Human CD33 antibody and glucocorticoid conjugate

Abstract

The present disclosure provides a human CD33 antibody and an antibody glucocorticoid receptor agonist conjugate, and methods of using the conjugate for the treatment of myeloid cell-related diseases.

Description

Technical Field

[0001] The present disclosure provides a human CD33 antibody and an antibody glucocorticoid receptor agonist conjugate, methods of using the conjugate for the treatment of myeloid cell-related diseases, processes for preparing the conjugate, and pharmaceutical compositions comprising the human CD33 antibody glucocorticoid conjugate.

Background Art

[0002] Anti-human CD33 antibodies and conjugates have been described previously. Many of the antibodies are myelosuppressive antibodies that modulate CD33-mediated responses. For example, rituximab, a humanized antibody that has been reported to deplete myeloid cells via ADCC and CDC activities and modulate CD33-mediated cytokine responses, was terminated as a monotherapy in AML due to lack of efficacy, vadastuximab talirine, a rituximab conjugate, was terminated due to safety concerns, and rituximab Ac225 is currently in Phase I / II in AML. (Perl, A., Hematology Am Soc Hematol Educ Program.,(1):54-65,2017; Bothell, Wash.(BUSINESS WIRE)., June 19,2017; Abedin, S., Blood., 136(Supplement 1):9-10,2020). Other such anti-human CD33 antibody therapeutics include BI836858, which was unable to demonstrate clinical activity in a Phase I AML study but has been evaluated in combination with other compounds in AML, IMGN779, which is currently in a Phase I trial in AML, and gemtuzumab ozogamicin, the only approved anti-human CD33 antibody conjugate for the treatment of AML. (Vasu S, et al., Haematologica., 107(3):770-773,2022; Mol.Cancer Ther., 17(6):1271-1279,2018). AL003, a humanized CD33 antibody that has been reported to modulate CD33-mediated responses, is in a Phase II trial for the treatment of Alzheimer's disease. (Alector, Inc., GlobeNewswire, Nov. 10,2021).

[0003] International Publication No. WO 2017 / 210471 discloses certain glucocorticoid receptor agonists (GCs) and immune complexes thereof that are useful for treating inflammatory diseases. International Publication No. WO 2018 / 089373 discloses novel steroids, their protein conjugates, and methods of treating diseases, disorders, and conditions including administering the steroids and conjugates. To date, there are no human CD33 GC conjugates approved for the treatment of diseases including myeloid cell-related diseases.

Summary of the Invention

[0004] The present disclosure provides certain novel fully human CD33 antibodies that bind to human CD33, do not deplete myeloid cells (a “non-depleting anti-human CD33 antibody”), internalize into myeloid cells, do not modulate CD33-mediated responses (e.g., cytokine expression, inflammatory response), and do not significantly degrade CD33 (a “non-degrading anti-human CD33 antibody”). The present disclosure further provides compositions comprising such anti-human CD33 antibodies, as well as methods of using such anti-human CD33 antibodies and their compositions. Such anti-human CD33 antibodies can be conjugated to therapeutic agents (e.g., inflammatory agents, glucocorticoids, cytotoxic agents, siRNA, saRNA, peptides, small molecules, antibodies and their binding fragments) for use in targeted delivery of therapeutic agents to human CD33-expressing myeloid cells for the treatment of myeloid cell-related diseases. Furthermore, certain anti-human CD33 antibodies of the present disclosure do not significantly affect the availability of the CD33 receptor on the cell surface and thus can provide repeated CD33-targeted delivery of therapeutic agents to myeloid cells. The present disclosure further provides certain novel fully human CD33 antibody glucocorticoid (GC) conjugates in which the antibody binds to human CD33. The present disclosure further provides compositions comprising the novel anti-human CD33 antibody GC conjugates, as well as methods of using such anti-human CD33 antibody GC conjugates and their compositions. The present disclosure further provides certain novel anti-human CD33 GC conjugates useful for the treatment of fibrotic diseases such as rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, atopic dermatitis, psoriasis, inflammatory bowel disease, multiple sclerosis, Sjögren's syndrome, scleroderma, and macrophage activation syndrome.Accordingly, the specific anti-human CD33 antibodies and / or anti-human CD33 antibody-GC conjugates provided herein have one or more of the following properties: 1) bind to human CD33 and cynomolgus CD33 with desirable binding affinity and / or association and dissociation rates, 2) internalize into myeloid cells upon binding to CD33, 3) do not deplete myeloid cells, 4) do not induce effector function activity (e.g., ADCC), 5) do not degrade cell surface or intracellular CD33, 6) do not significantly affect the availability of the CD33 cell surface receptor on myeloid cells, 7) modulate glucocorticoid receptor agonist-mediated cytokine responses in vitro (e.g., inhibit IL-6 and TNFα), 8) modulate target-specific glucocorticoid receptor agonist-mediated responses in vivo (e.g., inhibit target-site specific tissue inflammation and gene expression (e.g., FKBP5)), 9) have a low immunogenicity risk, 10) inhibit plasmacytoid dendritic cell differentiation and IFNα production in vitro, and / or 11) have a favorable development potential profile, e.g., acceptable stability, solubility, viscosity, low aggregability, hydrophobicity, and / or a favorable pharmacokinetic profile that facilitates development, manufacture, and / or formulation.

[0005] Accordingly, in some embodiments, an antibody that binds to human CD33 (an "anti-human CD33 antibody") is provided herein, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 4, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. In some embodiments, the anti-human CD33 antibody comprises a heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO: 10.

[0006] In some embodiments, an antibody that binds to human CD33 is provided herein, the antibody comprising a VH and a VL, the VH comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 14. In some embodiments, the anti-human CD33 antibody comprises a HC comprising SEQ ID NO: 9 and a LC comprising SEQ ID NO: 15.

[0007] In some embodiments, antibodies that bind to human CD33 are provided herein, the antibodies comprising a VH and a VL, the VH comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, the VL comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 26, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In such embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8 or 14. In a further embodiment, the anti-human CD33 antibody comprises an HC comprising SEQ ID NO: 9 and an LC comprising SEQ ID NO: 10 or 15.

