dose-dependent manner.

[0119] FIG. 11A to FIG. 11B Figure 4 shows that low-fucose IgG1 anti-CD33 mAb mediates ADCC activity. In response to increasing concentrations of IgG1 anti-CD33 mAb, human NK cells against MOLM-13 ( Fig.11A ) and MV4-11( Fig. 11B )ADCC of target cells.

[0120] Fig.12 Shown are CD33-positive and CD33-negative cell lines stained with various concentrations of C3CB189 for 4 h to characterize the surface binding curves of the bispecific antibodies.

[0121] Fig.13 Shown are T cells from six healthy donors tested in T cell redirection assays with the indicated cell lines and percent cytotoxicity determined by FACS. Mean ± SD are plotted.

[0122] Fig.14A and Fig. 14B C3CB189 binds to the C2 domain and mediates cytotoxicity in primary samples regardless of their SNP12459419 genotype status. Fig.14A ) in CD33 + KG-1 cell line and CD33 - T cell-mediated cytotoxicity and activation assays using CD33×CD3 or CD123×CD3 bispecific antibodies in the KG1ΔCD33 cell line. Fig. 14B ) T cells alone were incubated with increasing concentrations of C3CB189 for 48 hours and T cell activation was measured by flow cytometry. Mean values ​​± SD are plotted.

[0123] FIG. 15A to FIG. 15C . Fig.15A EC showing cytotoxicity readout from six healthy donors is shown. 20 ,EC 50 and EC 90 The median value of . Fig. 15B Similar to Fig.13 , but here T cell activation was measured. Fig. 15C Similar to Fig.15A , but here the median EC readout of T cell activation from six healthy donors is shown. 20 ,EC 50 and EC 90 The value of .

[0124] FIG. 16A to FIG. 16E : In vivo C3CB189 mediates potent tumor activity in two established murine AML models. Fig.16A Shown are T cell humanized NSG mice bearing established KG-1 tumors injected intraperitoneally with 0.1 mg / kg, 0.5 mg / kg and 1 mg / kg of C3CB189. Tumor volumes were measured twice a week and the results are expressed as mean tumor volume ± SEM for each group. Fig. 16B T cell humanized NSG mice carrying diffuse MOLM-13Luc cells were shown to be injected intraperitoneally with 0.005 mg / kg, 0.05 mg / kg and 0.5 mg / kg of C3CB189. Kaplan Meier survival analysis was used to determine survival. Fig. 16C and Fig. 16B Same, but here bioluminescence was measured twice weekly, and representative images (ventral and dorsal views of n=3-5 animals) of live animal imaging of bioluminescence on days 9, 13, and 16 are shown (control group n=3 on day 16 due to mortality). Fig.16D Similar to Fig. 16B , but here mice were administered C3CB189 at 0.005 mg / kg and 0.05 mg / kg for three doses. T cell infiltration in the bone marrow was measured by flow cytometry analysis and the results were expressed as percentage of tumor cells (top) or CD3 + The percentage of T cells (lower panel) is shown. Fig.16E and Fig.16D Same as above, but here T cell infiltration in the bone marrow was measured by IHC staining and the results were expressed as CD33 + Tumor cells (above) or CD8 + T cells (lower panel) are shown.

[0125] Fig.17 C3CB189 is shown to mediate anti-tumor responses in a disseminated murine AML model. Tumor cells were implanted on day 0, T cells were implanted on day 5, and dosing was performed as indicated by the bars below the X-axis. Group bioluminescence is plotted as mean ± SEM. * indicates a significant difference (p≤0.05) between treatment with C3CB189 and blank × CD3 control on day 13 (n=9-10 / group on day 13).

[0126] Fig.18A and Fig.18B The results show that C3CB189 induced CD33 in whole blood + T cell-dependent cytotoxicity of cell lines. Fig.18A Shown are T cells from ten healthy donors (one point per donor) tested in a T cell redirection assay with the indicated cell lines. Mean ± SD are plotted. Fig.18B and Fig.18A Same as above, but here cytotoxic and T cell activating ECs are shown 20 ,EC 50 and EC 90 ND means not determined, that is, Prism either provides an approximate value or the curve is ambiguous. Values ​​are medians from 10 healthy donors.

[0127] Fig.19A and Fig.19B C3CB189 was shown to mediate cytotoxicity of AML blasts in primary patient samples. Fig.19A Figure 48 shows the expression of CD33 in fresh whole blood of AML patients mediated by C3CB189 after 48 hours. + In vitro evaluation of cytotoxicity in progenitor cells. The individual EC values ​​for each patient sample are shown. 50 value. Fig.19B and Fig.19A Same, but here T cell activation is measured.

[0128] Fig. 20 CD33 is expressed in cynomolgus macaque immune subsets. Whole blood from six normal healthy cynomolgus macaque donors was stained with a monoclonal antibody to CD33 and analyzed by flow cytometry. Mean values ​​± SD are plotted.

[0129] Fig.21 It shows that C3CB189 mediates MOLM-13 cells, normal CD33 + Cytotoxicity of monocytes and neutrophils of cynomolgus monkeys and activated cynomolgus monkey T cells. In vitro evaluation of C3CB189-mediated cytotoxicity in AML cell line MOLM-13 cells exogenously added to normal healthy cynomolgus monkey whole blood (n=6). The percentage of cytotoxicity, T cell activation and CD33 of MOLM-13 cells using C3CB189 and blank × CD3 bispecific antibodies are shown. + Monocyte and neutrophil cytotoxicity. Mean ± SEM are plotted.

[0130] FIG. 22A to FIG. 22D C3CB189 was shown to mediate CD33 + Leukocyte Reduction Cynomolgus monkeys were treated with a single intravenous injection of control (vehicle), 0.05 mg / kg, 0.2 mg / kg, or 1 mg / kg of C3CB189. Fig.22A The C3CB189 concentration versus time curve is shown. Fig. 22B T cell activation (CD8 + %CD25 + ). Fig. 22C The effect of C3CB189 on granulocytes (neutrophils) is shown. Fig.22D Shown are the effects of C3CB189 on monocytes.

[0131] Fig.23 Cytokine release after administration of C3CB189 to cynomolgus macaques is shown. Mean (SD) cytokine levels in cynomolgus macaques after a single intravenous injection of C3CB189. (A) IFNγ, (B) IL-10, (C) IL-2, (D) IL-6, (E) MCP, (F) TNFα. All following LLOQ values ​​are considered half of the LLOQ for plotting and averaging purposes.

[0132] FIG. 24A to FIG. 24C It was shown that C3CB189 binds to the C2 domain and mediates cytotoxicity in primary samples regardless of their SNP 12459419 genotype status. Fig.24A HDX mapping and subsequent illustration of epitope regions in the IgC and IgV domains of the CD33 ECD protein for the V and C2 binder mAbs are shown. The V epitope region is colored in blue and the C2 epitope region is colored in red. Fig. 24B CD33 in fresh whole blood of AML patients at 27 nM bispecific antibody concentration is shown. + In vitro evaluation of cytotoxicity of parent cells. Mean values ​​± SD are plotted. Fig.24C Shown is the evaluation of C3CB189-mediated cytotoxicity of frozen purified monocytes from 25 normal donors at 0.27 nM bispecific antibody concentration. Mean ± SD are plotted.

[0133] FIG. 25A to FIG. 25C The genotyping results of SNP rs12459419 are shown. Fig.25A Shown are the genotyping results of SNP rs12459419 in cell lines from Taqman assay. Fig.25B and Fig.25A Same, but here primary patient samples were genotyped. Fig.25C and Fig.25A Same as above, but here frozen mononuclear cells from healthy donors were genotyped using Taqman assay as well as Sanger sequencing. DETAILED DESCRIPTION

[0134] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification.

[0136] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0137] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include the value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0138] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0139] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, shall be understood to mean including the specified integer or group of integers but not excluding any other integer or group of integers, and to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such compositions, mixtures, processes, methods, articles, or apparatuses. Furthermore, unless expressly indicated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0140] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0141] As used herein, the term "consisting of" used throughout the specification and claims means including any listed integer or group of integers, but additional integers or groups of integers cannot be added to the specified method, structure, or composition.

[0142] As used herein, the term "consisting essentially of" used throughout the specification and claims means including any recited integer or group of integers, and optionally including any recited integer or group of integers that does not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0143] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0144] It should also be understood that when referring to dimensions or characteristics of components of the preferred invention, the use of "about," "approximately," "substantially," and similar terms herein indicates that the described dimensions / characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) without changing the least significant digits.

[0145] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-CD33 antibodies and polynucleotides encoding the same, anti-CD33 anti-CD3 bispecific antibodies and polynucleotides encoding the same, CD33 polypeptides and CD33 polynucleotides encoding the same) refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0146] For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and program parameters for the sequence algorithm are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage for the test sequence relative to the reference sequence based on the specified program parameters.

[0147] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman (Adv. Appl. Math. Vol. 2: p. 482 (1981)), by using the homology alignment algorithm of Needleman & Wunsch (J. Mol. Biol. Vol. 48: p. 443 (1970)), by searching the similarity method of Pearson & Lipman (Proc. Nat'l. Acad. Sci. USA Vol. 85: p. 2444 (1988)), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology). Biology, FM Ausubel et al., eds., Laboratory Manual, a joint venture between Greene Publishing Associates and John Wiley & Sons, Inc. (1995 supplement) (Ausubel).

[0148] Examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. 1990 J. Mol. Biol. Vol. 215, pp. 403 to 410 and Altschul et al. 1997 Nucleic Acids Res. Vol. 25: pp. 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighboring word score threshold (Altschul et al., supra). These initial neighboring word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0149] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score goes to zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, Vol. 89: p. 10915 (1989)).

[0150] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, Vol. 90: pp. 5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0151] Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is generally substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

[0152] Antibody

[0153] The present invention generally relates to isolated anti-CD33 antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions comprising the antibodies. The present invention further relates to isolated anti-CD33 / anti-CD3 bispecific antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions comprising the bispecific antibodies. Methods for preparing these antibodies are also provided, as well as methods for using these antibodies to treat diseases including cancer. The antibodies of the present invention have one or more desired functional properties, including but not limited to high affinity binding to CD33 and / or CD3, high specificity to CD33 and / or CD3, and the ability to treat cancer or prevent cancer when administered alone or in combination with other anti-cancer therapies.

[0154] In one general aspect, the present invention relates to an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds to CD33. In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof binds to the C2 domain of CD33. In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof binds to the V domain of CD33.

[0155] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, composite antibodies and chimeric antibodies and monoclonal or polyclonal antibody fragments. Generally speaking, an antibody is a protein or peptide chain that exhibits binding specificity for a specific antigen. The structure of an antibody is well known. According to the amino acid sequence of the heavy chain constant domain, immunoglobulins can be designated as five major classes (i.e., IgA, IgD, IgE, IgG, and IgM). IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibody of the present invention can be any of the five main species or corresponding subclasses. Preferably, the antibody of the present invention is IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of a vertebrate species can be designated as one of two completely different types, i.e., κ and λ. Therefore, the antibody of the present invention can contain κ or λ light chain constant domains. According to a specific embodiment, the antibody of the present invention includes a heavy chain and / or light chain constant region from a rat or human antibody. In addition to the heavy and light chain constant domains, the antibody also comprises an antigen binding region consisting of a light chain variable region and a heavy chain variable region, each variable region comprising three domains (i.e., complementary determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region is alternatively referred to as LCDR1, LCDR2, and LCRD3, and the heavy chain variable region is alternatively referred to as HCDR1, HCRD2, and HCDR3.

[0156] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to CD33 is substantially free of antibodies that do not bind to CD33). Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0157] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical except for possible naturally occurring mutations that may be present in small amounts. The monoclonal antibodies of the present invention can be prepared by hybridoma methods, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, a monoclonal antibody can be produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal such as a transgenic mouse or rat, the transgenic non-human animal having a genome comprising a human heavy chain transgene and a light chain transgene.

[0158] As used herein, the term "antigen binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab), a scFv dimer (bivalent diabody), a multispecific antibody formed by a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen binding fragment is capable of binding to the same antigen as the antigen to which the parent antibody or parent antibody fragment binds. According to specific embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain. According to other specific embodiments, the antigen binding fragment comprises Fab and F(ab').

[0159] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region or only a heavy chain variable region.

[0160] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human prepared using any technique known in the art. This definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain polypeptide and / or light chain polypeptide.

[0161] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with a human antibody, such that the antigen-binding properties of the antibody are retained but its antigenicity in the human body is reduced.

[0162] As used herein, the term "chimeric antibody" refers to an antibody whose amino acid sequence of an immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody with desired specificity, affinity and capacity derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions correspond to sequences in antibodies derived from another mammalian species (e.g., human) to avoid eliciting an immune response in that species.

[0163] As used herein, the term "multi-specific antibody" refers to an antibody comprising multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the multiple immunoglobulin variable domain sequences has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence in the multiple immunoglobulin variable domain sequences has binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, such as the same protein (or the subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope do not overlap or substantially do not overlap. In one embodiment, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multi-specific antibody comprises the third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multi-specific antibody is a bispecific antibody molecule, a tri-specific antibody molecule or a tetra-specific antibody molecule.

[0164] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. The bispecific antibody is characterized by a first immunoglobulin variable domain sequence with binding specificity to a first epitope and a second immunoglobulin variable domain sequence with binding specificity to a second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, such as the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a half antibody or a fragment thereof with binding specificity to the first epitope and a half antibody or a fragment thereof with binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a scFv or fragment thereof having binding specificity for a first epitope and a scFv or fragment thereof having binding specificity for a second epitope. In one embodiment, the first epitope is located on CD33 and the second epitope is located on CD3. In one embodiment, the first epitope is located on CD33 and the second epitope is located on PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD3 and / or other tumor-associated immunosuppressive factors or surface antigens.

[0165] As used herein, the term "CD33" refers to a 67 kD single-pass transmembrane glycoprotein that is a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family. CD33 is also known as Siglec-3, gp67, or p67. The structure of CD33 consists of an amino-terminal V-set Ig-like domain (encoded by exon 2 of CD33) that mediates sialic acid binding and a C2-set IG-like domain (encoded by exon 4) in its extracellular portion (Laszlo et al., 2016). Alternative splicing of CD33 RNA can result in a shorter isoform expressed on the cell surface that lacks the V-set but retains the C2-set Ig-like domain (Laszlo, Estey, & Walter, 2014; Laszlo et al., 2016). The biological relevance of this splicing process was largely unknown until recent studies showed that the single nucleotide polymorphism (SNP) rs12459419 is present in approximately 50% of the AML population and causes a skipping deletion of exon 2 of CD33, resulting in a deletion of the V domain of CD33 (Lamba et al., 2017). Full-length human CD33 was provided by Uniprot P20138 (SEQ ID NO: 1).

[0166] As used herein, an antibody that "specifically binds to CD33" refers to an antibody that specifically binds to CD33 at a concentration of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 An antibody that binds to CD33, preferably human CD33, preferably the C2 domain of CD33, with a KD of M or less. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). According to the present disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD of an antibody can be determined using surface plasmon resonance, such as by using a biosensor system (e.g., systems), or by using bio-layer interferometry techniques such as the Octet RED96 system.

[0167] The smaller the KD value of an antibody, the higher the affinity of the antibody for binding to the target antigen.