[0008] In some embodiments, antibodies that bind to human CD33 are provided herein, the antibodies comprising a VH and a VL, the VH comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, the VL comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 17, LCDR1 comprising SEQ ID NO: 18, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20. In some embodiments, the anti-human CD33 antibody comprises an HC comprising SEQ ID NO: 21 and an LC comprising SEQ ID NO: 22.

[0009] In some embodiments, the anti-human CD33 antibody is a fully human antibody. In some embodiments, the anti-human CD33 antibody is an endogenous antibody. In a further embodiment, the anti-human CD33 antibody has a human IgG1 or human IgG4 isotype.

[0010] In a further embodiment, the anti-human CD33 antibody has a modified human IgG1 Fc region comprising L234A, L235A and / or P329A (EU numbering), also referred to as IgG1AAA, which has a reduced or abolished binding to Fcγ and C1q receptors. In such an embodiment, the anti-human CD33 antibody having the IgG1AAA modified Fc region has a reduced or abolished Fc effector function activity such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (all residues are numbered according to EU numbering). Such an antibody is referred to as an "IgG1 effector null" antibody. In such an embodiment, the anti-human CD33 antibody having the IgG1AAA modified Fc region does not deplete CD33-expressing cells (e.g., myeloid cells). In such an embodiment, the anti-human CD33 antibody is a non-depleting antibody. In a further embodiment, the anti-human CD33 antibody having an IgG1AAA backbone has a significantly reduced and / or abolished degradation of the CD33 receptor compared to IgG1. In such an embodiment, the anti-human CD33 antibody is a non-degrading antibody.

[0011] In some embodiments, antibody fragments that bind to human CD33 (e.g., Fab or scFv) are provided herein, the antibody fragments comprising VH and VL, VH comprising HCDR1, HCDR2, and HCDR3, VL comprising LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 4, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8.

[0012] In some embodiments, an antibody fragment that binds to human CD33 (e.g., Fab or scFv) is provided herein, the antibody fragment comprising VH and VL, VH comprising HCDR1, HCDR2, and HCDR3, VL comprising LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 14.

[0013] In some embodiments, an antibody fragment that binds to human CD33 (e.g., Fab or scFv) is provided herein, the antibody fragment comprising VH and VL, VH comprising HCDR1, HCDR2, and HCDR3, VL comprising LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 17, LCDR1 comprising SEQ ID NO: 18, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20.

[0014] In some embodiments, the anti-human CD33 antibody has a modified human IgG1 or human IgG4 constant domain that contains engineered cysteine residues for use in the production of an antibody conjugate compound (also referred to as a bioconjugate) (see WO 2018 / 232088 (A1)). More specifically, in such embodiments, the anti-human CD33 antibody comprises cysteine at amino acid residue 124 (EU numbering), or cysteine at amino acid residue 378 (EU numbering), or cysteine at amino acid residue 124 (EU numbering) and cysteine at amino acid residue 378 (EU numbering).

[0015] In some embodiments, the present disclosure provides a nucleic acid encoding the heavy chain (HC) or light chain (LC), or variable heavy domain (VH) or variable light domain (VL) of a novel antibody that binds to anti-human CD33, or a vector comprising such a nucleic acid.

[0016] In some embodiments, the present disclosure provides a nucleic acid comprising the sequence of SEQ ID NO: 11, 12, 16, 23, or 24.

[0017] In some embodiments, a nucleic acid encoding the heavy chain or light chain of an antibody that binds to anti-human CD33 is provided. In some embodiments, a nucleic acid comprising a sequence encoding SEQ ID NO: 9, 10, 15, 21, or 22 is provided. In some embodiments, a nucleic acid comprising a sequence encoding an antibody heavy chain comprising SEQ ID NO: 9 or 21 is provided. For example, the nucleic acid can comprise the sequence of SEQ ID NO: 11 or 23. In some embodiments, a nucleic acid comprising a sequence encoding an antibody light chain comprising SEQ ID NO: 10, 15, or 22 is provided. For example, the nucleic acid can comprise the sequence of SEQ ID NO: 12, 16, or 24.

[0018] In some embodiments of the present disclosure, a nucleic acid encoding the VH or VL of an anti-human CD33 antibody is provided. In some embodiments, a nucleic acid comprising a sequence encoding SEQ ID NO: 7, 8, 14, 19, or 20 is provided. In some embodiments, a nucleic acid comprising a sequence encoding an antibody VH comprising SEQ ID NO: 7 or 19 is provided. In some embodiments, a nucleic acid comprising a sequence encoding an antibody VL comprising SEQ ID NO: 8, 14, or 20 is provided.

[0019] Some embodiments of the present disclosure provide a vector comprising a nucleic acid sequence encoding an antibody heavy chain or light chain. For example, such a vector can comprise a nucleic acid sequence encoding SEQ ID NO: 9 or 21. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22.

[0020] Also provided herein are vectors comprising a nucleic acid sequence encoding an antibody VH or VL. For example, such a vector can comprise a nucleic acid sequence encoding SEQ ID NO: 7 or 19. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 8, 14, or 20.

[0021] Also provided herein are vectors comprising a first nucleic acid sequence encoding an antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 15. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 21 and a second nucleic acid sequence encoding SEQ ID NO: 22.

[0022] Also provided herein are compositions comprising a first vector comprising a nucleic acid sequence encoding an antibody heavy chain and a second vector comprising a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 15. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 21 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 22.

[0023] Also provided herein is a composition comprising a vector comprising a nucleic acid sequence encoding an antibody heavy chain and a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22.

[0024] The nucleic acids of the present disclosure can be expressed, for example, in a host cell after the nucleic acid is operably linked to an expression control sequence. Expression control sequences capable of expressing the operably linked nucleic acid are well known in the art. The expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. An expression vector containing a nucleic acid of interest (e.g., a nucleic acid encoding the heavy or light chain of an antibody) can be introduced into a host cell by well-known methods such as stable or transient transfection, transformation, transduction, or infection. Additionally, the expression vector can include one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to assist in the detection of host cells transformed with the desired nucleic acid sequence.

[0025] In another aspect, provided herein are cells, such as host cells, comprising a nucleic acid, vector, or nucleic acid composition described herein. The host cell can be a cell that has been stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express all or a portion of an antibody described herein. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with an expression vector that expresses the HC and LC polypeptides of an antibody of the disclosure. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with a first vector that expresses the HC polypeptide of an antibody described herein and a second vector that expresses the LC polypeptide. Such host cells, such as mammalian host cells, can express an antibody that binds to anti-human CD33 as described herein. Mammalian host cells known to be capable of expressing an antibody include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0026] In some embodiments, a cell, such as a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22.

[0027] In some embodiments, a cell, such as a host cell, comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22.

[0028] In some embodiments, a cell, such as a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22. In some embodiments, a cell, such as a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, a cell, such as a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 15. In some embodiments, a cell, such as a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 21 and a second nucleic acid sequence encoding SEQ ID NO: 22.

[0029] The present disclosure further provides a process for generating an antibody that binds to human CD33 described herein by culturing the above-described host cell, such as a mammalian host cell, under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. The medium in which the antibody is secreted can be purified by conventional techniques. Various methods of protein purification can be employed, such methods being known in the art, for example, as described in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3 rd Edition, Springer, NY (1994).

[0030] The present disclosure further provides an antibody or an antigen-binding fragment thereof produced by any of the processes described herein.

[0031] In another aspect, pharmaceutical compositions comprising the antibodies, nucleic acids, or vectors described herein are provided herein. Such pharmaceutical compositions may also include one or more pharmaceutically acceptable excipients, diluents, or carriers. The pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).

[0032] Considering that the anti-human CD33 antibodies described herein do not deplete bone marrow cells, they offer advantages over bone marrow cell-depleting antibodies for treating bone marrow cell-related immune diseases, for example, avoiding the problematic concomitant immune deficiency, long-term immunosuppression, and other complications resulting from bone marrow cell depletion, and enhancing / inducing the immunomodulatory functions of CD33-expressing bone marrow cells. Furthermore, as shown below, the anti-human CD33 antibodies described herein are internalized into bone marrow cells. Thus, the anti-human CD33 antibodies described herein can be conjugated to therapeutic agents for targeted delivery of the therapeutic agents to human bone marrow cells to induce immunomodulation or other therapeutic effects by the therapeutic agents for treating bone marrow cell-related diseases. Additionally, the anti-human CD33 antibodies described herein do not significantly degrade cell surface or intracellular CD33 and do not significantly affect the availability of the CD33 receptor on the cell surface. Thus, the anti-human CD33 antibodies described herein may enable repeated CD33-targeted delivery of therapeutic agents to bone marrow cells for treating bone marrow cell-related diseases.

[0033] In some embodiments, a method is provided herein for treating a myeloid cell-related disease (e.g., an immune disease, a neurodegenerative disease, or a myeloid cell-related cancer) in a subject (e.g., a human patient) in need of treatment for the myeloid cell-related disease by administering to the subject an effective amount of an anti-human CD33 antibody conjugated to a therapeutic agent, or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure provides an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, or a pharmaceutically acceptable salt thereof, for use in therapy. In one embodiment, the present disclosure provides an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, or a pharmaceutically acceptable salt thereof, for use in the treatment of a myeloid cell-related disease. In one embodiment, the present disclosure provides the use of an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a myeloid cell-related disease. The conjugates, nucleic acids, vectors, or pharmaceutical compositions described herein can be administered by parenteral routes (e.g., subcutaneous and intravenous). In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to CD33-expressing myeloid cells for the treatment of a myeloid cell-related disease, wherein the therapeutic agent is conjugated to the anti-human CD33 antibody of the present disclosure and the therapeutic agent induces an immunomodulatory or other therapeutic effect.

[0034] Accordingly, in one embodiment, the present disclosure provides a conjugate of Formula I:

[0035] [Chemical Formula] wherein Ab is an antibody that binds to human CD33 (an "anti-human CD33 antibody"),

[0036] [Chemical Formula] is

[0037] [Chemical Formula] and n is 1 to 5.

[0038] In a further embodiment, the present invention provides a conjugate of formula I:

[0039] [ka] wherein Ab is an anti-human CD33 antibody, Ab comprises a VH and a VL, VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO: 3 or 17; LCDR1 comprises SEQ ID NO: 4, 13, 18, or 26; LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO: 6, During the ceremony,

[0040] [ka] teeth,

[0041] [ka] and n is 1 to 5.

[0042] In a further embodiment, the present invention provides a conjugate of formula I:

[0043] [ka] wherein Ab is an anti-human CD33 antibody, Ab comprises a VH and a VL, VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; HCDR1 contains SEQ ID NO: 1, HCDR2 contains SEQ ID NO: 2, HCDR3 contains SEQ ID NO: 3 or 17, LCDR1 contains SEQ ID NO: 4, 13, 18, or 26, LCDR2 contains SEQ ID NO: 5, LCDR3 contains SEQ ID NO: 6, wherein,

[0044]

Chemical formula

[0045]

Chemical formula

[0046] In a further embodiment, the present invention provides a conjugate of formula I:

[0047]

Chemical formula

[0048]

Chemical formula

[0049]

Chem.

[0050] In a further embodiment, the present invention provides a conjugate of formula I:

[0051]

Chem.

[0052]

Chem.

[0053]

Chem.

[0054] In a further embodiment, the present invention provides a conjugate of formula I:

[0055]

Chem.

[0056]

Chemical formula

[0057]

Chemical formula

[0058] In a further embodiment, the present disclosure provides a conjugate of formula I:

[0059]

Chemical formula

[0060]

Chem.

[0061]

Chem.

[0062] In a further embodiment, the present disclosure provides a conjugate of formula I:

[0063]

Chem.

[0064]

Chem.

[0065]

Chem.

[0066] In one embodiment, n is from 2 to 5.

[0067] In one embodiment, n is from 3 to 5.

[0068] In one embodiment, n is from 3 to 4.

[0069] In one embodiment, n is 4.

[0070] In one embodiment, n is 3.