[0168] According to a specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0169] a. SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively;

[0170] b. SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively;

[0171] c. SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively;

[0172] d. SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively;

[0173] e. SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively;

[0174] f. SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively;

[0175] g. SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively;

[0176] h. SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively;

[0177] i. SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively;

[0178] j. SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively;

[0179] k. SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively;

[0180] l. SEQ ID NO: 402, 403, 404, 522, 523 and 524, respectively;

[0181] m. SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively;

[0182] n. SEQ ID NO: 423, 424, 425, 543, 544 and 545, respectively;

[0183] o. SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively;

[0184] p. SEQ ID NO: 351, 352, 353, 474, 475 and 476, respectively;

[0185] q. SEQ ID NOs: 357, 358, 359, 480, 481 and 482, respectively;

[0186] r. SEQ ID NOs: 372, 373, 374, 495, 496 and 497, respectively;

[0187] s. SEQ ID NOs: 375, 376, 377, 498, 499 and 500, respectively;

[0188] t. SEQ ID NO: 381, 382, ​​383, 504, 505 and 506, respectively;

[0189] u. SEQ ID NOs: 384, 385, 386, 507, 508 and 509, respectively;

[0190] v. SEQ ID NO: 390, 391, 392, 510, 511 and 512, respectively;

[0191] w. SEQ ID NOs: 393, 394, 395, 513, 514 and 515, respectively;

[0192] x. SEQ ID NOs: 396, 397, 398, 516, 517 and 518, respectively;

[0193] y. SEQ ID NOs: 399, 400, 401, 519, 520 and 521, respectively;

[0194] z. SEQ ID NOs: 405, 406, 407, 525, 526 and 527, respectively;

[0195] aa. SEQ ID NOs: 414, 415, 416, 534, 535 and 536, respectively;

[0196] bb. SEQ ID NOs: 417, 418, 419, 537, 538 and 539, respectively;

[0197] cc. SEQ ID NOs: 420, 421, 422, 540, 541 and 542, respectively;

[0198] dd. SEQ ID NO: 429, 430, 431, 549, 550 and 551, respectively;

[0199] ee. SEQ ID NOs: 432, 433, 434, 552, 553 and 554, respectively;

[0200] ff. SEQ ID NOs: 435, 436, 437, 555, 556 and 557, respectively;

[0201] gg. SEQ ID NOs: 438, 439, 440, 558, 559 and 560, respectively;

[0202] hh. SEQ ID NOs: 441, 442, 443, 561, 562 and 563, respectively;

[0203] ii. SEQ ID NOs: 450, 451, 452, 570, 571 and 572, respectively;

[0204] jj. SEQ ID NOs: 453, 454, 455, 573, 574 and 575, respectively;

[0205] kk. SEQ ID NOs: 456, 457, 458, 576, 577 and 578, respectively;

[0206] 11. SEQ ID NO: 459, 460, 461, 579, 580 and 581, respectively;

[0207] mm. SEQ ID NOs: 378, 379, 380, 582, 583 and 584, respectively;

[0208] nn. SEQ ID NO: 414, 415, 416, 585, 586 and 587, respectively; or

[0209] oo. SEQ ID NOs: 429, 430, 431, 480, 481 and 482, respectively;

[0210] The antibody or antigen-binding fragment thereof specifically binds to CD33, preferably human CD33.

[0211] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 259-296, or a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 300-338. According to a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 259-296 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 300-338.

[0212] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, said isolated monoclonal antibody or antigen-binding fragment thereof comprising:

[0213] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332;

[0214] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331;

[0215] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302;

[0216] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310;

[0217] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322;

[0218] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300;

[0219] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304;

[0220] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305;

[0221] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306;

[0222] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307;

[0223] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307;

[0224] 1. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317;

[0225] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319;

[0226] n. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324;

[0227] o. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325;

[0228] p. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 301;

[0229] q. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303;

[0230] r. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 308;

[0231] s. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 309;

[0232] t. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 311;

[0233] u. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 312;

[0234] v. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 313;

[0235] w. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 314;

[0236] x. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 315;

[0237] y. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 316;

[0238] z. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 318;

[0239] aa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 321;

[0240] bb. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322;

[0241] cc. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 323;

[0242] dd. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 326;

[0243] ee. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 327;

[0244] ff. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 328;

[0245] gg. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 329;

[0246] hh. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 330;

[0247] ii. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 333;

[0248] jj. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 334;

[0249] kk. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 335;

[0250] 11. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 336;

[0251] mm. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 337;

[0252] nn. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 338; or

[0253] oo. A heavy chain variable region having a polypeptide sequence of SEQ ID NO:286 and a light chain variable region having a polypeptide sequence of SEQ ID NO:303.

[0254] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 300. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:259; and a light chain variable region having the polypeptide sequence of SEQ ID NO:300.

[0255] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 351, 352, 353, 474, 475 and 476, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 301. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:260; and a light chain variable region having the polypeptide sequence of SEQ ID NO:301.

[0256] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 302. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:261; and a light chain variable region having the polypeptide sequence of SEQ ID NO:301.

[0257] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 357, 358, 359, 480, 481 and 482, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 303. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:262; and a light chain variable region having the polypeptide sequence of SEQ ID NO:303.

[0258] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 304. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:263; and a light chain variable region having the polypeptide sequence of SEQ ID NO:304.

[0259] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 305. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:264; and a light chain variable region having the polypeptide sequence of SEQ ID NO:305.

[0260] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 306. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:265; and a light chain variable region having the polypeptide sequence of SEQ ID NO:306.

[0261] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 307. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:266; and a light chain variable region having the polypeptide sequence of SEQ ID NO:307.

[0262] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 372, 373, 374, 495, 496 and 497, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 308. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:267; and a light chain variable region having the polypeptide sequence of SEQ ID NO:308.

[0263] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 375, 376, 377, 498, 499 and 500, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 309. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:268; and a light chain variable region having the polypeptide sequence of SEQ ID NO:309.

[0264] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 310. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:269; and a light chain variable region having the polypeptide sequence of SEQ ID NO:310.

[0265] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 381, 382, ​​383, 504, 505 and 506, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 311. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:270; and a light chain variable region having the polypeptide sequence of SEQ ID NO:311.

[0266] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 384, 385, 386, 507, 508 and 509, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 312. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:271; and a light chain variable region having the polypeptide sequence of SEQ ID NO:312.

[0267] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 307. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:272; and a light chain variable region having the polypeptide sequence of SEQ ID NO:307.

[0268] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 390, 391, 392, 510, 511 and 512, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 313. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:273; and a light chain variable region having the polypeptide sequence of SEQ ID NO:313.

[0269] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 393, 394, 395, 513, 54 and 515, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 314. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:274; and a light chain variable region having the polypeptide sequence of SEQ ID NO:314.

[0270] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 396, 397, 398, 516, 517 and 518, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 315. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:275; and a light chain variable region having the polypeptide sequence of SEQ ID NO:315.

[0271] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 399, 400, 401, 519, 520 and 521, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 316. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:276; and a light chain variable region having the polypeptide sequence of SEQ ID NO:316.

[0272] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 402, 403, 404, 522, 523 and 524, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 317. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:277; and a light chain variable region having the polypeptide sequence of SEQ ID NO:317.

[0273] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 405, 406, 407, 525, 526 and 527, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 318. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:278; and a light chain variable region having the polypeptide sequence of SEQ ID NO:318.

[0274] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 319. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:279; and a light chain variable region having the polypeptide sequence of SEQ ID NO:319.

[0275] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 320. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:280; and a light chain variable region having the polypeptide sequence of SEQ ID NO:320.

[0276] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 414, 415, 416, 534, 535 and 536, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 321. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:281; and a light chain variable region having the polypeptide sequence of SEQ ID NO:321.

[0277] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 417, 418, 419, 537, 538 and 539, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 322. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:282; and a light chain variable region having the polypeptide sequence of SEQ ID NO:322.

[0278] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 420, 421, 422, 540, 541 and 542, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 323. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:283; and a light chain variable region having the polypeptide sequence of SEQ ID NO:323.

[0279] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 423, 424, 425, 543, 544 and 545, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 324. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:284; and a light chain variable region having the polypeptide sequence of SEQ ID NO:324.

[0280] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 325. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:285; and a light chain variable region having the polypeptide sequence of SEQ ID NO:325.

[0281] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 429, 430, 431, 549, 550 and 551, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 326. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:286; and a light chain variable region having the polypeptide sequence of SEQ ID NO:326.

[0282] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 432, 433, 434, 552, 553 and 554, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 327. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:287; and a light chain variable region having the polypeptide sequence of SEQ ID NO:327.

[0283] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 435, 436, 437, 555, 556 and 557, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 328. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:288; and a light chain variable region having the polypeptide sequence of SEQ ID NO:328.

[0284] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 438, 439, 440, 558, 559 and 560, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 329. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:289; and a light chain variable region having the polypeptide sequence of SEQ ID NO:329.

[0285] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 441, 442, 443, 561, 562 and 563, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 330. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:290; and a light chain variable region having the polypeptide sequence of SEQ ID NO:330.

[0286] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 331. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:291; and a light chain variable region having the polypeptide sequence of SEQ ID NO:331.

[0287] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 332. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:292; and a light chain variable region having the polypeptide sequence of SEQ ID NO:332.

[0288] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 450, 451, 452, 570, 571 and 572, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 333. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:293; and a light chain variable region having the polypeptide sequence of SEQ ID NO:333.

[0289] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 453, 454, 455, 573, 574 and 575, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 334. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:294; and a light chain variable region having the polypeptide sequence of SEQ ID NO:334.

[0290] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 456, 457, 458, 576, 577 and 578, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 335. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:295; and a light chain variable region having the polypeptide sequence of SEQ ID NO:335.

[0291] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 459, 460, 461, 579, 580 and 581, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 336. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:296; and a light chain variable region having the polypeptide sequence of SEQ ID NO:336.

[0292] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 378, 379, 380, 582, 583 and 584, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 337. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:269; and a light chain variable region having the polypeptide sequence of SEQ ID NO:337.

[0293] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs: 414, 415, 416, 585, 586 and 587, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 338. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:281; and a light chain variable region having the polypeptide sequence of SEQ ID NO:338.

[0294] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 429, 430, 431, 480, 481 and 482, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 303. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:286; and a light chain variable region having the polypeptide sequence of SEQ ID NO:303.

[0295] Also provided herein is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, comprising an anti-CD33 antibody or antigen-binding fragment thereof and an anti-CD3 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof is an anti-CD33 monoclonal antibody or antigen-binding fragment thereof of the present invention, and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequences, (1) SEQ ID NO: 342, 343, 344, 465, 466 and 467, respectively, or (2) SEQ ID NO: 345, 346, 347, 468, 469 and 470, respectively.

[0296] The term "CD3" refers to the CD3 protein multi-subunit complex. CD3 may also be referred to as "cluster of differentiation 3". The CD3 protein multi-subunit complex consists of six (6) different polypeptide chains, including the CD3γ chain (SwissProt P09693) (SEQ ID NO: 588), the CD3δ chain (SwissProt P04234) (SEQ ID NO: 589), two CD3ε chains (SwissProt P07766) (SEQID NO: 590) and a CD3 chain homodimer (SwissProt 20963) (SEQ ID NO: 591), which are associated with the T cell receptor α and β chains. CD3 is a T cell co-receptor for activating both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). The CD3γ, CD3δ and CD3ε polypeptide chains of the CD3 multi-subunit complex are associated with the T cell receptor (TCR) and the CD3 chain to generate an activation signal in T lymphocytes, and the interaction between CD3 and the T cell receptor constitutes the TCR complex. Unless otherwise indicated, the term "CD3" includes any CD3 variants, isoforms and species homologs that are naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNAs encoding those polypeptides, preferably "CD3" is a human CD3 protein multi-subunit complex.

[0297] Redirecting T lymphocytes to cancer cells expressing CD33 via the TCR / CD3 complex represents an attractive alternative treatment approach. The TCR / CD3 complex of T lymphocytes consists of TCRα / β or TCRγ / δ heterodimers co-expressed on the cell surface and the invariant subunits of γ, δ, ε, ζ and η marked by CD3. Human CD3ε is described by UniProt P07766 (CD3E_HUMAN). The anti-CD3ε antibody described in the prior art is SP34 (Yang SJ, The Journal of Immunology, (1986) 137; 1097-1100), which reacts with both primate and human CD3 and is commercially available from Pharmingen. Other anti-CD3 antibodies described in the prior art include UCHT-1 (see WO2000041474) and BC-3 (Fred Hutchinson Cancer Research Institute; Phase I / II trial for GvHD, Anasetti et al., Transplantation, 54:844 (1992)). SP34 differs from UCHT-1 and BC-3 in that SP-34 recognizes an epitope present only on the epsilon chain of CD3 (see Salmeron et al., 1991, J. Immunol., Vol. 147, p. 3047), whereas UCHT-1 and BC-3 recognize epitopes contributed by both the epsilon chain and the gamma chain. Antibody sequences with the same sequence as SP34 are described in at least WO2008119565, WO2008119566, WO2008119567, WO2010037836, WO2010037837 and WO2010037838. An antibody sequence with 96% identity to SP34 VH is described in US8236308 (WO2007042261).

[0298] Different formats of bispecific antibodies have been described and recently reviewed by Chames and Baty in Curr Opin Drug Disc Dev, 2009, vol. 12, p. 276.

[0299] In some embodiments, the bispecific antibodies of the invention are diabodies, crossbodies or bispecific antibodies obtained by controlled Fab arm exchange, such as those described herein.

[0300] In some embodiments, bispecific antibodies include IgG-like molecules with complementary CH3 domains to force heterodimerization; recombinant IgG-like dual-targeting molecules, wherein each side of the molecule contains a portion of the Fab fragment or Fab fragment of at least two different antibodies; IgG fusion molecules, in which the full-length IgG antibody is fused with an additional Fab fragment or a portion of the Fab fragment; Fc fusion molecules, in which a single-chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; Fab fusion molecules, in which different Fab fragments are fused together; heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and diabodies, in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0301] In some embodiments, IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A / S).

[0302] In some embodiments, recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK / Domantis), two-in-one antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX bodies (CovX / Pfizer).

[0303] In some embodiments, IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), IgG-like bispecific antibodies (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

[0304] In some embodiments, Fc fusion molecules include ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART) (MacroGenics), and Dual (ScFv).sub.2-Fab (National Research Center for Antibody Medicine--China).

[0305] In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-based, diabody-based domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (Affimed), dual affinity retargeting molecules (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), dual-targeting heavy chain domain antibodies only.

[0306] The full-length bispecific antibodies of the present invention can be produced, for example, using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies in the following manner: a substitution is introduced at the heavy chain CH3 junction in each half molecule to promote the formation of heterodimers of two antibody half molecules with different specificities in a cell-free environment in vitro or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of a second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and re-formed by dissociation-association. The CH3 domain of the Fab arm can be engineered to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each of which binds to a different epitope, i.e., an epitope on CD33 and an epitope on CD3.

[0307] As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains having the same CH3 amino acid sequence.

[0308] As used herein, "heterodimerization" refers to the interaction of two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody with two heavy chains containing different CH3 amino acid sequences.

[0309] The "button" technology (see, e.g., PCT International Dimeric Antibodies WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the junction of the CH3 domains in human IgG can be mutated at positions that affect the interaction of the CH3 domains, thereby promoting heterodimer formation. Amino acids with small side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After the two antibodies are co-expressed, heterodimers are formed due to the preferential interaction of the heavy chain with the "button" with the heavy chain with the "button". Exemplary CH3 substitution pairs that form knobs and buckles (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0310] Other strategies may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in U.S. Patent Publication Nos. 2010 / 0015133; 2009 / 0182127; 2010 / 028637 or 2011 / 0123532. In other techniques, heterodimerization can be promoted by the following substitutions (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W, as described in US Patent Publication No. US2012 / 0149876 or US Patent Publication No. US2013 / 0195849.

[0311] In addition to the above methods, the bispecific antibodies of the present invention can also be produced in an in vitro cell-free environment by the following manner: introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies, and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions, thereby isomerizing disulfide bonds according to the method described in International Patent Publication No. WO2011 / 131746. In the method, the first monospecific bivalent antibody (e.g., anti-CD33 antibody) and the second monospecific bivalent antibody (e.g., anti-CD3 antibody) are modified to have certain substitutions at the CH3 domain that promote the stability of the heterodimer; these antibodies are incubated together under reducing conditions sufficient to isomerize disulfide bonds of cysteine ​​in the hinge region; thereby generating bispecific antibodies by Fab arm exchange. The incubation conditions can be optimally restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine ​​and β-mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions can be used: in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5-8, such as pH 7.0 or pH 7.4, at a temperature of at least 20° C., incubated for at least 90 minutes.

[0312] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0313] a. SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively;

[0314] b. SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively;

[0315] c. SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively;

[0316] d. SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively;

[0317] e. SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively;

[0318] f. SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively;

[0319] g. SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively;

[0320] h. SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively;

[0321] i. SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively;

[0322] j. SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively;

[0323] k. SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively;

[0324] l. SEQ ID NO: 402, 403, 404, 522, 523 and 524, respectively;

[0325] m. SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively;

[0326] n. SEQ ID NO: 423, 424, 425, 543, 544 and 545, respectively;

[0327] o. SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively;

[0328] p. SEQ ID NOs: 357, 358, 359, 480, 481 and 482, respectively;

[0329] q. SEQ ID NOs: 372, 373, 374, 495, 496 and 497, respectively;

[0330] r. SEQ ID NO: 375, 376, 377, 498, 499 and 500, respectively;

[0331] s. SEQ ID NO: 381, 382, ​​383, 504, 505 and 506, respectively;

[0332] t. SEQ ID NO: 384, 385, 386, 507, 508 and 509, respectively;

[0333] u. SEQ ID NOs: 390, 391, 392, 510, 511 and 512, respectively;

[0334] v. SEQ ID NOs: 393, 394, 395, 513, 514 and 515, respectively;

[0335] w. SEQ ID NOs: 396, 397, 398, 516, 517 and 518, respectively;

[0336] x. SEQ ID NOs: 399, 400, 401, 519, 520 and 521, respectively;

[0337] y. SEQ ID NOs: 405, 406, 407, 525, 526 and 527, respectively;

[0338] z. SEQ ID NOs: 414, 415, 416, 534, 535 and 536, respectively;

[0339] aa. SEQ ID NOs: 417, 418, 419, 537, 538 and 539, respectively;

[0340] bb. SEQ ID NOs: 420, 421, 422, 540, 541 and 542, respectively;

[0341] cc. SEQ ID NOs: 429, 430, 431, 549, 550 and 551, respectively;

[0342] dd. SEQ ID NO: 432, 433, 434, 552, 553 and 554, respectively;

[0343] ee. SEQ ID NOs: 435, 436, 437, 555, 556 and 557, respectively;

[0344] ff. SEQ ID NOs: 438, 439, 440, 558, 559 and 560, respectively;

[0345] gg. SEQ ID NOs: 441, 442, 443, 561, 562 and 563, respectively;

[0346] hh. SEQ ID NOs: 450, 451, 452, 570, 571 and 572, respectively;

[0347] ii. SEQ ID NO: 453, 454, 455, 573, 574 and 575, respectively;

[0348] jj. SEQ ID NOs: 456, 457, 458, 576, 577 and 578, respectively;

[0349] kk. SEQ ID NOs: 459, 460, 461, 579, 580 and 581, respectively;

[0350] 11. SEQ ID NO:378, 379, 380, 582, 583 and 584, respectively;

[0351] mm. SEQ ID NO: 414, 415, 416, 585, 586 and 587, respectively; or

[0352] nn. SEQ ID NOs: 429, 430, 431, 480, 481 and 482, respectively;

[0353] The anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0354] 1) SEQ ID NO: 342, 343, 344, 465, 466 and 467, respectively; or

[0355] 2) are SEQ ID NOs: 345, 346, 347, 468, 469 and 470, respectively.

[0356] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 259-296 or a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 300-338; and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: 257 or 258 or a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to one of SEQ ID NOs: NO:298 or 299 has a light chain variable region of a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical.

[0357] In certain embodiments, the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof comprises:

[0358] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0359] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0360] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0361] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0362] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0363] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0364] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0365] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0366] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0367] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0368] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0369] 1. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0370] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0371] n. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0372] o. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0373] p. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 301; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0374] q. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0375] r. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 308; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0376] s. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 309; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0377] t. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 311; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0378] u. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 312; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0379] v. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 313; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0380] w. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 314; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0381] x. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 315; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0382] y. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 316; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0383] z. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 318; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0384] aa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 321; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0385] bb. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0386] cc. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 323; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0387] dd. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 326; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0388] ee. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 327; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0389] ff. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 328; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0390] gg. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 329; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0391] hh. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 330; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0392] ii. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 333; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0393] jj. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 334; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0394] kk. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 335; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0395] 11. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 336; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0396] mm. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 337; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0397] nn. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 338; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0398] oo. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0399] pp. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0400] qq. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0401] rr. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0402] ss. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0403] tt. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0404] uu. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0405] vv. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0406] ww. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0407] xx. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0408] yy. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0409] zz. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0410] aaa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0411] bbb. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0412] ccc. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299; or

[0413] ddd. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0414] eee. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 301; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0415] fff. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0416] ggg. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 308; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0417] hhh. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 309; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0418] iii. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 311; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0419] jjj. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 312; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0420] kkk. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 313; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0421] lll. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 314; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0422] mmm. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 315; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0423] nnn. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 316; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0424] ooo. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 318; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0425] ppp. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 321; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0426] qqq. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0427] rrr. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 323; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0428] sss. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 326; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0429] ttt. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 327; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0430] uuu. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 328; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0431] vvv. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 329; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0432] www. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 330; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0433] xxx. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 333; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0434] yyy. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 334; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0435] zzz. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 335; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0436] aaaa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 336; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0437] bbbb. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 337; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0438] cccc. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 338; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299; or

[0439] dddd. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299.

[0440] According to another specific aspect, the present invention relates to an isolated anti-CD33 monoclonal antibody or antigen-binding fragment thereof that induces antibody-dependent cell-mediated cytotoxicity (ADCC). The monoclonal antibody or antigen-binding fragment thereof can, for example, induce ADCC in vitro. The monoclonal antibody or antigen-binding fragment thereof can have an EC of less than about 2 nM. 50 Inducing ADCC. In certain embodiments, EC 50 In some embodiments, the CD33 monoclonal antibody or antigen-binding fragment thereof comprises an IgG1 low-fucose backbone.

[0441] In some embodiments described herein, the immune effector properties of CD33-specific antibodies can be enhanced or silenced via Fc modification by techniques known to those skilled in the art. For example, Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc., can be provided and / or controlled by modifying residues in Fc that contribute to these activities.

[0442] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0443] The ability of monoclonal antibodies to induce ADCC can be enhanced by modifying their oligosaccharide components. Most of the glycans of human IgG1 or IgG3 are N-glycosylated at Asn297 in the form of well-known bibranched G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unengineered CHO cells typically have a glycan fucose content of about at least 85%. Removing core fucose from bibranched complex-type oligosaccharides attached to the Fc region can enhance the ADCC of antibodies via improved Fc.γ.RIIIa binding without changing antigen binding or CDC activity. Such mAbs can be achieved using different methods reported to result in the successful expression of relatively highly defucosylated antibodies with bibranched complex-type Fc oligosaccharides, such as controlling the culture osmotic pressure (Konno et al., Cytotechnology, 64:249-65, 2012), using the variant CHO cell line Lec13 as a host cell line (Shields et al., J Biol Chem, 277:26733-26740, 2002), using the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs, 2(4), 2010; Epub ahead of print; PMID: 20562582), using the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa ...). Chem, Vol. 278, pp. 3466-3473, 2003), introduction of small interfering RNA specific for the α-1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng, Vol. 88, pp. 901-908, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or the potent α-mannosidase I inhibitor, kifnosine (Ferrara et al., J Biol Chem, Vol. 281, pp. 5032-5036, 2006; Ferrara et al., Biotechnol Bioeng, Vol. 93, pp. 851-861, 2006; Xhou et al., Biotechnol Bioeng, 99: 652-65, 2008).

[0444] In some embodiments described herein, ADCC elicited by CD33 antibodies can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residues are numbered according to the EU index), as described in U.S. Pat. No. 6,737,056.

[0445] According to another specific aspect, the present invention relates to an isolated anti-CD33 monoclonal antibody or antigen-binding fragment thereof that is capable of translating to an isolated monoclonal antibody or antigen-binding fragment thereof at a concentration of less than about 5×10 -8 The dissociation constant (KD) of M binds to CD33. In certain embodiments, the dissociation constant is less than about 5×10 -8 M, less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1×10 -10 M, less than 5×10 -11 M or less than 1×10 -11 M.

[0446] According to another specific aspect, the present invention relates to an isolated anti-CD33 monoclonal antibody or antigen-binding fragment thereof that is capable of binding to CD33 at an EC of less than about 2 nM. 50 Binds to CD33 and induces internalization of CD33. In certain embodiments, EC 50 Less than about 2.0 nM, less than about 1.9 nM, less than about 1.8 nM, less than about 1.7 nM, less than about 1.6 nM, less than about 1.5 nM, less than about 1.4 nM, less than about 1.3 nM, less than about 1.2 nM, less than about 1.1 nM, less than about 1.0 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM and less than about 0.1 nM.

[0447] According to another specific aspect, the present invention relates to an isolated anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, which is capable of inducing T cell-dependent cytotoxicity in cells expressing CD33. The bispecific antibody or antigen-binding fragment thereof can, for example, be activated in vitro in cells expressing CD33 at an EC of less than about 2 nM. 50 In some embodiments, EC 50Less than about 2.0 nM, less than about 1.9 nM, less than about 1.8 nM, less than about 1.7 nM, less than about 1.6 nM, less than about 1.5 nM, less than about 1.4 nM, less than about 1.3 nM, less than about 1.2 nM, less than about 1.1 nM, less than about 1.0 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM and less than about 0.1 nM.

[0448] According to another specific aspect, the present invention relates to an isolated anti-CD33 monoclonal antibody and / or an isolated anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof, wherein the anti-CD33 monoclonal antibody or the anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof is chimeric.

[0449] According to another specific aspect, the present invention relates to an isolated anti-CD33 monoclonal antibody and / or an isolated anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof, wherein the anti-CD33 monoclonal antibody or the anti-CD33 / anti-CD3 bispecific antibody or the antigen-binding fragment thereof is human or humanized.

[0450] In another general aspect, the present invention relates to isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof of the present invention. In another general aspect, the present invention relates to isolated nucleic acids encoding bispecific antibodies or antigen-binding fragments thereof of the present invention. It will be appreciated by those skilled in the art that the coding sequence of a protein may be altered (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, it will be appreciated by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibodies and / or bispecific antibodies of the present invention may be altered without changing the amino acid sequence of the protein.

[0451] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or its antigen-binding fragment of the present invention. In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a bispecific antibody or its antigen-binding fragment of the present invention. According to the present disclosure, any vector known to those skilled in the art, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element of the conventional function of establishing an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce antibodies or their antigen-binding fragments in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention. According to the present disclosure, such techniques are well known to those skilled in the art.

[0452] In another general aspect, the present invention relates to a host cell comprising a nucleic acid encoding a monoclonal antibody and / or bispecific antibody of the present invention or an isolated antigen-binding fragment thereof. In view of the present disclosure, any host cell known to those skilled in the art can be used for recombinantly expressing the antibody of the present invention or its antigen-binding fragment. In some embodiments, the host cell is an Escherichia coli TG1 or BL21 cell (for expressing, for example, scFv or Fab antibody), a CHO-DG44 or CHO-K1 cell or a HEK293 cell (for expressing, for example, a full-length IgG antibody). According to a specific embodiment, a recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein the recombinant expression vector is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0453] In another general aspect, the present invention relates to a method for producing a monoclonal antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing a cell comprising glycosidic acid encoding a monoclonal antibody or antigen-binding fragment thereof under conditions that produce the monoclonal antibody or antigen-binding fragment thereof of the present invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). In another general aspect, the present invention relates to a method for producing a bispecific antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing a cell comprising glycosidic acid encoding a bispecific antibody or antigen-binding fragment thereof under conditions that produce the bispecific antibody or antigen-binding fragment thereof of the present invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art and as described herein.

[0454] Pharmaceutical composition

[0455] In another general aspect, the present invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In another general aspect, the present invention relates to a pharmaceutical composition comprising a bispecific antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising an antibody of the present invention and a pharmaceutically acceptable carrier. The antibodies of the present invention and compositions comprising the same can also be used to manufacture drugs for therapeutic applications mentioned herein.

[0456] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation or other materials known in the art for use in pharmaceutical preparations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration of the specific application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effect of the composition according to the present invention or the biological activity of the composition according to the present invention. According to the present disclosure, according to a specific embodiment, any pharmaceutically acceptable carrier suitable for antibody drug compositions can be used in the present invention.

[0457] The preparation of pharmaceutically active ingredients and pharmaceutically acceptable carriers is known in the art, such as Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulators, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present invention.

[0458] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e. a formulation comprising water. The liquid formulation may comprise a solution, a suspension, an emulsion, a microemulsion, a gel, etc. The aqueous formulation typically comprises at least 50% w / w of water, or at least 60% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, or at least 95% w / w of water.

[0459] In one embodiment, the pharmaceutical composition can be formulated as an injectable, which can be injected, for example, via an injection device (eg, a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0460] In another embodiment, the pharmaceutical composition is a solid preparation, such as a freeze-dried or spray-dried composition, which can be used as is, or a solvent and / or diluent can be added by a physician or patient before use. Solid dosage forms can include tablets, such as compressed and / or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical composition can also be in the form of a sachet, dragee, powder, granule, lozenge, or powder, for example, for reconstitution.

[0461] Dosage forms may be immediate release, in which case they may contain a water soluble or dispersible carrier, or they may be delayed release, sustained release or modified release, in which case they may contain a water insoluble polymer that modulates the rate of dissolution of the dosage form in the gastrointestinal tract or subcutaneously.

[0462] In other embodiments, the pharmaceutical composition may be delivered intranasally, intrabuccally, or sublingually.

[0463] The pH in the aqueous formulation may be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0464] In another embodiment of the present invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, sodium dihydrogen phosphate, fumaric acid, glycine, glycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine and tris (hydroxymethyl) aminomethane and mixtures thereof. Buffers may exist alone or in aggregates with a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments of the present invention.

[0465] In another embodiment of the present invention, pharmaceutical composition comprises preservative.The non-limiting example of buffer comprises: benzethonium chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, 4-hydroxy butyl benzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin ether, o-cresol, meta-cresol, p-cresol, 4-hydroxy ethyl benzoate, imidourea, 4-hydroxy methyl benzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, 4-hydroxy propyl benzoate, sodium dehydroacetate, thimerosal and their mixture.Preservative can exist alone or in aggregate with about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml concentration exists.The pharmaceutical composition comprising each in these specific preservatives constitutes the alternative embodiment of the present invention.