[0071] In one embodiment, n is 2.

[0072] In some embodiments, Ab in the conjugate of Formula 1 comprises VH and VL, VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. In some embodiments, Ab is Ab2, and Ab2 comprises an HC comprising SEQ ID NO: 9 and an LC comprising SEQ ID NO: 10.

[0073] In some embodiments, Ab in the conjugate of Formula 1 comprises VH and VL, VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 13, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 14. In some embodiments, Ab is Ab3, and Ab3 comprises an HC comprising SEQ ID NO: 9 and an LC comprising SEQ ID NO: 15.

[0074] In some embodiments, Ab in the conjugate of Formula 1 comprises VH and VL, VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 17, LCDR1 comprises SEQ ID NO: 18, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab comprises VH comprising SEQ ID NO: 19 and VL comprising SEQ ID NO: 20. In some embodiments, Ab is Ab1, and Ab1 comprises HC comprising SEQ ID NO: 21 and LC comprising SEQ ID NO: 22.

[0075] As used herein, the formula:

[0076]

Chemical formula

[0077]

Chemical formula

[0078] As used herein, the formula:

[0079]

Chemical formula

[0080]

Chemical formula

[0081] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IV:

[0082] [Chemical Formula]

[0083] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVa:

[0084] [Chemical Formula]

[0085] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVb:

[0086] [Chemical Formula]

[0087] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVc:

[0088] [Chemical Formula]

[0089] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVd:

[0090] [Chemical Formula]

[0091] In one embodiment, the present disclosure provides a conjugate of Formula V:

[0092]

Chem.

[0093] In a further embodiment, the present disclosure provides a conjugate of Formula Va:

[0094]

Chem.

[0095] In some embodiments, the conjugate of Formula I modulates a CD33-target specific glucocorticoid receptor agonist-mediated response. In such embodiments, the conjugate of Formula I modulates a CD33-target specific glucocorticoid receptor agonist-mediated response such as an inflammatory response, cytokine expression, and / or glucocorticoid receptor agonist-mediated gene expression.

[0096] Due to their important roles in regulating the immune response, dysregulation of myeloid cells is associated with various myeloid cell-related immune diseases including autoimmune / inflammatory diseases and is caused by abnormal activation (increased pro-inflammatory cytokines) of myeloid and lymphoid cells. Examples of myeloid cell-mediated immune diseases include rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjogren's syndrome, macrophage activation syndrome, and fibrotic diseases such as scleroderma.

[0097] In one embodiment, the present disclosure provides a method of treating a subject in need of treatment for a myeloid cell-related disease, the method comprising administering to the subject (e.g., a human patient) an effective amount of a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, e.g., a conjugate of Formula I, or a pharmaceutically acceptable salt thereof. In certain embodiments, the myeloid cell-related disease is an immune disease, a neurodegenerative disease, or cancer. In certain embodiments, the myeloid cell-related disease is an immune disease, e.g., rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjogren's syndrome, macrophage activation syndrome, or a fibrotic disease such as scleroderma. In one embodiment, the present disclosure further provides a method of treating a subject in need of treatment for rheumatoid arthritis, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating a subject in need of treatment for systemic lupus erythematosus, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating a subject in need of treatment for lupus nephritis, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating a subject in need of treatment for cutaneous lupus, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating a subject in need of treatment for giant cell arteritis, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating a subject in need of treatment for polymyalgia rheumatica, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof.In one embodiment, the present disclosure further provides a method of treating psoriatic arthritis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating atopic dermatitis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating psoriasis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating ulcerative colitis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating Crohn's disease in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present invention further provides a method of treating asthma in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating juvenile idiopathic arthritis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating multiple sclerosis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method of treating Sjogren's syndrome in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof.In one embodiment, the present disclosure further provides a method for treating chronic spontaneous urticaria in a subject in need of treatment for macrophage activation syndrome, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method for performing the same in a subject in need of treatment for a fibrotic disease such as scleroderma, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the myeloid cell-related disease is a neurodegenerative disease (e.g., Alzheimer's disease). In one embodiment, the present disclosure further provides a method for performing the same in a subject in need of treatment for Alzheimer's disease, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the myeloid cell-related disease is a myeloid cell-related cancer (e.g., AML). In one embodiment, the present invention further provides a method for performing the same in a subject in need of treatment for AML, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof.

[0098] In one embodiment, the present disclosure further provides a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, for use in therapy, e.g., a conjugate of Formula I, or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure provides a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, for use in the treatment of myeloid cell-related diseases, e.g., a conjugate of Formula I, or a pharmaceutically acceptable salt thereof. In certain embodiments, the myeloid cell-related disease is an immune disease, a neurodegenerative disease, or cancer. In certain embodiments, the myeloid cell-related disease is an immune disease, e.g., rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjogren's syndrome, macrophage activation syndrome, and fibrotic diseases such as scleroderma. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of rheumatoid arthritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of systemic lupus erythematosus. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of lupus nephritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of cutaneous lupus. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of giant cell arteritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of polymyalgia rheumatica. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of psoriatic arthritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of atopic dermatitis.In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of psoriasis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of ulcerative colitis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of Crohn's disease. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of dermatomyositis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of juvenile idiopathic arthritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of multiple sclerosis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of Sjogren's syndrome. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of macrophage activation syndrome. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of fibrotic diseases such as scleroderma. In some embodiments, the myeloid cell-related disease is a neurodegenerative disease (e.g., Alzheimer's disease). In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of Alzheimer's disease. In some embodiments, the myeloid cell-related disease is a myeloid cell-related cancer (e.g., AML). In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the treatment of AML.

[0099] In one embodiment, the present disclosure also provides the use of a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein for the manufacture of a medicament for the treatment of myeloid cell-related diseases, for example, a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the myeloid cell-related disease is an immune disease, a neurodegenerative disease, or cancer. In certain embodiments, the myeloid cell-related disease is an immune disease, such as rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjogren's syndrome, macrophage activation syndrome, and fibrotic diseases such as scleroderma. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of rheumatoid arthritis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of systemic lupus erythematosus. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of lupus nephritis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cutaneous lupus. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of giant cell arteritis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of polymyalgia rheumatica. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of psoriatic arthritis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of atopic dermatitis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of psoriasis.In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of ulcerative colitis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of Crohn's disease. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of dermatomyositis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of juvenile idiopathic arthritis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of multiple sclerosis. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of Sjögren's syndrome. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of macrophage activation syndrome. In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of fibrotic diseases such as scleroderma. In some embodiments, the myeloid cell-related disease is a neurodegenerative disease (e.g., Alzheimer's disease). In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of Alzheimer's disease. In some embodiments, the myeloid cell-related disease is a myeloid cell-related cancer (e.g., AML). In one embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of AML.

[0100] The present disclosure provides a method for generating a conjugate, the method comprising conjugating a compound of the present disclosure with an anti-human CD33 antibody. The present disclosure provides a method for generating a conjugate, the method comprising conjugating a compound of the present disclosure with an anti-human CD33 antibody or an antigen-binding fragment thereof.

[0101] The present disclosure provides a method for generating a conjugate, which comprises conjugating a compound of formula IV with an anti-human CD33 antibody. The present disclosure provides a method for generating a conjugate, which comprises conjugating a compound of formula IVa with an anti-human CD33 antibody. The present disclosure provides a method for generating a conjugate, which comprises conjugating a compound of formula IVb with an anti-human CD33 antibody. The present disclosure provides a method for generating a conjugate, which comprises conjugating a compound of formula IVc with an anti-human CD33 antibody. The present disclosure provides a method for generating a conjugate, which comprises conjugating a compound of formula IVd with an anti-human CD33 antibody.

[0102] In some embodiments, the conjugate generated is a conjugate of formula I.

[0103] The present disclosure provides a method for generating a conjugate, which comprises (a) reducing an anti-human CD33 antibody with a reducing agent to generate a reduced anti-human CD33 antibody, wherein the anti-human CD33 antibody contains one or more engineered cysteine residues; (b) oxidizing the reduced anti-human CD33 antibody with an oxidizing agent to generate an oxidized anti-human CD33 antibody; and (c) contacting the oxidized anti-human CD33 antibody with a compound of the present disclosure to generate a conjugate.

[0104] The present disclosure provides a method for generating a conjugate, which comprises (a) reducing an anti-human CD33 antibody with a reducing agent to generate a reduced anti-human CD33 antibody, wherein the anti-human CD33 antibody contains one or more engineered cysteine residues; (b) oxidizing the reduced anti-human CD33 antibody with an oxidizing agent to generate an oxidized anti-human CD33 antibody; and (c) oxidizing the anti-human CD33 antibody with a compound of the formula

[0105]

Chem.

[0106] In some embodiments, the reducing agent is dithiothreitol. In some embodiments, the oxidizing agent is dehydroascorbic acid. In some embodiments, the reducing agent is dithiothreitol and the oxidizing agent is dehydroascorbic acid.

[0107] In one embodiment, the present disclosure further provides a pharmaceutical composition comprising an anti-human CD33 antibody conjugated to a therapeutic agent disclosed herein, or a pharmaceutically acceptable salt thereof, or an antibody, nucleic acid, or vector described herein, together with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure further provides a pharmaceutical composition comprising a conjugate of Formula I or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure further provides a pharmaceutical composition comprising a conjugate of Formula I together with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure further provides a process for preparing a pharmaceutical composition comprising mixing a conjugate of Formula I or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure also encompasses novel intermediates and processes for the synthesis of conjugates of Formula I.

Modes for Carrying Out the Invention

[0108] As used herein, the term "CD33" or "CD33 receptor", unless otherwise indicated, refers to the human myeloid cell surface antigen CD33 (also known as sialic acid binding Ig-like lectin 3, SIGLEC-3, SIGLEC3 FLJ00391, or p67) belonging to the sialic acid binding Ig-like lectin (SIGLEC) family of the immunoglobulin superfamily. This term also includes naturally occurring variants of CD33, such as splice variants or allelic variants. The amino acid sequence of human CD33 is known in the art and is, for example, NCBI reference sequence XP_011525833.1 (SEQ ID NO: 25). The amino acid sequence of cynomolgus monkey CD33 is known in the art and is, for example, sequence XP_045235686.1 (SEQ ID NO: 27). The term "CD33" is used herein to collectively refer to all known human CD33 isoforms and polymorphic forms.

[0109] As used herein, the term "myeloid cell-related disease" refers to a disease associated with CD33-expressing myeloid cells. Such myeloid cell-related diseases can include, for example, immune diseases, neurodegenerative diseases, or myeloid cell-related cancers. Myeloid cell-related immune diseases can be, for example, rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjogren's syndrome, macrophage activation syndrome, or fibrotic diseases such as scleroderma. Myeloid cell-related neurodegenerative diseases can be, for example, Alzheimer's disease. Myeloid cell-related cancers can be, for example, acute myeloid leukemia (AML).

[0110] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugate antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, and the term "antibody fragment or antigen-binding fragment" refers to, for example, Fab, Fab’, F(ab’)2, Fv fragment, scFv, scFab, disulfide-bonded Fv (sdFv), Fd fragment, and linear antibodies, etc., which may be fused to the Fc region or the IgG heavy chain constant region, and includes at least a portion of an antibody that retains the ability to interact with an antigen.

[0111] An exemplary antibody is an immunoglobulin G (IgG) type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via inter-chain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is mainly involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is mainly involved in effector functions. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region refers to the region of the antibody that includes the Fc region and the CH1 domain of the antibody heavy chain. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The IgG isotype can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The numbering of amino acid residues in the constant region is based on the EU index as in Kabat. Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health (1991). The term EU index numbering or EU numbering is used interchangeably herein.

[0112] The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3", and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be carried out according to well-known schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The definition of the North CDR is used for the exemplified anti-human CD33 antibodies described herein.

[0113] Exemplary embodiments of the antibodies of the present disclosure also include antibody fragments or antigen-binding fragments that include at least a portion of an antibody that retains the ability to specifically interact with an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-bonded Fv (sdFv), Fd fragments, and linear antibodies, which may be fused, for example, to an Fc region or an IgG heavy chain constant region.