[0466] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of this embodiment include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), sugar alcohols (such as glycerol, 1,2-propylene glycol, propylene glycol), 1,3-propylene glycol and 1,3-butylene glycol), polyethylene glycol (e.g., PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars can be monosaccharides, disaccharides or polysaccharides, or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucan, amylopectin, dextrin, cyclodextrin, α and β-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is sugar alcohol, wherein the term "sugar alcohol" is defined as a C (4-8) hydrocarbon with at least one -OH group. The non-limiting example of sugar alcohol comprises mannitol, sorbitol, inositol, galactitol, hexadecene, xylitol and arabitol.The pharmaceutical composition comprising every kind of isotonic agent listed in this paragraph constitutes alternative embodiment of the present invention.Isotonic agent can exist alone or in aggregate with about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml concentration exists.The pharmaceutical composition comprising each in these specific isotonic agents constitutes alternative embodiment of the present invention.

[0467] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. Chelating agents may exist alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the present invention.

[0468] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants and / or one or more protease inhibitors.

[0469] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy- / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as thioglycerol or thioglycolic acid. The stabilizer may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the present invention.

[0470] In another embodiment of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble portion (hydrophilic) and a fat-soluble portion (lipophilic). For example, the surfactant is selected from: anionic surfactants, cationic surfactants, nonionic surfactants and / or zwitterionic surfactants. The surfactant can exist alone or in aggregates with a concentration of about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical composition comprising each of these specific surfactants constitutes an alternative embodiment of the present invention.

[0471] In another embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present alone or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the invention.

[0472] In another general aspect, the present invention relates to a method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof of the present invention, comprising combining the monoclonal antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. In another general aspect, the present invention relates to a method for producing a pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof of the present invention, comprising combining the bispecific antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0473] How to use

[0474] In another general aspect, the present invention relates to a method of targeting CD33 on the surface of cancer cells in a subject, the method comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds CD33, or an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the present invention.

[0475] Also contemplated herein is a therapeutic anti-CD33 antibody immunoconjugate comprising a therapeutic agent selected from a radionuclide, a boron, a gadolinium or a uranium atom, an immunomodulator such as a cytokine, a stem cell growth factor, a lymphotoxin such as a tumor necrosis factor (TNF), a hematopoietic factor such as an interleukin (IL), a colony stimulating factor (CSF) such as a granulocyte colony stimulating factor (G-CSF) or a granulocyte macrophage colony stimulating factor (GM-CSF), an interferon (IFN) such as an interferon-α, -β or -γ and a stem cell growth factor such as the one designated "S1 factor", a hematopoietic factor, an erythropoietin, a thrombopoietin, an antibody, a hormone, a hormone antagonist, an enzyme, an enzyme inhibitor, a photosensitizing therapeutic agent, a cytotoxic drug such as an antimitotic agent, an alkylating agent, an angiopoietic inhibitor, an apoptotic agent, an alkaloid agent, a COX-2 inhibitor and an antibiotic, a cytotoxin such as a plant toxin, a microbial toxin and an animal toxin and synthetic variants thereof, an angiogenesis inhibitor, different antibodies and combinations thereof. In a preferred embodiment, the cytokine is selected from IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-γ, TNF-α and combinations thereof, and the radionuclide is selected from Auger emitters, beta-emitters and alpha-emitters, such as P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, I-125, I-131, Pr-142, Pr-143, Pm-149, Sm-153, Tb-161, Ho-166, Er-169, Lu- -177, Re-186, Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212 and Bi-213, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Fm-255, B-10, Gd-157, U-235 and combinations thereof. Preferably, the energy of the radionuclide is between 20 keV and 10,000 keV.

[0476] The functional activity of antibodies and antigen-binding fragments thereof that bind to CD33 can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind to CD33 include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and OctetRed analysis; binding assays by FACS detection of antibody binding to CD33 on cancer cells. According to specific embodiments, methods for characterizing antibodies and antigen-binding fragments thereof that bind to CD33 include those described below.

[0477] In another general aspect, the present invention relates to a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds to CD33 or a pharmaceutical composition of the present invention. The cancer may be, for example, a cancer that expresses CD33. The cancer may be, for example, selected from, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors. The cancer may be, for example, a hematological cancer. Hematological cancers may be, for example, leukemias, lymphomas and myelomas. . In certain embodiments, the hematological cancer can be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low risk or high risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0478] According to an embodiment of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of an anti-CD33 antibody or an antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that induces a desired biological or pharmacological response in a subject. The therapeutically effective amount can be determined empirically and in a conventional manner relative to a specified purpose.

[0479] As used herein, with reference to an anti-CD33 antibody or antigen-binding fragment thereof, a therapeutically effective amount means an amount of the anti-CD33 antibody or antigen-binding fragment thereof that modulates an immune response in a subject in need thereof.

[0480] According to specific embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder, or condition being treated or symptoms associated therewith; (ii) reduce the duration of the disease, disorder, or condition being treated or symptoms associated therewith; (iii) prevent the development of the disease, disorder, or condition being treated or symptoms associated therewith; (iv) cause regression of the disease, disorder, or condition being treated or symptoms associated therewith; (v) prevent the development or onset of the disease, disorder, or condition being treated or symptoms associated therewith; vi) prevent recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of subjects suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospital stay of subjects suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (ix) improve survival of subjects suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder or condition being treated, or symptoms associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect of another therapy.

[0481] The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder or condition to be treated, the mode of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0482] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for administration by intravenous, subcutaneous or intramuscular injection.

[0483] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer that is not necessarily identifiable in a subject, but is capable of being identified in a subject. The terms "treat" and "treatment" may also refer to causing regression, preventing development, or at least delaying the development of a disease, disorder, or condition. In specific embodiments, "treat" and "treatment" refer to alleviating, preventing development or onset, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition, such as a tumor or, more preferably, cancer. In specific embodiments, "treat" and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In specific embodiments, "treat" and "treatment" refer to improved survival of a subject suffering from a disease, disorder, or condition. In specific embodiments, "treat" and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0484] According to a specific embodiment, a composition for treating cancer is provided. For cancer treatment, the composition can be used in combination with another treatment, including but not limited to chemotherapy, anti-CD20mAb, anti-TIM-3mAb, anti-CTLA-4 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, PD-1 / PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADC), targeted therapy or other anticancer drugs.

[0485] As used herein, in the context of two or more therapies being applied to a subject, the term "combination" refers to the use of more than one therapy. The use of the term "combination" does not limit the order in which the therapy is applied to a subject. For example, the first therapy (e.g., compositions as described herein) can be applied to a subject before the second therapy (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks ago), at the same time, or afterwards (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later) is applied.

[0486] Implementation

[0487] The present invention provides the following non-limiting embodiments.

[0488] Embodiment 1 is an isolated monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to the C2 domain of CD33.

[0489] Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0490] a. SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively;

[0491] b. SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively;

[0492] c. SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively;

[0493] d. SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively;

[0494] e. SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively;

[0495] f. SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively;

[0496] g. SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively;

[0497] h. SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively;

[0498] i. SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively;

[0499] j. SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively;

[0500] k. SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively;

[0501] l. SEQ ID NO: 402, 403, 404, 522, 523 and 524, respectively;

[0502] m. SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively;

[0503] n. SEQ ID NO: 423, 424, 425, 543, 544 and 545, respectively; or

[0504] o. SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively;

[0505] The antibody or antigen-binding fragment thereof specifically binds to CD33, preferably human CD33.

[0506] Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 292, 291, 261, 269, 280, 259, 263, 264, 265, 266, 272, 277, 279, 284 or 285, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 332, 331, 302, 310, 320, 300, 304, 305, 306, 307, 317, 319, 324 or 325.

[0507] Embodiment 4 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising:

[0508] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332;

[0509] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331;

[0510] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302;

[0511] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310;

[0512] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322;

[0513] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300;

[0514] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304;

[0515] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305;

[0516] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306;

[0517] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307;

[0518] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307;

[0519] 1. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317;

[0520] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319;

[0521] n. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; or

[0522] o. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 325.

[0523] Embodiment 5 is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0524] a. SEQ ID NOs: 357, 358, 359, 480, 481 and 482, respectively;

[0525] b. SEQ ID NOs: 372, 373, 374, 495, 496 and 497, respectively;

[0526] c. SEQ ID NOs: 375, 376, 377, 498, 499 and 500, respectively;

[0527] d. SEQ ID NOs: 381, 382, ​​383, 504, 505 and 506, respectively;

[0528] e. SEQ ID NOs: 384, 385, 386, 507, 508 and 509, respectively;

[0529] f. SEQ ID NOs: 390, 391, 392, 510, 511 and 512, respectively;

[0530] g. SEQ ID NOs: 393, 394, 395, 513, 514 and 515, respectively;

[0531] h. SEQ ID NOs: 396, 397, 398, 516, 517 and 518, respectively;

[0532] i. SEQ ID NOs: 399, 400, 401, 519, 520 and 521, respectively;

[0533] j. SEQ ID NOs: 405, 406, 407, 525, 526 and 527, respectively;

[0534] k. SEQ ID NOs: 414, 415, 416, 534, 535 and 536, respectively;

[0535] l. SEQ ID NOs: 417, 418, 419, 537, 538 and 539, respectively;

[0536] m. SEQ ID NOs: 420, 421, 422, 540, 541 and 542, respectively;

[0537] n. SEQ ID NO: 429, 430, 431, 549, 550 and 551, respectively;

[0538] o. SEQ ID NOs: 432, 433, 434, 552, 553 and 554, respectively;

[0539] p. SEQ ID NO: 435, 436, 437, 555, 556 and 557, respectively;

[0540] q. SEQ ID NOs: 438, 439, 440, 558, 559 and 560, respectively;

[0541] r. SEQ ID NOs: 441, 442, 443, 561, 562 and 563, respectively;

[0542] s. SEQ ID NOs: 450, 451, 452, 570, 571 and 572, respectively;

[0543] t. SEQ ID NO: 453, 454, 455, 573, 574 and 575, respectively;

[0544] u. SEQ ID NOs: 456, 457, 458, 576, 577 and 578, respectively;

[0545] v. SEQ ID NOs: 459, 460, 461, 579, 580 and 581, respectively;

[0546] w. SEQ ID NOs: 378, 379, 380, 582, 583 and 584, respectively;

[0547] x. SEQ ID NOs: 414, 415, 416, 585, 586 and 587, respectively; or

[0548] y. SEQ ID NOs: 429, 430, 431, 480, 481 and 482, respectively;

[0549] The antibody or antigen-binding fragment thereof specifically binds to CD33, preferably human CD33.

[0550] Embodiment 6 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 5, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NO: 260, 262, 267, 268, 270, 271, 273, 274, 275, 276, 278, 281, 282, 283, 286, 287, 288, 289, 290, 293, 294, 295 or 296, or a polypeptide sequence at least 95% identical to one of SEQ ID NO: 260, 262, 267, 268, 270, 271, 273, 274, 275, 276, 278, 281, 282, 283, 286, 287, 288, 289, 290, 293, 294, 295 or 296. NO: 301, 303, 308, 309, 311, 312, 313, 314, 315, 316, 318, 321, 322, 323, 326, 327, 328, 329, 330, 333, 334, 335, 336, 337 or 338 has a light chain variable region of a polypeptide sequence that is at least 95% identical.

[0551] Embodiment 7 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 5 or 6, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0552] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 301;

[0553] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303;

[0554] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 308;

[0555] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 309;

[0556] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 311;

[0557] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 312;

[0558] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 313;

[0559] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 314;

[0560] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 315;

[0561] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 316;

[0562] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 318;

[0563] 1. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 321;

[0564] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322;

[0565] n. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 323;

[0566] o. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 326;

[0567] p. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 327;

[0568] q. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 328;

[0569] r. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 329;

[0570] s. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 330;

[0571] t. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 333;

[0572] u. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 334;

[0573] v. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 335;

[0574] w. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 336;

[0575] x. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 337;

[0576] y. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 338; or

[0577] z. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 303.

[0578] Embodiment 8 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof has an EC of less than about 2 nM in vitro. 50 Induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0579] Embodiment 9 is the isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 8, wherein the monoclonal antibody or antigen-binding fragment thereof comprises an IgG1 low-fucose backbone.

[0580] Embodiment 10 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein the monoclonal antibody or antigen-binding fragment thereof has a density of less than about 5×10 -9 The dissociation constant (KD) of M binds to CD33.

[0581] Embodiment 11 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein the monoclonal antibody or antigen-binding fragment thereof binds to CD33 and has an EC of less than about 2 nM. 50 Induce internalization.

[0582] Embodiment 12 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof inhibits the activity of CD33.

[0583] Embodiment 13 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 12, wherein the antibody or antigen-binding fragment thereof is chimeric.

[0584] Embodiment 14 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13, wherein the antibody or antigen-binding fragment thereof is human or humanized.

[0585] Embodiment 15 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13, wherein the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent.

[0586] Embodiment 16 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14.

[0587] Embodiment 17 is a vector comprising the isolated nucleic acid according to embodiment 16.

[0588] Embodiment 18 is a host cell comprising the vector according to embodiment 17.

[0589] Embodiment 19 is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14 and a pharmaceutically acceptable carrier.

[0590] Embodiment 20 is a method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to Embodiment 19.

[0591] Embodiment 21 is a method according to embodiment 20, wherein the cancer is a hematological cancer.

[0592] Embodiment 22 is a method according to embodiment 21, wherein the hematological cancer is selected from leukemia, lymphoma or multiple myeloma.

[0593] Embodiment 23 is a method according to embodiment 22, wherein the hematological cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0594] Embodiment 24 is a method of producing a monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions in which the monoclonal antibody or antigen-binding fragment is produced, and recovering the antibody or antigen-binding fragment from the cell or culture.

[0595] Embodiment 25 is a method of producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1 to 14, comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0596] Embodiment 26 is an anti-CD33 / anti-CD3 bispecific antibody comprising an anti-CD33 antibody or an antigen-binding fragment thereof and an anti-CD3 antibody or an antigen-binding fragment thereof,

[0597] The anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0598] a. SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively;

[0599] b. SEQ ID NOs: 444, 445, 446, 564, 565 and 566, respectively;

[0600] c. SEQ ID NOs: 354, 355, 356, 477, 478 and 479, respectively;

[0601] d. SEQ ID NOs: 378, 379, 380, 501, 502 and 503, respectively;

[0602] e. SEQ ID NOs: 411, 412, 413, 531, 532 and 533, respectively;

[0603] f. SEQ ID NOs: 348, 349, 350, 471, 472 and 473, respectively;

[0604] g. SEQ ID NOs: 360, 361, 362, 483, 484 and 485, respectively;

[0605] h. SEQ ID NOs: 363, 364, 365, 486, 487 and 488, respectively;

[0606] i. SEQ ID NOs: 366, 367, 368, 489, 490 and 491, respectively;

[0607] j. SEQ ID NOs: 369, 370, 371, 492, 493 and 494, respectively;

[0608] k. SEQ ID NOs: 387, 388, 389, 492, 493 and 494, respectively;

[0609] l. SEQ ID NO: 402, 403, 404, 522, 523 and 524, respectively;

[0610] m. SEQ ID NOs: 408, 409, 410, 528, 529 and 530, respectively;

[0611] n. SEQ ID NO: 423, 424, 425, 543, 544 and 545, respectively; or

[0612] o. SEQ ID NOs: 426, 427, 428, 546, 547 and 548, respectively;

[0613] And the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0614] 1) SEQ ID NO: 342, 343, 344, 465, 466 and 467, respectively; or

[0615] 2) are SEQ ID NOs: 345, 346, 347, 468, 469 and 470, respectively.

[0616] Embodiment 27 is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to Embodiment 25, wherein the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 292, 291, 261, 269, 280, 259, 263, 264, 265, 266, 272, 277, 279, 284, or 285, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 332, 331, 302, 310, 320, 300, 304, 305, 306, 307, 317, 319, 324, or 325; and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 257 or 258, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 259. A light chain variable region having a polypeptide sequence that is at least 95% identical to that of NO:298 or 299.

[0617] Embodiment 28 is the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to embodiment 25 or 26, comprising:

[0618] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0619] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0620] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0621] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0622] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0623] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0624] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0625] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0626] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0627] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0628] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0629] 1. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0630] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0631] n. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0632] o. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0633] p. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0634] q. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 291 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 331; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0635] r. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 261 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 302; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0636] s. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 310; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0637] t. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 280 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0638] u. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 259 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 300; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0639] v. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 263 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 304; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0640] w. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 264 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 305; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0641] x. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 265 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 306; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0642] y. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 266 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0643] z. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 272 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 307; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0644] aa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 277 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 317; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0645] bb. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 279 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 319; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0646] cc. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 284 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 324; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299; or

[0647] dd. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 285 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 325; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299.

[0648] Embodiment 29 is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 28, wherein the anti-CD33 antibody or antigen-binding fragment thereof specifically binds to the C2 domain of the CD33.

[0649] Embodiment 30 is an anti-CD33 / anti-CD3 bispecific antibody comprising an anti-CD33 antibody or an antigen-binding fragment thereof and an anti-CD3 antibody or an antigen-binding fragment thereof,

[0650] The anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0651] a. SEQ ID NOs: 357, 358, 359, 480, 481 and 482, respectively;

[0652] b. SEQ ID NOs: 372, 373, 374, 495, 496 and 497, respectively;

[0653] c. SEQ ID NOs: 375, 376, 377, 498, 499 and 500, respectively;

[0654] d. SEQ ID NOs: 381, 382, ​​383, 504, 505 and 506, respectively;

[0655] e. SEQ ID NOs: 384, 385, 386, 507, 508 and 509, respectively;

[0656] f. SEQ ID NOs: 390, 391, 392, 510, 511 and 512, respectively;

[0657] g. SEQ ID NOs: 393, 394, 395, 513, 514 and 515, respectively;

[0658] h. SEQ ID NOs: 396, 397, 398, 516, 517 and 518, respectively;

[0659] i. SEQ ID NOs: 399, 400, 401, 519, 520 and 521, respectively;

[0660] j. SEQ ID NOs: 405, 406, 407, 525, 526 and 527, respectively;

[0661] k. SEQ ID NOs: 414, 415, 416, 534, 535 and 536, respectively;

[0662] l. SEQ ID NOs: 417, 418, 419, 537, 538 and 539, respectively;

[0663] m. SEQ ID NOs: 420, 421, 422, 540, 541 and 542, respectively;

[0664] n. SEQ ID NO: 429, 430, 431, 549, 550 and 551, respectively;

[0665] o. SEQ ID NOs: 432, 433, 434, 552, 553 and 554, respectively;

[0666] p. SEQ ID NO: 435, 436, 437, 555, 556 and 557, respectively;

[0667] q. SEQ ID NOs: 438, 439, 440, 558, 559 and 560, respectively;

[0668] r. SEQ ID NOs: 441, 442, 443, 561, 562 and 563, respectively;

[0669] s. SEQ ID NOs: 450, 451, 452, 570, 571 and 572, respectively;

[0670] t. SEQ ID NO: 453, 454, 455, 573, 574 and 575, respectively;

[0671] u. SEQ ID NOs: 456, 457, 458, 576, 577 and 578, respectively;

[0672] v. SEQ ID NOs: 459, 460, 461, 579, 580 and 581, respectively;

[0673] w. SEQ ID NOs: 378, 379, 380, 582, 583 and 584, respectively;

[0674] x. SEQ ID NOs: 414, 415, 416, 585, 586 and 587, respectively; or

[0675] y. SEQ ID NOs: 429, 430, 431, 480, 481 and 482, respectively;

[0676] And the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0677] 1) SEQ ID NO: 342, 343, 344, 465, 466 and 467, respectively; or

[0678] 2) are SEQ ID NOs: 345, 346, 347, 468, 469 and 470, respectively.

[0679] Embodiment 31 is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to Embodiment 29, wherein the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 95% identical to one of SEQ ID NO: 260, 262, 267, 268, 270, 271, 273, 274, 275, 276, 278, 281, 282, 283, 286, 287, 288, 289, 290, 293, 294, 295 or 296, or a polypeptide sequence that is at least 95% identical to one of SEQ ID NO: NO: 301, 303, 308, 309, 311, 312, 313, 314, 315, 316, 318, 321, 322, 323, 326, 327, 328, 329, 330, 333, 334, 335, 336, 337 or 338; and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 257 or 258 or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 298 or 299.

[0680] Embodiment 32 is the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to embodiment 29 or 30, comprising:

[0681] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 301; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0682] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0683] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 308; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0684] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 309; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0685] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 311; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0686] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 312; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0687] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 313; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0688] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 314; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0689] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 315; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0690] j. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 316; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0691] k. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 318; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0692] 1. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 321; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0693] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0694] n. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 323; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0695] o. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 326; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0696] p. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 327; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0697] q. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 328; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0698] r. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 329; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0699] s. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 330; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0700] t. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 333; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0701] u. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 334; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0702] v. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 335; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0703] w. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 336; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0704] x. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 337; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0705] y. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 338; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0706] z. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298;

[0707] aa. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 260 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 301; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0708] bb. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 262 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0709] cc. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 308; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0710] dd. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 268 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 309; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0711] ee. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 270 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 311; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0712] ff. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 271 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 312; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0713] gg. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 273 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 313; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0714] hh. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 274 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 314; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0715] ii. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 275 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 315; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0716] jj. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 276 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 316; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0717] kk. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 278 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 318; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0718] 11. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 321; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0719] mm. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 282 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 322; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0720] nn. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 283 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 323; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0721] oo. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 286 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 326; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0722] pp. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 287 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 327; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0723] qq. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 288 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 328; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0724] rr. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 289 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 329; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0725] ss. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 290 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 330; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0726] tt. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 293 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 333; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0727] uu. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 294 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 334; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0728] vv. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 335; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0729] ww. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 296 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 336; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0730] xx. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 269 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 337; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299;

[0731] yy. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 338; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 299; or

[0732] zz. A heavy chain variable region having a polypeptide sequence of SEQ ID NO:286 and a light chain variable region having a polypeptide sequence of SEQ ID NO:303; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:258 and a light chain variable region having a polypeptide sequence of SEQ ID NO:299.

[0733] Embodiment 33 is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 26 to 32, wherein the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof has an EC of less than about 1 nM in cells expressing CD33 in vitro. 50 Values ​​induce T cell-dependent cytotoxicity.

[0734] Embodiment 34 is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 33, wherein the bispecific antibody or antigen-binding fragment thereof is chimeric.

[0735] Embodiment 35 is an anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 34, wherein the bispecific antibody or antigen-binding fragment thereof is human or humanized.

[0736] Embodiment 36 is an isolated nucleic acid encoding the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 35.

[0737] Embodiment 37 is a vector comprising the isolated nucleic acid according to embodiment 36.

[0738] Embodiment 38 is a host cell comprising the vector according to embodiment 37.

[0739] Embodiment 39 is a pharmaceutical composition comprising the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 26 to 35 and a pharmaceutically acceptable carrier.

[0740] Embodiment 40 is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to Embodiment 39.

[0741] Embodiment 41 is a method according to embodiment 40, wherein the cancer is a hematological cancer.

[0742] Embodiment 42 is a method according to embodiment 41, wherein the hematological cancer is selected from leukemia, lymphoma or multiple myeloma.

[0743] Embodiment 43 is a method according to embodiment 42, wherein the hematological cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0744] Embodiment 44 is a method of producing an anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof according to any one of Embodiments 26 to 35, comprising culturing a cell comprising a nucleic acid encoding the anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof under conditions in which the anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof is produced, and recovering the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment from the cell or culture.

[0745] Embodiment 45 is a method of producing a pharmaceutical composition comprising the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 26 to 35, comprising combining the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0746] Example

[0747] Reagents

[0748] Antigen generation

[0749] Human and cynomolgus CD33 proteins were produced in the presence of human serum albumin (HSA) in monomeric form with or without mutation (UniprotP02768 with C58S mutation) fused at the C-terminus for immunization and assays. A cDNA encoding the CD33 protein antigen with a six-histidine tag was synthesized and cloned into a mammalian secretory expression vector under the actin promoter using standard molecular biology techniques.

[0750] The full-length human CD33 extracellular domain (ECD) derived from Uniprot P20138 (SEQ ID NO: 1) (human CD33ECD) was fused to the signal sequence at the N-terminus, with or without HSA, followed by a six-histidine tag at the C-terminus, hCD33ECD with only HSA and only hCD33ECD. Human CD33ECD expression constructs were transiently transfected into HEK293 derivative cells Expi293 (Gibco / Thermo Fisher Scientific; Waltham, MA, USA) using Expifectamine according to the manufacturer's protocol. Cells were incubated at 37°C on an orbital shaker with 8% CO2 for 5 days before harvesting. Expressing cells were removed by centrifugation, and soluble CD33 was purified from the culture medium using immobilized metal affinity chromatography using Ni Sepharose 6 Fast Flow resin (GE Healthcare; Little Chalfont, United Kingdom) followed by Superdex 200 preparative size exclusion chromatography (SEC) (GE Healthcare) in Dubelcco's phosphate saline buffer, pH 7.2 (1×DPBS). SEC elution fractions, excluding any disulfide aggregates, were combined and sterile filtered to yield the final protein for immunoassays and CD33 assays. Protein concentration was determined by A280, and the quality of purified protein was assessed by SDS-PAGE and analytical SEC (Phenomenex; Torrance, CA, USA). Endotoxin measurements were performed using EndoSafe-PTS columns, a chromogenic LAL assay (Charles River; Wilmington, MA, USA).

[0751] Human CD33 ECD subdomain proteins, hCD33 V domain-HSA, hCD33 V domain-his, hCD33 C2 domain-HSA and hCD33 C2 domain-His were similarly constructed, expressed and purified as full-length human CD33 ECD.

[0752] Based on Genbank sequence XP_005590138.1, cynomolgus monkey CD33 constructs cynomolgus monkey CD33 ECD-HSA, cynomolgus monkey CD33-His were also generated for immunization and cross-selectivity assays. The expression and purification of cynomolgus monkey CD33 protein was identical to human CD33 protein.

[0753] The CD33 antigen for screening was biotinylated in 50 mM sodium phosphate (pH 7.2) using the SureLink Chromagenic Biotin Labeling kit (SeraCare KPL) according to the manufacturer's conditions. Briefly, a 25 mM stock solution of biotin was added to the CD33 protein at a 4:1 molar ratio of biotin to protein and incubated at room temperature for 30 minutes with gentle rotation, then switched to 4 ° C for another 2 hours. Unincorporated biotin was removed by buffer exchange into 1×DPBS. Protein concentration and biotin incorporation were determined by measuring at A280 nm and A354 nm using NanoDrop. The sequence of each of the above antigens is shown in Table 1.

[0754] Table 1: Antigen sequences

[0755] Protein name Protein ID SEQ ID NO Cynomolgus macaque CD33 ECD-HSA C33W1 2 Human CD33 ECD-HSA C33W2 3 Human CD33-V-HSA C33W3 4 HumanCD33-C2-HSA C33W4 5 Human CD33-V-His C33W8 6 Human CD33 C2-His C33W9 7 Human CD33 ECD-His C33W49 8 Cynomolgus macaque CD33 ECD-His C33W50 9 Human CD33 full length 10 Cynomolgus macaque CD33 full length 11

[0756] Generation of isogenic cell lines expressing CD33

[0757] Cell lines expressing human and cynomolgus macaque CD33 were generated using lentivirus containing full-length human CD33 or cynomolgus macaque CD33 and puromycin (Genecopoeia, Rockville, MD, USA) for selection of CD33-positive cells. HEK293F cells (ATCC), which are negative for CD33, were transduced with lentiviral particles to overexpress human CD33 and cynomolgus macaque CD33. After transduction, cells positively expressing CD33 and resistance markers were selected by treating the pooled cells and growing them in DMEM + 10% HI FBS (Life Technologies, Carlsbad, CA, USA) and supplemented with different concentrations of puromycin (Life Technologies, USA).

[0758] In addition to the cell lines produced by HEK, several commercial cell lines were used for binding and cytotoxicity assays. These include MOLM13, KG1, SH2, OCIAML3 and MV411, and can be purchased from the American Type Culture Collection or the German Microbiological Culture Collection (Deutsche Sammlungvon Mikrooranismen und Zellkulturen), and cultured at 37°C, 5% CO2 in complete RPMI medium with 10% FBS.

[0759] Example 1: Immune Activity

[0760] OmniRat

[0761] Human immunoglobulin transgenic rat strain ( Ligand Pharmaceuticals; San Diego, CA) was used to culture hybridoma cells expressing human CD33 monoclonal antibody. Contains a chimeric human / rat IgH locus (comprising 22 human V H , all people D and J H The fragment was linked to rat C H locus) together with a complete human IgL locus (12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ). (See, e.g., Osborn et al., 2013, J Immunol, Vol. 190, No. 4, pp. 1481-1490). As a result, the rats exhibit reduced expression of rat immunoglobulins, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity chimeric human / mouse IgG monoclonal antibodies with fully human variable regions. PCT Publication WO 2014 / 093908 to Bruggemann et al. describes Preparation and use of rats and genome modifications carried by such rats.

[0762] When immunized with recombinant human and cynomolgus macaque CD33 (human CD33 ECD-HSA and cynomolgus macaque CD33 ECD-HSA, respectively), the transgenic rats produced chimeric human-rat IgG antibodies against human CD33, some of which also bound to cynomolgus macaque CD33.

[0763] Eight OmniRats were immunized alternately with human CD33 ECD-HSA and cynomolgus macaque CD33 ECD-HSA. After a 46-day immunization schedule, lymph nodes were harvested from all eight OmniRats and used to generate hybridomas. Eighty-one 96-well hybridoma supernatant plates were screened via combined ELISA and AlphaLISA using standard techniques, of which 128 hybridoma supernatants were selected for specific binding to human CD33 ECD-HSA and cynomolgus macaque CD33 ECD-HSA. Most of the 128 supernatants were also positive for binding to cells overexpressing human CD33 or cynomolgus macaque CD33.

[0764] Six additional OmniRats were immunized with rhuCD33 alone. After the 31-day immunization schedule, lymph nodes were harvested from all six OmniRats and used to generate hybridomas. Thirty 96-well plates of hybridoma supernatants were screened via binding ELISA using standard techniques, of which 94 hybridoma supernatants were selected for specific binding to human CD33 ECD-HSA and cynomolgus CD33 ECD-HSA. Hybridoma lysates were prepared from positive clones and proceeded to v-region cloning as described below.