[0114] As used herein, the term "Fc region" refers to the region of an antibody that includes the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion or the entire hinge region of the antibody heavy chain. Biological activities such as effector functions are due to the Fc regions that vary depending on the antibody isotype. Examples of antibody effector functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, complement-dependent cytotoxicity (CDC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and activation of B cells.

[0115] The terms "Fc receptor" and "FcR" represent receptors that bind to the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. "Fc gamma receptor" or "FcγR" is an FcR that binds to IgG antibodies and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor") that have similar amino acid sequences that mainly differ in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92 (1991); 25 Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med., 126:330-41 (1995).

[0116] As used herein, the term "non-depleting antibody" refers to an antibody that does not significantly reduce the number of CD33-expressing cells (e.g., myeloid cells) in a subject after treatment as compared to the number of myeloid cells before treatment. The number of myeloid cells and survival rate can be measured using well-known assays such as trypan blue staining and a Vi cell counter. Non-depleting antibodies typically do not induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis of myeloid cells.

[0117] As used herein, the term "receptor availability" refers to CD33 receptor expression on the cell surface. CD33 cell surface receptor expression can refer to de novo expression of CD33 receptors on the cell surface and / or CD33 receptors that are recycled to the cell surface after internalization.

[0118] As used herein, the term "non-degrading antibody" refers to an antibody that does not significantly reduce CD33 receptor expression on the cell surface and / or intracellular (internalized) CD33. Non-degrading CD33 antibodies, for example when used herein, do not significantly affect receptor availability.

[0119] Terms such as "modulate" as used herein refer to modifying or changing a measurable value, and include modifying or changing such a measurable value upward (i.e., upmodulating or upregulating) or downward (i.e., downmodulating or downregulating).

[0120] As used herein, the term "binds" is intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in the proximity of two proteins or molecules as determined by common methods known in the art.

[0121] As used herein, the term "therapeutic agent" refers to a therapeutic composition such as an anti-inflammatory agent, glucocorticoid, cytotoxic agent, siRNA, saRNA, peptide, oligonucleotide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or fragment thereof, or a combination thereof, which can be conjugated to an anti-human CD33 antibody disclosed herein to form a conjugate (e.g., an antibody-drug conjugate). In some embodiments, such conjugates induce an immunomodulatory effect or other therapeutic effect for the treatment of myeloid cell-related diseases by specific targeting of the CD33 receptor on myeloid cells by an anti-human CD33 antibody disclosed herein and subsequent delivery of the therapeutic agent to myeloid cells.

[0122] Furthermore, the therapeutic agent can be conjugated to the anti-human CD33 antibody disclosed herein in various ways at various positions or portions of the antibody such that such conjugation does not interfere with the binding of the antibody to the CD33 receptor and does not interfere with the therapeutic properties of the therapeutic agent when conjugated. The terms "linked" and "conjugated" are used interchangeably herein and refer to a first molecule or compound, such as an antibody or fragment thereof, being associated, bound, connected, covalently bound or joined, or joined in some other way, to a second molecule or compound, such as a therapeutic agent described herein.

[0123] As used interchangeably herein, the term "nucleic acid" refers to a polymer of nucleotides that includes single-stranded and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules incorporating natural nucleotides, modified nucleotides, and / or nucleotide analogs. The polynucleotides of the present disclosure may also include, for example, substrates incorporated therein by DNA or RNA polymerase or synthetic reactions.

[0124] Embodiments of the present disclosure include conjugates in which a polypeptide (e.g., an anti-human CD33 antibody) is conjugated to one or more drug moieties, e.g., two drug moieties, three drug moieties, four drug moieties, five drug moieties, or more drug moieties. The drug moieties can be conjugated to the polypeptide at one or more sites in the polypeptide as described herein. In certain embodiments, the conjugate has an average drug-to-antibody ratio (DAR) (molar ratio) in the range of 2-5, or 3-5, or 3-4. In a particular embodiment, the conjugate has an average DAR of 3-4. In certain embodiments, the conjugate has an average DAR of about 3. In certain embodiments, the conjugate has an average DAR of about 4.

[0125] As further understood by those skilled in the art, the conjugate of Formula I, as used herein, can also be referred to as an anti-human CD33 antibody glucocorticoid conjugate (“anti-human CD33 Ab GC conjugate”).

[0126] Anti-human CD33 antibody conjugates, e.g., the anti-human CD33 antibody GC conjugates of the present disclosure, can be formulated as pharmaceutical compositions, which are administered by any route that makes the conjugate bioavailable, such routes including, for example, intravenous injection or subcutaneous administration. Such pharmaceutical compositions can be prepared using techniques and methods known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, A. Adejare, Editor, 23 rd Edition, published 2020, Elsevier Science).

[0127] As used herein, the terms "treating", "treatment", or "for treating" include, but are not necessarily limited to, inhibiting, slowing down, stopping, controlling, delaying, or reversing the progression or severity of an existing symptom or disorder, or improving an existing symptom or disorder, and do not necessarily indicate complete elimination of the existing symptom or disorder. Treatment includes administration of a protein, nucleic acid, vector, or composition for treating a symptom or disorder in a patient, particularly a human.

[0128] As used herein, the term "inhibiting" or "inhibition" refers to, for example, reducing, decreasing, slowing, lessening, halting, disrupting, suppressing, antagonizing, or blocking a biological response or activity, and does not necessarily indicate complete elimination of the biological response.

[0129] As used herein, the term "subject" refers to a mammal including, but not limited to, human, chimpanzee, ape, monkey, cow, horse, sheep, goat, pig, rabbit, dog, cat, rat, mouse, guinea pig, etc. Preferably, the subject is a human.