[0765] OmniMouse

[0766] Human immunoglobulin transgenic mouse strain ( Ligand Pharmaceuticals) was used to culture hybridoma cells expressing human CD33 monoclonal antibody. Contains a chimeric human / rat IgH locus as well as a fully human IgL locus. The mice exhibit reduced expression of mouse immunoglobulins, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity chimeric human / mouse IgG monoclonal antibodies with fully human variable regions.

[0767] When immunized with recombinant human and cynomolgus macaque CD33 (human CD33 ECD-HSA and cynomolgus macaque CD33 ECD-HAS, respectively), the transgenic mice produced chimeric human-rat IgG antibodies against human CD33, some of which also bound to cynomolgus macaque CD33.

[0768] Four OmniMice were immunized alternately with human CD33 ECD-HSA and cynomolgus macaque CD33 ECD-HSA. After a 53-day immunization schedule, spleens and lymph nodes were harvested from all four OmniMice and used to generate hybridomas. Forty-eight 96-well hybridoma supernatant plates were screened via combined ELISA and AlphaLISA, of which 8 hybridoma supernatants were selected for specific binding to human CD33 ECD-HSA and cynomolgus macaque CD33 ECD-HSA. Hybridoma lysates were prepared from positive clones and proceeded to the v region cloning described below.

[0769] V region cloning

[0770] Total RNA from hybridoma cell lysates was purified using the RNeasy 96 kit (Qiagen; Hilden, Germany) according to the manufacturer's protocol, and the resulting RNA was quantified using Drop Sense and stored at -80°C or cDNA was synthesized using the Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen; Carlsbad, CA, USA). First-strand cDNA synthesis was performed using gene-specific primers annealed to the constant regions of the heavy chain, kappa chain, and lambda chain, respectively. The RT-PCR reaction mixture consisted of up to 3 g of purified RNA, gene-specific primers, dNTP mix, reaction buffer, 25 mM MgCl2, DTT, RNaseOUT TM (40 U / μl, Invitrogen) and SuperScript TMIII RT (200U / μl, Invitrogen catalog number 18080-051), and incubated at 50°C for 50 minutes and at 85°C for 5 minutes. The resulting single-stranded cDNA was stored at -20°C, or the single-stranded DNA was PCR amplified. Platinum Pfx polymerase (Invitrogen) was used for PCR reaction. Using optimized PCR conditions, the v region fragment was amplified by forward primers and reverse primers annealing to the leader sequence and the heavy chain, k chain and lambda chain constant regions, respectively. The resulting PCR fragment was run on a gel and sequenced on GENEWIZ using pre-designed primers to obtain the V region sequence. The resulting .abi files of the V region sequence were collected and analyzed by the Sanger V region sequence analysis program created by Janssen Biologics Discovery. The AA sequence of the recovered V region was recorded in an internal database, codons were optimized and cloned into a pUnder-based expression vector carrying the appropriate constant region of the desired human antibody isotype IgG1 F405L and IgG4 PAA. A total of 76 OMNIRat antibodies and 8 OMNIMouse antibodies were successfully cloned and continued to be further characterized. The following table summarizes the top 42 sequences identified from the OMNIRat campaign (see Table 2) and the top 16 sequences identified from the OMNIMouse campaign (see Table 3), where several OMNIRat antibodies were cloned into IgG1 as well as IgG4 PAA, and all antibodies from the OMNIMouse campaign were cloned into both IgG1 and IgG4 PAA.

[0771] Table 2: Antibody sequences identified by CD33 immunization in OMNIRat

[0772]

[0773]

[0774] HC: heavy chain; LC: light chain

[0775] Table 3: Antibody sequences identified by CD33 immunization in OMNIMouse

[0776]

[0777] HC: heavy chain; LC: light chain

[0778] Expi293 small-scale transfection and purification

[0779] Antibodies identified in the immunization campaign and subsequent v-region cloning (to IgG1 F405L and IgG4 PAA) were expressed and purified via a small 2 ml scale. TMCells (from Thermo Fisher Scientific) were cultured at 1.25 × 10 5 to 2.25×10 5 The density of viable cells / mL was inoculated into Expi293 TM Expression Medium and cultured in polycarbonate, disposable, sterile, ventilated, unbaffled conical shake flasks in a 37°C, 7% CO2 shaking incubator (INFORS HT Multitron Pro). For routine cell growth in 125 mL to 2 L shake flasks, set the shaking speed to 130 rpm for a shaker with a 19 mm shaking diameter. When the density reaches 3× with 98% to 99% viability, 6 10 to 5 × 10 6 When the cells reached the logarithmic growth phase with viable cells / mL, the cells were subcultured.

[0780] On the day of transfection, the viable cell density and viability percentage were determined. 6 For optimal transfection, sterile heavy and light chain plasmid DNA was used at a concentration of 0.1 mg / mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0781] Expi293 was transfected according to the manufacturer's transfection protocol (Thermo Fisher publication number MAN0007814). TM Cells were transfected in 24-well deep-well plates (GE Healthcare). Briefly, 0.1 mL of OptiMEM TM Dilute plasmid DNA in 5 μL of ExpiFectamine 5 μL of culture medium (ThermoFisher Scientific) at the following ratios: 0.250 μg heavy chain DNA: 0.750 μg light chain DNA: 0.5 μg pAdvantage. TM 293 transfection reagent and mix it with 95 μL OptiMEM TM The culture medium was gently mixed and incubated for 1 minute. TM Add 293 reagent to the diluted DNA, mix gently, and add ExpiFectamine TM 293 / plasmid DNA complex was incubated at room temperature for 40 minutes. After incubation, 1.8 mL of Expi293 TM Cells were added to the complexes and incubated overnight in a shaking incubator at 37°C, 7% CO2.

[0782] On day 1 after transfection, add 10 μL of ExpiFectamine TM 293 Enhancer 1 and 100 Lμ of Expifectamine 293 TM Enhancer 2 was added and the plates were returned to the incubator for another 5 days. The cultures were harvested on day 6 post-transfection by centrifugation at 850×G for 15 minutes and then purified.

[0783] The 1.7ml clear expression supernatant prepared above is transferred to a new 962ml deep well plate. By moving the 1:4 mixture of 800μl mAb Select Sure resin (GE Healthcare) and DPBS- / -slurry to each hole of 96-well Acroprep Advance 1μM glass filter plates (Pall Corporation (Pall)) to prepare the purification plate. Apply a vacuum pressure of 200mbar to remove excess PBS to the plate, then wash with the fresh PBS of 800μl. Apply a vacuum pressure of 200mbar to remove wash buffer. Then the clarified supernatant is transferred to the resin washed with PBS, gently mixed and incubated for 15 minutes. After incubation, apply a vacuum pressure of 200mbar to remove supernatant. The mAbSelect Sure resin was washed three times with PBS and once with 25 mM sodium acetate (pH 5) (TEKNOVA; Hollister, CA, USA), applying a vacuum pressure of 200 mbar between washes to remove excess buffer. The mAb bound to the resin was eluted with 0.1 M sodium acetate (pH 3.5) and incubated for 10 minutes for effective dissociation. The filter plate was placed on top of a 96-deep well plate, and the eluted mAb was collected in the bottom plate via centrifugation at 1000 g for 2 minutes. 80 μl of 2.5 M Tris-acetate (pH 7.2) was added to neutralize the mAb. mAbs were soaked in PBS overnight in 96-well DispoDIALYZER plates (Harvard Apparatus; Holliston, MA), transferred to 96-well Acroprep Advance 0.2 μm Supor filter plates (Pall; Port Washington, NY), placed on top of 96 deep-well plates, and the protein solution was filtered via centrifugation at 1,500 g for 15 minutes in a benchtop centrifuge. Protein concentration was determined by A280 measurement on the filtrate using a DropSense Instrument (Trinean).

[0784] Example 2: Characteristics of anti-CD33 mAbs

[0785] The OMNIRat antibodies, which were immunoidentified, cloned, and subsequently expressed and purified, were further characterized for binding to cells expressing CD33 and binding to recombinant antigens. The purified antibodies were evaluated for binding to HEK293F cells stably transfected to express human CD33 or cynomolgus macaque CD33 (generated as described above) and parental HEK293F as a negative control. Cells were harvested from tissue culture flasks using non-enzymatic dissociation buffer (Thermo Scientific). The flask was rinsed twice with PBS, and dissociation buffer was added to the flask, and the flask was incubated at 37°C for 10 minutes until the cells became non-adherent. The cells were centrifuged at 300g for 5 minutes and then centrifuged at 1.0×10 6 Cells / ml were resuspended in staining buffer (Becton Dickinson; Franklin Lakes, NJ). 50,000 cells / well of each cell type were placed in 50 μl of staining buffer in a round bottom plate (Becton Dickinson). 50 μl of 2X concentration of test mAb or isotype control was added at 3 dilutions and zero (120nM, 12nM, 1.2nM and 0nM), and the resulting solution was incubated at 4°C for 30 minutes. 100 μl of staining buffer was added to all wells of each plate, the plate was centrifuged at 300g for 5 minutes, the buffer was removed, 200 μl of staining buffer was added to all wells of each plate, the plate was centrifuged at 300g for 5 minutes, and the buffer was removed. 50 μl of 2 μg / ml goat anti-human Fc AF647 secondary antibody (Jackson Immunoresearch; West Grove, PA) was added to all wells of the plate, and the plate was incubated at 4°C for 30 minutes. 100 μl of staining buffer was added to all wells of the plate, the plate was centrifuged at 300g for 5 minutes, and the buffer was removed. 200 μl of running buffer (running buffer is staining buffer, 1 mM EDTA, 0.1% guluronic acid) was added to all wells of the plate, the plate was centrifuged at 300g for 5 minutes, and the buffer was removed. 30 μl of running buffer containing Sytox Green live / dead dye (ThermoFisher) was added to all wells with cells, and the plate was read on the iQue IntelliCyt flow cytometer. Cells were gated on forward and side scatter to eliminate debris, then gated on singlets, then gated on live cells excluding Sytox staining. Antibody binding was assessed by mean fluorescence intensity in the AF647 channel.

[0786] To begin evaluating the biophysical binding properties of the purified mAbs, off-rate screening was performed. 76 OMNIRat anti-CD33 mAbs were tested for binding to recombinant human CD33 ECD-HSA (C33W2) and cynomolgus macaque CD33 ECD-HSA (C33W1) proteins (Janssen) and off-rates were measured using an IBIS MX96 SPRi array platform (Carterra; Newton, PA, USA). CMD50m sensor chips (Xantec, lot CMD50m) with an association time of 10 minutes were used in the IBIS instrument. 0415 .a), goat anti-human Fc IgG (Jackson Immunoresearch catalog number 109-005-098) was directly immobilized in acetate buffer (pH 4.5) via amine coupling at 100 μg / mL. An average GAH-Fc immobilization level of approximately 9000 Rus was achieved. The sensor chip was transferred to a continuous flow microdroplet (CFM) unit to capture each anti-CD33 mAb at 10 μg / ml for 10 minutes. Binding to IBIS SPRi was measured by single cycle kinetics without regeneration. Using PBST (PBS with 0.005% Tween) as the running buffer, each antigen concentration series (3 μM, 3-fold dilution series) was injected sequentially from low concentration (0.46 nM) to high concentration (μ3 M) to bind the captured mAb with an association time of 5 minutes and a dissociation time of 15 minutes. The raw binding data (.trix file format) were referenced and aligned using SprintX software (Wasatch, version 1.9.3.2), and then exported (.ibmx file format) to Scrubber software (version 2.0) for 1:1 binding kinetic analysis (Wasatch, version 2.0.0.33) to extract k off result.

[0787] Table 4 below summarizes the top 32 clones evaluated by binding to cell lines expressing human and cynomolgus macaque CD33 and to recombinant antigens where the off-rate of one of the antigens was at least >10e-3. Of these 32 clones, all but 4 showed measurable binding to cells expressing either human or cynomolgus macaque. All 32 clones were further characterized via epitope binning and full kinetic analysis.

[0788] Table 4. Cellular binding and dissociation rate analysis of anti-CD33 antibodies derived from OMNIRat

[0789]

[0790]

[0791]

[0792] This batch of mAbs was then further characterized for full affinity analysis and epitope binning. Binding of anti-CD33 mAbs to recombinant human CD33 ECD-HSA (C33W2) and cynomolgus macaque CD33 ECD-HSA (C33W1) was measured by ProteOn SPR (Bio-Rad). Goat anti-human Fc IgG (Jackson Immunoresearch, catalog number 109-005-098) was directly immobilized in acetate buffer (pH 5.0) via amine coupling at 30 μg / mL, on all 6 ligand channels oriented vertically on a GLC sensor chip (Bio-Rad, catalog number 176-5011), with a flow rate of 30 μL / min in PBS containing 0.005% Tween-20. The immobilization density averaged approximately 5000 response units (RU) with less than 5% variation between different channels. Different mAbs were captured on anti-human Fc IgG surfaces at 0.25 μg / ml or 0.5 μg / ml (160 RU to 300 RU) in a vertical ligand orientation, with the 6th ligand channel as a no-ligand surface control. In a 3-fold dilution series of 5-fold concentrations, 0.3 μM concentrations of human and cynomolgus macaque CD33-HSA proteins were flown in as analytes to bind to the captured mAbs in a horizontal orientation. Buffer samples were also injected in the 6th channel to monitor the dissociation and baseline stability of the captured mAbs. The dissociation phase of human and cynomolgus macaque CD33-HSA at all concentrations was monitored at a flow rate of 100 μL / min for 15 minutes to bind to C33B782, 60 minutes to bind to C33B912 (same as C33B904 with hIgG4), and then regenerated using an 18-second pulse of 0.85% phosphoric acid to remove the antigen and bound mAbs. The raw binding data were processed by double referencing after subtracting the response data from: 1) the midpoint to correct for nonspecific interactions between the Ag and the empty chip surface; and 2) the buffer channel to correct for baseline drift due to dissociation of the capture mAb surface over time. The processed data for each mAb at all antigen concentrations were globally fit to a 1:1 simple Langmuir binding model to extract the kinetics (k on ,k off ) and an estimate of the affinity (KD) constant.

[0793] In order to determine whether this batch of mAbs all bind to 1 different epitope or whether there is a wide range of epitope coverage, epitope binning experiments were performed. Competitive antigen epitope binning of CD33 mAb was performed on an IBIS SPRi instrument (Carterra) using a CMD-200M sensor prism chip. Each anti-CD33 antibody was directly immobilized on the chip in acetate buffer (pH 4.5) at 10 μg / ml via amine coupling using a separate continuous flow microdropper (CFM). The printed sensor chip was then transferred to the IBIS instrument and binning analysis was performed using a classic or "sandwich" binning format. By sequentially injecting 50nM human CD33 ECD-HSA (C33W2), a single anti-CD33mAb was then injected as a competitive analyte in a 133nM solution to regenerate the fixed anti-CD33 mAb to the surface after each sequential injection cycle of antigen and antibody for binning.