[0130] As used herein, the term "effective amount" refers to the amount or dosage of a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof that, when administered to a subject singly or in multiple doses, provides a desired effect in a subject being diagnosed or treated. The term "effective amount" as used herein further refers to the amount of a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof that elicits a desired biological or medical response in a subject, e.g., elicits a decrease or inhibition of the activity of a protein, or ameliorates a condition, alleviates a state, decelerates or delays the progression of a disease, or prevents a disease or the like. In non-limiting embodiments, the term "effective amount" refers to the amount (dosage and duration and means of administration) of a conjugate or a pharmaceutically acceptable salt thereof that is effective to at least partially alleviate, inhibit, prevent, and / or ameliorate a condition, or disorder, or disease in order to achieve a desired therapeutic result when administered to a subject. The effective amount is also an amount where the beneficial effect outweighs the toxic or detrimental effects of the conjugate of the present disclosure or the pharmaceutically acceptable salt thereof of the present disclosure.

[0131] The effective amount can be determined by one of ordinary skill in the art using known techniques and observing the results obtained under similar circumstances. In determining the effective amount for a patient, several factors are considered by the attending physician, including, but not limited to, the species of the patient; its size, age, and general health; the particular disease or disorder involved; the degree or involvement or severity of the disease or disorder; the response of the individual patient; the particular conjugate administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances.

[0132] Also included within the scope of the present invention are pharmaceutically acceptable salts of the conjugates of Formula I. Pharmaceutically acceptable salts of the conjugates of the present invention, such as the conjugates of Formula I, can be formed under standard conditions known in the art. See, e.g., Berge, S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66:1-19, (1977).

[0133]

Table 1

[0134] The conjugate of the present disclosure or a salt thereof can be readily prepared by various procedures known to those skilled in the art, some of which are illustrated in the following preparations and examples. Those skilled in the art will recognize that they can combine the specific synthetic steps for each of the described routes in different ways or combine them with steps from different schemes to prepare the conjugate of the present disclosure or a salt thereof. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, milling, and crystallization. All substituents are as defined unless otherwise indicated. Reagents and starting materials are readily available to those skilled in the art. The following preparations, examples, and assays further illustrate the invention but should in no way be construed as limiting the scope of the invention.

Brief Description of the Drawings

[0135]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4A

Figure 4B

Figure 4C

[0136] Preparation 1 6-Bromo-2-fluoro-3-methoxybenzaldehyde

[0137]

Chemical formula

[0138] Two reactions were carried out in parallel. To a solution of 4-bromo-2-fluoro-1-methoxybenzene (250 g, 1.2 mol) in THF (1500 mL) was slowly added LDA (2 M, 730 mL) at -78 °C over 30 minutes. After an additional 30 minutes, DMF (140 mL, 1.8 mol) was slowly added at -78 °C over 30 minutes. After 1 hour, the two reaction mixtures were combined, and the mixture was diluted with aqueous citric acid solution (2000 mL) and extracted with EtOAc (1500 mL × 2). The combined organic layers were washed with saturated aqueous NaCl solution (1000 mL) and concentrated under reduced pressure to obtain a residue. The residue was triturated with petroleum ether (1000 mL) at room temperature for 12 hours to obtain the title compound (382 g, 67% yield). ES / MS m / z 233.9 (M+H).

[0139] Preparation 2 2-Fluoro-3-methoxy-6-methylbenzaldehyde

[0140] [Chem.]

[0141] Three reactions were carried out in parallel. 6-Bromo-2-fluoro-3-methoxybenzaldehyde (120 g, 5.3 mol), methylboronic acid (47 g, 7.9 mol), Pd(dppf)Cl2 (12 g, 0.02 mol), and Cs2CO3 (340 g, 1.1 mol) were added to a mixture of 1,4-dioxane (600 mL) and water (120 mL). The mixture was stirred at 120 °C. After 12 h, the three reactants were combined, the mixture was diluted with saturated aqueous NH4Cl solution (1000 mL), and extracted with MTBE (1500 mL × 2). The combined organic layers were washed with saturated aqueous NaCl solution (1000 mL) and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography, eluting with 40:1 Pet ether:EtOAc to give the title compound (180 g, 59%). ES / MS m / z 169.3 (M+H).

[0142] Preparation 3 2-Fluoro-3-hydroxy-6-methylbenzaldehyde

[0143] [Chem.]

[0144] 2-Fluoro-3-methoxy-6-methylbenzaldehyde (175 g, 1.0 mol) was added into DCM (1050 mL). BBr3 (200 mL, 2.1 mol) was slowly added into the solution at 0 °C. The reactant was stirred at room temperature. After 1 h, the mixture was diluted with saturated aqueous NaHCO3 solution (1000 mL) until pH = 7 - 8 and extracted with MTBE (1500 mL × 2). The combined organic layers were washed with saturated aqueous NaCl solution (1000 mL) and concentrated under reduced pressure to give the title compound (110 g, 68%). ES / MS m / z 154.9 (M+H).

[0145] Preparation 4 tert-Butyl N-[3-[(2-Fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate

[0146]

Chem.

[0147] 2-Fluoro-3-hydroxy-6-methylbenzaldehyde (130 g, 0.84 mol), tert-butyl (3-(bromomethyl)phenyl)carbamate (200 g, 0.70 mol), and potassium carbonate (350 g, 2.5 mol) were added to acetonitrile (780 mL) at room temperature and then heated to 50 °C. After 5 hours, the reaction mixture was diluted with water (600 mL) and extracted with EtOAc (800 mL × 2). The combined organic layers were washed with saturated NaCl aqueous solution (800 mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography, eluting with 50:1 Pet ether:EtOAc to obtain a crude product. The crude product was triturated with MTBE (500 mL) at room temperature for 30 minutes to obtain the title compound (103 g, 32%). ES / MS m / z 382.1 (M+Na + ).

[0148] Preparation 5 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-Aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-4-one

[0149]

Chem.