[0794] In order to monitor the activity of immobilized mAb before and after regeneration, the buffer injection without any competing mAb was carried out at the beginning and end of the experiment to measure the binding activity of the antigen alone. The antibody in the response indication solution relative to the combination of buffer (individual antigen) is blocked or the antigen is clamped and combined with the fixed mAb. The original binning data (.trix file format) is quoted and zeroed using SprintX software (Wasatch, 1.9.3.2 version), and then exported (.ibmx file format) to binning software HtTools.exe (Wasatch, 2.0.0.33 version) for analysis. Data is processed by removing the antibody that the antigen response is lower than 20RU and the antibody that is not self-blocking. The competing mAb response is normalized relative to the independent antigen binding response. The antibody of normalized response <0.25 represents a blocker, and the antibody of normalized response ≥0.25 represents a non-blocker / clamping agent. The incision at height 2.5 is used to predict different boxes in the combined dendrogram.

[0795] The following table summarizes the complete kinetic analysis and epitope binning of the 32 selected mAbs. In total, 8 anti-CD33 mAbs had subnanomolar affinities for both human and cynomolgus CD33, and these mAbs corresponded to 3 different epitope bins, while a larger batch had a range of affinities and 7 different epitope bins.

[0796] Table 5. Full kinetic analysis and epitope binning of OMNIRat-derived mAbs

[0797]

[0798]

[0799]

[0800] OmniMouse batches (8 mAbs total) were generated separately and further characterized for binding to cells. Cell binding was performed as described above and summarized in the table below. Of the 8 mAbs tested, 6 directly bound to cells expressing CD33, while 2 did not bind to cells expressing CD33.

[0801] Table 6. OMNIMouse-derived mAbs binding to cells expressing human and cynomolgus macaque cell lines

[0802]

[0803]

[0804] Using the methods described above and summarized in the table below, six mAbs binding to CD33 on cells were further biophysically characterized via full kinetic analysis of recombinant antigens. Of the six mAbs tested, one bound to human CD33 with picomolar affinity (C33B912), had submolar affinity for cynomolgus CD33, and one had very strong affinity for human CD33 but only nanomolar affinity for cynomolgus CD33 (C33B911). Two other clones had subnanomolar affinity for both human and cynomolgus CD33 (C33B913 and C33B916), but neither had an affinity within the range of C33B912.

[0805] Table 7. Complete kinetic analysis of mAbs derived from OMNIMouse

[0806]

[0807] Epitope binning experiments were performed on 6 cell-binding mAbs derived from the OMNIMouse, as well as several control mAbs previously identified in early OMNIRat campaigns. Control mAbs were selected based on subnanomolar affinity for human CD33 and the number of different epitope bins. The binning software HtTools assigns epitope bin numbers on a per-experiment basis, so having several controls for already defined epitope bins is critical for cross-comparisons. Both human CD33 high affinity clones derived from the OMNIMouse (C33B911 and C33B912) were binned with clones from bin 4 above (bin 4 in this experiment), while the subnanomolar clone (C33B916) was binned with C33B836 (bin 1 in the above experiment) into bin 2.

[0808] Table 8: Epitope bins of OMNIMouse anti-CD33 mAbs

[0809] mAbs V Zone ID Table Box C33B915 C33F553 1 C33B916 C33F554 2 C33B836 C33F53 2 C33B914 C33F552 2 C33B913 C33F551 3 C33B806 C33F106 3 C33B911 C33F549 4 C33B912 C33F550 4 C33B778 C33F78 4 C33B830 C33F130 4 C33B782 C33F82 5 C33B792 C33F92 5 C33B799 C33F99 5 C33B760 C33F60 6 C33B777 C33F77 7

[0810] CD33 consists of two IgG domains, a membrane distal V domain and a membrane proximal C2 domain. SNP rs12459419 can lead to alternative splicing of CD33 pre-mRNA transcripts to produce only the C2 form expressed on cells, so targeting this domain may provide clinical benefit. To determine which of the two domains the mAb is able to bind, six mAbs with the highest binding capacity covering four different epitope bins were screened for off-rates according to the above protocol using human CD33 ECD-HSA, human CD33 V-HSA, and human CD33 C2-HSA as binding antigens. As shown in the table below, both clones previously grouped in bin 4 bind to the human CD33 C2 domain but not the human CD33 V domain, while clones in bins 2 and 3 bind to the V domain but not the C2 domain. The two clones grouped in bin 5 do not bind to either domain, so their exact binding locations may span the two domains. Three (3) commercially available mAbs (WM53 (EMD Millipore; Darmstadt, Germany), P67.7 (Biolegend, San Diego, CA), and LSBio clone 906 (LifeSpan Biosciences, Seattle, WA)) were included in this experiment and all showed binding to the V domain, but not the C2 domain. Reviewing the epitope bins in Tables 5 and 8 relative to the C2 domain binding data in Table 9, a total of 15 mAbs were able to potentially bind to the C2 domain in the range of about 25 nM to about 30 pM on the human full-length protein.

[0811] Table 9. Dissociation rate domain binding

[0812]

[0813] To support further in vivo and in vitro studies, select clones (C33B836, C33B782, C33B778, C33B904, C33B806, C33B830, C33B937, C33B792, C33B760, and C33B777) were selected for amplification and fab arm exchange to generate bispecific DuoBody molecules with anti-CD3 antibodies. TM Cells (from ThermoFisher Scientific) were cultured at 1.25 × 10 5 to 2.25×10 5viable cells / mL were inoculated into ExpiCHO TM Expression Medium and cultured in polycarbonate, disposable, sterile, ventilated, unbaffled conical shake flasks in a 37°C, 7% CO2 shaking incubator (INFORS HT MultitronPro). For routine cell growth in 125 mL to 2 L shake flasks, set the shaking speed to 130 rpm for a shaker with a 19 mm shaking diameter. When the density reaches 4× with 98% to 99% viability, 6 10 to 6 × 10 6 When the cells reached the logarithmic growth phase with viable cells / mL, the cells were subcultured.

[0814] Two days before transfection, place ExpiCHO-S TM The cells were 1.5×10 6 viable cells / mL were inoculated into the required culture volume. On the day of transfection, the viable cell density and viability percentage were determined. 6 For optimal transfection, use sterile heavy and light chain plasmid DNA at a concentration of ≥1 mg / mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0815] ExpiCHO-S were transfected according to the manufacturer’s Max Titer transfection protocol (Thermo Fisher Publication No. MAN0014337). TM All amounts and volumes shown below are per mL of final transfection culture volume. Briefly, 0.04 mL of cold OptiPRO TM Medium (ThermoFisher Scientific) to dilute plasmid DNA at the following ratio: 0.125 μg heavy chain DNA: 0.375 μg light chain DNA: 0.5 μg pAdvantage. Dilute 6.4 μL of ExpiFectamine TM CHO transfection reagent and mixed with 0.04 mL of OptiPRO TM The culture medium was gently mixed and incubated for 1 minute. TM Add CHO reagent to the diluted DNA, mix gently, and add ExpiFectamine TM The CHO / plasmid DNA complex was incubated at room temperature for 5 minutes. After incubation, the complex was added to the ExpiCHO-S TM The cells were incubated overnight in a shaking incubator at 37°C with 7% CO2.

[0816] For the Max Titer protocol, add 6 μL of ExpiFectamine on day 1 after transfection. TM CHO Enhancer and 160 μL of ExpiCHO TM Feed and transfer the flask to a 32°C, 7% CO2 shaking incubator. On day 5 after transfection, add 160 μL of ExpiCHO to the flask again. TM Feed and return to the incubator at 32° C. with shaking. Cultures were harvested on day 12 post-transfection, centrifuged at 5000 rpm for 15 minutes and clarified through 0.2 um Acropak 1500 filter capsules (Pall).

[0817] The expressed antibodies were purified from the clarified supernatants using mAbSelect Sure resin (GE Healthcare). The MabSelectSuRe Protein A column was equilibrated with 1× D-PBS (pH 7.2) prior to loading each culture supernatant. Unbound proteins were removed by extensive washing with 1× D-PBS (pH 7.2). Bound proteins were eluted with 0.1 M sodium acetate (pH 3.5). Peak fractions were neutralized with 2.5 M Tris (pH 7.2) and combined. The neutralized fraction pool was dialyzed into 1× dPBS for assay and biophysical characterization, or for bispecific DuoBody assembly.

[0818] The protein concentration of each elution pool was determined by measuring the absorbance at OD280 nm and calculated using the absorbance extinction coefficient based on the amino acid sequence.

[0819] Example 3: Fab arm exchange using purified parent mAb

[0820] The formation of CD33×CD3 bispecific antibodies requires two parent mAbs, one specific for the target arm (e.g., CD33) and one specific for the effector arm (e.g., CD3). The CD33 mAb was recombined with either a high affinity (CD3B219) or low affinity CD3 arm (CD3B376) arm. These parent mAbs were IgG4 PAA formats (Labrijn et al., 2013), where the target parent (CD33) contained the K409R mutation (the native amino acid of IgG4), while the killer parent (CD3) contained the F405L mutation and R409K. The monospecific anti-CD3 antibodies were expressed as IgG4 with the Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arm) in their Fc region (numbering according to the EU index). The monospecific antibodies were expressed and purified as described above. After purification, the parent CD33 antibody is mixed with the desired parent CD3 antibody in 75mM cysteamine-HCl under reducing conditions and incubated at 31°C for 5 hours. The recombination reaction is based on a molar ratio, in which a set amount of CD33 antibody (e.g., 10 mg or about 74.6 nanomoles) is combined with a CD3 antibody (e.g., about 67.8 nanomoles), wherein the amount of CD33 antibody added is 6% higher than the CD3 antibody. The concentration of the CD33 antibody stock solution varies between 0.8 mg / mL and 6 mg / mL, and the volume of the recombination reaction is different in each pairing. The recombination reaction is then dialyzed overnight with PBS to remove the reducing agent. The CD33×CD3 bispecific antibody reaction is performed with an excess of CD33 antibody (ratio) to minimize the amount of unreacted CD3 parent antibody remaining after recombination.

[0821] The final CD33×CD3 bispecific antibodies generated are listed in Table 10 together with the parental mAbs (ie, CD33, CD3 or blank) used for the reconstitution reactions.

[0822] Selected CD33 hits were also paired with a non-killer arm (blank) to generate a negative control for testing purposes. For control bispecific antibodies, B2M1 (an IgG4 PAA format RSV antibody) was generated, purified, and combined with the CD3 arms CD3B219 and CD3B376-F405L, R409K to generate CD3B288 (CD3×blank) and CD3B510 (CD3B376×blank) or with the CD33 arms C33B836, C33B806, C33B782, C33B792, C33B760, C33B830, C33B799, C33B778, C33B777 to generate C33B941, C33B943, C33B946, C33B945, C33B949, C33B942, C33B944, C33B947, C33B948 (CD33×blank), respectively.

[0823] Table 10: CD33×CD3 bispecific antibodies

[0824]

[0825]

[0826] Pep: peptide; Nuc: nucleotide; SEQ ID: SEQ ID NO

[0827] Example 4: In vitro T cell-mediated cytotoxicity assay using CD33×CD3 bispecific antibodies

[0828] In vitro T cell-mediated cytotoxicity assays were performed to assess whether CD33 hits paired with a CD3 arm (CD3B219) mediate killing of CD33 expressing AML cell line OCI-AML5. Briefly, effector cells (pan T cells, purchased from Biological Speciality) were harvested, counted, washed, and resuspended in RPMI (Invitrogen) containing 10% FBS (Invitrogen) cell culture medium to 1×10 6 Target cells (MOLM13) were labeled with CFSE (Invitrogen) and resuspended in RPMI containing 10% FBS to 2×10 5 cells / mL. Effector cells and CFSE-labeled target cells were mixed in a sterile 96-well round-bottom plate at a ratio of 5:1. 10 μL of Fc blocker (ReoPro Fc fragment) and 5 μL of bispecific antibody aliquots were added to each well containing various concentrations. The culture was incubated at 37°C under 5% CO2 for 48 hours. After 48 hours, the samples were added Fixable Near-IR Dead Cell Stain buffer (Life Technologies), and the culture was incubated in the dark for 20 minutes at room temperature, then washed and resuspended in 100 μL to 200 μL of FAC buffer. Drug-induced cytotoxicity was determined using a CANTO II flow cytometer (BD Biosciences; Franklin Lakes, NJ, USA) and analyzed using FlowJo software or Dive software (BD Biosciences). The population of interest is double positive CFSE+ / live / dead+ cells. As shown in Figure 1, all CD33×CD3 multispecific antibodies induced CD33 after 48 hours. +T cell redirected cytotoxicity of MOLM-13 cells. Table 11 summarizes the ECs generated using CD33×CD3 multispecific antibodies. 50 The top four antibodies, C3CB10, C3CB12, C3CB7 and C3CB88, were used for further characterization.

[0829] Table 11: CD33×CD3 T-cell mediated cytotoxicity assay. EC 50 Aggregation of values

[0830]

[0831] Example 5: In vitro CD33×CD3-mediated reduction of AML blasts and T cell activation in primary AML samples

[0832] To further evaluate the cytotoxic potential of the CD33×CD3 bispecific antibodies, an ex vivo cytotoxicity assay was performed using whole blood from AML patients using the first four antibodies (Figure 2). In this assay, various bispecific antibodies (CD33 antibodies paired with CD3 arms CD3B219 and CD3B376) were added to diluted whole blood from AML patients for a period of 48 hours without providing additional T cells because the assay relies on the presence of autologous T cells in the patient's blood. After 48 hours, the samples were stained with CD3 PerCPCy5.5, CD25 PE, CD33 FITC, and CD38 APC (all antibodies were purchased from Biolegend, San Diego, CA, USA). The samples were then washed at least 3 times in 1X LyseRBC Lysis Buffer (eBioscience). They were then incubated with The samples were stained with FixableNear-IR Dead Cell Stain Buffer (Life Technologies). - CD38 + Live CD33 in cells + The degree of tumor cytotoxicity was determined by PCR using the following formula: (CD33 in PBS / untreated control) + %-CD33 in treated samples + %) / (PBS / CD33 in untreated control + %). T cell activation was calculated as CD3 + CD25 + The percentage of events.

[0833] As shown in Figure 2, all CD33 lead antibodies paired with either CD3 arm (CD3B376 and CD3B219) promoted a dose-dependent reduction in total cytotoxicity associated with T cell activation after 48 hours. The blank arm control antibodies (blank × CD3B219 and blank × CD3B376) failed to show tumor cytotoxicity or T cell activation. This result also suggests that the CD33 × CD3 bispecific antibodies work in an autologous setting. These results are representative of 4 additional AML donor samples (data not shown). Table 12 summarizes the ECs generated with the CD33 × CD3 multispecific antibodies. 50 Value. From EC 50 As can be seen from the values, C33B904 paired with CD3 arms (C3CB88, C3CB189) and C33B836 paired with CD3 arms (C3CB7, C3CB97) are the most potent antibodies. Therefore, these 4 antibodies are the focus of further characterization.

[0834] Table 12: CD33×CD3 T-cell mediated in vitro cytotoxicity assay. 8 CD33×CD3 bispecific antibodies EC 50 Aggregation of values

[0835] Bispecific AbID <![CDATA[Primary AML cells kill EC 50 (nM)]]> C3CB11 3.958 C3CB12 2.635 C3C7 0.3315 C3CB88 0.6722 C3CB103 4.186 C3CB102 4.973 C3CB97 ～0.2316 C3CB189 0.5782

[0836] Example 6: CD33×CD3 bispecific antibody binds to the C2 domain of CD33 and induces expression of CD33 mononucleotide Confirmation of cytotoxicity in cell lines expressing SNPs

[0837] In vitro T cell-mediated cytotoxicity assay using CD33×CD3 bispecific antibodies

[0838] Recent studies have shown that single nucleotide polymorphism (SNP) rs12459419 is present in about 50% of the AML population and causes a skipping deletion of exon 2 of CD33, resulting in a deletion of the V domain of CD33. The study also showed that Mylotarg, which binds to the V domain of CD33, is ineffective in patients expressing the SNP, and thus reduces the risk of relapse in about 50% of the AML population and improves survival (Lamba et al., 2017, JCO, CD33 Splicing Polymorphism Determines Gemtuzumab Ozogamicin Response in De Novo Acute Myeloid Leukemia: Report From Randomized Phase III Children's Oncology Group Trial AAML0531). In view of the data of Mylotarg in the above studies, an in vitro T cell-mediated cytotoxicity assay was performed to evaluate whether CD33 hits (V combined with C33B836 and C2 combined with C33B904) paired with CD3 arms (CD3B219 or CD3B376) mediated killing of cell lines expressing SNP rs12459419. Briefly, effector cells (pan T cells, purchased from Biological Speciality) were harvested, counted, washed and resuspended in RPMI (Invitrogen) containing 10% FBS (Invitrogen) cell culture medium to 1×10 6 Target cells (KG1, SH2, and OCIAML3) were labeled with CFSE (Invitrogen) and resuspended in RPMI with 10% FBS to 2×10 5 cells / mL. KG1, SH2, and OCIAML3 were selected to represent the wild type, heterozygous, and homozygous forms of the CD33 SNP rs12459419 mutation, respectively. Effector cells and CFSE-labeled target cells were mixed in a sterile 96-well round-bottom plate at an effector cell:CFSE-labeled target cell ratio (E:T) = 5:1. 10 μl of Fc blocker (ReoPro Fc fragment) was added to each well containing various concentrations along with a 5 μμL aliquot of the bispecific antibody. The cultures were incubated at 37°C under 5% CO2 for 48 hours. After 48 hours, the samples were added Fixable Near-IR Dead Cell Stain buffer (Life Technologies), and at room temperature, the culture was incubated in the dark for 20 minutes, then washed and resuspended in 100 μL to 200 μL of FAC buffer. Drug-induced cytotoxicity was determined using a CANTO II flow cytometer (BD Biosciences) and analyzed using FlowJo software or Dive software (BD Biosciences). The population of interest is double positive CFSE+ / live / dead+ cells. As shown in Figure 3, unlike the blank control (blank group × CD3B219 and blank group × CD3B376), V-bound and C2-bound CD33×CD3 multispecific antibodies induced T cell redirected cytotoxicity of CD33+WT of the SNP rs12459419 mutant cell line KG1 after 48 hours. In contrast, unlike the V binder C33B836 (C3CB97, C3CB7), only the C2-binding C33B904-paired bispecific antibodies (C3CB189, C3CB88) mediated cytotoxicity against SH2 and OCIAML3 cell lines with heterozygous or homozygous rs12459419 SNP mutations, respectively. For this reason, the C33B904-paired bispecific antibodies (C3CB189, C3CB88) were further analyzed and characterized. In summary, these data suggest that CD33C2-binding bispecific antibodies (such as C33B904-paired bispecific antibodies) have the potential to show efficacy in a wider range of AML patients compared to V-binding competing anti-CD33 antibodies.

[0839] Example 7: In vitro CD33×CD3-mediated MOLM-13 and Reduction of spike in monocytes

[0840] To evaluate the cytotoxic potential of the CD33 × CD3 bispecific antibody when spiked in MOLM-13 cells and normal human monocytes, we used a CD33 × CD3 bispecific antibody with exogenously added CD33 +In vitro cytotoxicity assay of normal healthy human whole blood of AML cell line MOLM-13.Similar to the above experiment, various bispecific antibodies (CD33 antibodies paired with CD3 arms CD3B219 and CD3B376) were added to the diluted whole blood from 6 different normal human donors for a period of 48 hours without providing additional T cells, because the assay depends on the presence of autologous homologous T cells in donor blood.Before dilution, the concentration of T cells in the blood of each donor was counted.Then the blood was diluted with MOLM-13 cells labeled with CFSE (Invitrogen) so that the effector cell: target cell ratio (E: T) was 1: 5 to simulate the effector cell: target cell in AML patient samples.After 48 hours, the sample was stained with CD3 PerCPCy5.5, CD25 PE, CD33 FITC and CD14 Pacific Blue (all antibodies purchased from Biolegend).Then the sample was washed at least 3 times in 1X Lyse RBC Lysis Buffer (eBioscience company).Then with The samples were stained with Fixable Near-IR Dead Cell Stain Buffer (Life Technologies). CD14 was first quantified in the presence of the bispecific antibody. + Live CD33 in the monocyte fraction + The degree of tumor cytotoxicity was determined by + The percentage of cells was counted to determine the cytotoxicity of MOLM-13 cells. The cytotoxicity of monocytes was calculated as a percentage relative to PBS / untreated controls using the following formula: (CD33 in PBS / untreated controls) + CD14 + %-CD33 in treated samples + CD14 + %) / (PBS / CD33 in untreated control + CD14 + The data in Figure 4 show that two CD33×CD3 bispecific antibodies (the same CD33 lead C33B904 paired with CD3 arms CD3B376 and CD3B219) specifically induced the proliferation of MOLM-13 cells and CD33 after 48 hours. + Cytotoxicity of monocytes. A blank arm control was used as a negative bispecific antibody control. The blank arm control showed MOLM-13 and CD33 + Monocytes have little cytotoxic activity. These data show the average of 6 different normal donors. MOLM-13 and CD14 +The average EC of monocyte cytotoxicity 50 The values ​​are shown in Table 13.

[0841] Table 13: CD33×CD3 T-cell mediated in vitro cytotoxicity assay. 2 CD33×CD3 bispecific antibodies EC 50 Aggregation of values .

[0842] Bispecific Ab ID <![CDATA[MOLM13 kills EC 50 (nM)]]> <![CDATA[CD33 + CD14 + Kill EC 50 (nM)]]> C3CB189 0.1677 1.156 C3CB88 0.671 0.506

[0843] Example 8: Confirmation of species cross-reactivity of CD33×CD3 bispecific antibody with cynomolgus monkeys

[0844] In vitro CD33×CD3-mediated monocyte activation in an in vitro cytotoxicity assay using whole blood from cynomolgus macaques The reduction

[0845] In order to confirm the functional cross-reactivity and assess the potential of the cytotoxicity of CD33×CD3 bispecific antibodies when eliminating normal cynomolgus macaque monocytes, the in vitro cytotoxicity assay using healthy cynomolgus macaque whole blood was utilized.Similar to the above-mentioned experiment, various bispecific antibodies (CD33 antibodies paired with CD3 arms CD3B219 and CD3B376) were added to the diluted whole blood from 6 different normal cynomolgus macaque donors for a period of 48 hours without providing additional T cells, because the assay depends on the presence of autologous T cells in donor blood.After 48 hours, the sample was stained with CD3 PerCPCy5.5, CD25 PE, CD33 FITC and CD14 Pacific Blue (except the CD33 antibody purchased from Miltenyi, Bergisch Gladbach, Germany, all antibodies were purchased from Biolegend). The samples were then washed at least three times in 1× Lyse RBC Lysis Buffer (eBioscience) and then Fixable Near-IR Dead Cell Stain Buffer (Life Technologies) was used to stain. CD14 was first quantified in the presence of the bispecific antibody. + Live CD33 in the monocyte fraction + The degree of monocyte cytotoxicity was determined by PBS / untreated cells. Cytotoxicity was calculated as a percentage relative to PBS / untreated controls using the following formula: (CD33 in PBS / untreated controls + CD14 + %-CD33 in treated samples + CD14 + %) / (PBS / CD33 in untreated control + CD14 + %). T cell activation was calculated as CD3 + CD25 +The data in Figure 5 show that the CD33×CD3 bispecific antibody (the same CD33 lead C33B904 paired with CD3 arms CD3B376 and CD3B219) specifically induced CD33 after 48 hours. + Monocyte cytotoxicity and T cell activation. A blank arm control was used as a negative bispecific antibody control and showed little cytotoxicity or T cell activity. Table 14 shows the average of 6 different cynomolgus monkey donors.

[0846] Table 14: CD33×CD3 T-cell mediated in vitro cytotoxicity assay. 2 CD33×CD3 bispecific antibodies EC 50 Aggregation of values .

[0847] Protein AA ID <![CDATA[CD33 + CD14 + Kill EC 50 (nM)]]> <![CDATA[T cell activation EC 50 (nM)]]> C3CB189 3.60 0.02 C3CB88 0.89 0.02

[0848] Example 9: C3CB189 and C3CB88 in MOLM-13 human AML xenografts in T cell humanized NSG mice Benefits

[0849] In a female NOD.Cg-Prkdc humanized with 20 million T cells sci Iq tm1Wjl The effect of C3CB189 and C3CB88 was evaluated in the MOLM-13 human acute myeloid leukemia (AML) xenografts of the established luciferase transfection in l / SzJ (NSG) mice. On the 5th day after intravenous tumor implantation, animals were randomly divided into n=10 / groups by live bioluminescence imaging (BLI). C3CB189 and C3CB88 were used at 0.005mg / kg, 0.05mg / kg and 0.5mg / kg or blank × CD3 antibody control was used at 0.5mg / kg, and once every 3 to 4 days for 6 weeks.

[0850] Tumor growth inhibition (TGI%) determined by bioluminescence was calculated on day 13 after tumor implantation when at least eight animals were retained per group. Figure 6 ) and C3CB88( Figure 8 ) Statistically significant tumor growth inhibition was observed in all groups. Compared with controls treated with blank×CD3, 0.005 mg / kg, 0.05 mg / kg and 0.5 mg / kg of C3CB189 induced 76%, 100% and 82% tumor growth inhibition, respectively, and 0.005 mg / kg, 0.05 mg / kg and 0.5 mg / kg of C3CB88 induced 100%, 100% and 91% tumor growth inhibition, respectively.

[0851] Treatment with C3CB189 and C3CB88 resulted in a reduction in tumor burden and an increase in lifespan (ILS) greater than the 16-day median survival of the blank × CD3 control group. The median survival of animals treated with C3CB189 ranged from 19 to 27.5 days ( Figure 7 ), while the median survival of animals treated with C3CB88 was 26 to 28.5 days ( Fig. 9 ). Compared with the control group, 0.005 mg / kg, 0.05 mg / kg and 0.5 mg / kg of C3CB189 resulted in 19%, 72% and 50% increase in lifespan, respectively, and C3CB88 resulted in 63%, 78% and 72% increase in lifespan, respectively.

[0852] Example 10: Demonstration of internalization of CD33 antibody in in vitro protein A drug conjugate cell viability assay

[0853] In vitro cell viability assays were performed using pre-loaded protein A drug conjugate A-MMAF to detect the internalization of ligand-bound target antibodies. This cell-based functional assay was performed in AML cell line MOLM13 using a batch of anti-CD33 antibodies, C33B782, C33B806, C33B836, C33B904, C33B937, and isotype control antibody CNTO9412. In this assay, only the target antibody was tested as a control to distinguish between the cytotoxicity caused by antibody internalization and the cytotoxicity caused by the activity of the test antibody itself. Fig.10 The cytotoxicity of the antibody bound to protein A-MMAF in MOLM13 after incubation for 72 hours at 37°C, 5% CO2 is shown. Concentration-dependent cytotoxicity of all five CD33 antibodies was observed in MOLM13 cells, indicating that all five antibodies were internalized in this cell line. Isotype control antibody CNTO9412 did not show significant concentration-dependent cytotoxicity, indicating that these CD33 antibodies were target-specifically internalized in MOLM13 cells. Table 15 shows the EC values ​​of five (5) CD33×CD3 bispecific antibodies. 50 value.

[0854] The results also showed that antibody C33B836 had better internalization in MOLM13 cells than the others.

[0855] Table 14: CD3...

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, which specifically binds to the C2 domain of CD33, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR) 1, HCDR2, HCDR3, and a light chain complementary determining region (LCDR) 1, LCDR2, and LCDR3 as shown in the polypeptide sequences of SEQ ID NOs: 447, 448, 449, 567, 568, and 569, respectively.

2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 292 or a light chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO:

332.

3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

332.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the monoclonal antibody or antigen-binding fragment thereof has an EC of less than 2 nM in vitro. 50 Induces antibody-dependent cell-mediated cytotoxicity (ADCC).

5. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 low-fucose backbone.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof has a concentration of less than 5×10 -9 The dissociation constant (KD) of M binds to CD33.

7. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to CD33 and has an EC of less than 2 nM. 50 Induce internalization.

8. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is chimeric.

9. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is human or humanized.

10. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent.

11. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A vector comprising the isolated nucleic acid according to claim 11.

13. A host cell comprising the vector according to claim 12.

14. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

15. Use of the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating acute myeloid leukemia (AML) in a subject in need thereof.

16. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the method comprising: Culturing cells containing nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the antibody or antigen-binding fragment thereof from the cells or culture.

17. A method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 10, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

18. An anti-CD33 / anti-CD3 bispecific antibody, comprising an anti-CD33 antibody or an antigen-binding fragment thereof and an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the anti-CD33 antibody or antigen-binding fragment thereof comprises heavy chain complementary determining region (HCDR) 1, HCDR2, HCDR3, light chain complementary determining region (LCDR) 1, LCDR2 and LCDR3 as shown in the polypeptide sequences of SEQ ID NOs: 447, 448, 449, 567, 568 and 569, respectively, And the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR) 1, HCDR2, HCDR3, a light chain complementary determining region (LCDR) 1, LCDR2 and LCDR3 as shown in the following polypeptide sequence: 1) SEQ ID NO: 342, 343, 344, 465, 466 and 467, respectively; or 2) are SEQ ID NOs: 345, 346, 347, 468, 469 and 470, respectively.

19. The anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to claim 18, wherein the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 292 or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 332; and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 257 or 258 or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 298 or 299.

20. The anti-CD33 / anti-CD3 bispecific antibody or the antigen-binding fragment thereof according to claim 18 or 19, wherein the anti-CD33 / anti-CD3 bispecific antibody or the antigen-binding fragment thereof comprises: a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 257 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 298; or b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 292 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 332; and a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 258 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

299.

21. The anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to claim 18 or 19, wherein the anti-CD33 antibody or antigen-binding fragment thereof specifically binds to the C2 domain of CD33.

22. The anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to claim 18 or 19, wherein the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof has an EC of less than 1 nM in vitro. 50 Values ​​induce T cell-dependent cytotoxicity of cells expressing CD33.

23. The anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to claim 18 or 19, wherein the bispecific antibody or antigen-binding fragment thereof is chimeric.

24. The anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to claim 18 or 19, wherein the bispecific antibody or antigen-binding fragment thereof is human or humanized.

25. An isolated nucleic acid encoding the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 18 to 24.

26. A vector comprising the isolated nucleic acid according to claim 25.

27. A host cell comprising the vector according to claim 26.

28. A pharmaceutical composition comprising the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 18 to 24, and a pharmaceutically acceptable carrier.

29. Use of the pharmaceutical composition according to claim 28 in the preparation of a medicament for treating acute myeloid leukemia (AML) in a subject in need thereof.

30. A method for producing an anti-CD33 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof according to any one of claims 18 to 24, the method comprising: Culturing cells comprising a nucleic acid encoding the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment under conditions in which the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment is produced, and recovering the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment from the cells or culture.

31. A method for producing a pharmaceutical composition comprising the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment according to any one of claims 18 to 24, the method comprising combining the anti-CD33 / anti-CD3 bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

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