[0150] Perchloric acid (70% in water, 4.8 mL) was added to a suspension of (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one (4.4 g, 12 mmol, also known as "16α-hydroxy prednisolone") and tert-butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate (4.0 g, 11 mmol, Preparation 4) in acetonitrile (110 mL) at -10 °C and the mixture was warmed to room temperature. After 1 hour, DMF (10 mL) was added to the suspension at room temperature. After 18 hours, the reaction was quenched with saturated aqueous NaHCO3 and extracted with 9:1 DCM:isopropanol. The organic layers were combined, dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by reverse phase chromatography eluting with 1:1 aqueous NH4HCO3 (10 mM + 5% MeOH):ACN to give the title compound, Peak 1 (1.72 g, 25%). ES / MS m / z 618.6 (M+H). 1 H NMR (400.13 MHz, DMSO-d6) δ 0.93 - 0.87 (m, 6H), 1.40 (s, 3H), 1.71 - 1.60 (m, 1H), 1.89 - 1.76 (m, 4H), 2.18 - 2.12 (m, 2H), 2.29 (s, 4H), 4.23 - 4.17 (m, 1H), 4.32 - 4.30 (m, 1H), 4.50 - 4.43 (m, 1H), 4.81 (d, J = 3.2 Hz, 1H), 4.98 - 4.95 (m, 3H), 5.16 - 5.10 (m, 3H), 5.61 (s, 1H), 5.95 (s, 1H), 6.18 - 6.15 (m, 1H), 6.53 - 6.48 (m, 2H), 6.58 (s, 1H), 6.90 - 6.86 (m, 1H), 6.99 (t, J = 7.7 Hz, 1H), 7.12 (t, J = 8.5 Hz, 1H), 7.33 - 7.30 (m, 1H).

[0151] Preparation 6 (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(3-((3-Aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-4-one (also referred to herein as "GC1")

[0152]

Chem.

[0153] From Preparation 5, the residue was purified by reverse-phase chromatography eluting with a 1:1 aqueous solution of NH4HCO3 (10 mM + 5% MeOH):ACN to give the title compound, Peak 2 (1.24 g, 18%). ES / MS m / z 618.6 (M+H). 1 H NMR (400.13 MHz, d6-DMSO) δ 0.88 (s, 3H), 1.24 - 1.12 (m, 2H), 1.40 (s, 3H), 1.69 - 1.56 (m, 1H), 1.91 - 1.76 (m, 4H), 2.08 - 2.01 (m, 2H), 2.22 (s, 3H), 2.39 - 2.29 (m, 1H), 3.18 (d, J = 5.2 Hz, 1H), 4.12 - 4.00 (m, 1H), 4.37 - 4.30 (m, 2H), 4.79 (d, J = 3.1 Hz, 1H), 5.00 - 4.93 (m, 2H), 5.10 - 5.06 (m, 3H), 5.31 (d, J = 6.7 Hz, 1H), 5.95 (s, 1H), 6.18 (dd, J = 1.8, 10.1 Hz, 1H), 6.34 (s, 1H), 6.53 - 6.48 (m, 2H), 6.58 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.99 (t, J = 7.7 Hz, 1H), 7.09 (t, J = 8.5 Hz, 1H), 7.33 (d, J = 10.1 Hz, 1H).

[0154] Preparation 7 (3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine

[0155]

Chem.

[0156] To a solution of N-succinimidyl 3-maleimidopropionate (5.0 g, 19 mmol) and L-alanyl-L-alanine (3.4 g, 21 mmol) in DMF (25 mL) was added DIPEA (3.1 mL, 18 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by silica gel chromatography eluting with 2% acetic acid in EtOAc to give the title compound (4.0 g, 69%). ES / MS m / z 312.3 (M+H).

[0157] Preparation 8 3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-Fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (also referred to herein as “GC-L”)

[0158]

Chem.

[0159] (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(3-((3-Aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-4-one (24 g, 39 mmol, see Preparation 6) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine (15 g, 47 mmol, see Preparation 7) in DMF (250 mL) were cooled to 0-5 °C, and 2,6-lutidine (11 mL, 97 mmol) and then HATU (17 g, 43 mmol) were added. The mixture was stirred at 0-5 °C for 5 minutes, then the cooling bath was removed and the mixture was stirred for 2 hours. The mixture was diluted with EtOAc. The organic solution was washed 3 times with water and once with saturated NaCl aqueous solution, dried over Na2SO4 (assisted solubility by adding MeOH), filtered and evaporated to give the crude product. The crude product was purified by silica gel chromatography using a gradient of 1-10% MeOH in DCM to give the title compound (24 g, 68%). ES / MS m / z 911.4 (M+H). 11H NMR (400.13 MHz, DMSO): δ 9.88 (s, 1H), 8.20 (d, J = 7.1 Hz, 1H), 8.11 (d, J = 7.2 Hz, 1H), 7.68 (s, 1H), 7.60 - 7.58 (m, 1H), 7.34 - 7.29 (m, 2H), 7.14 - 7.09 (m, 2H), 7.00 (s, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.34 (s, 1H), 6.18 (dd, J = 1.8, 10.0 Hz, 1H), 5.95 (s, 1H), 5.76 (s, 1H), 5.31 (d, J = 6.8 Hz, 1H), 5.13 - 5.04 (m, 3H), 4.78 (d, J = 3.1 Hz, 1H), 4.41 - 4.30 (m, 4H), 4.10 - 4.00 (m, 1H), 3.61 (t, J = 7.3 Hz, 2H), 2.42 - 2.31 (m, 3H), 2.22 (s, 3H), 2.11 - 2.01 (m, 2H), 1.91 - 1.78 (m, 5H), 1.40 (s, 3H), 1.31 (d, J = 7.2 Hz, 3H), 1.19 - 1.11 (m, 5H), 0.88 (s, 3H).

[0160] Preparation 9 3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-Fluoro-3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide

[0161]

Chemical Structure

[0162] In a manner similar to the procedure described for Preparation 8, the compound of Preparation 9 was prepared from (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-4-one (see Preparation 5) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine (see Preparation 7). ES / MS m / z 911.4 (M+H). 1 H NMR (500.11 MHz, DMSO): δ 9.88 (s, 1H), 8.23 - 8.20 (m, 1H), 8.11 (d, J = 7.2 Hz, 1H), 7.69 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.33 - 7.28 (m, 2H), 7.15 - 7.08 (m, 2H), 7.00 (s, 2H), 6.91 - 6.89 (m, 1H), 6.17 (dd, J = 1.7, 10.1 Hz, 1H), 5.94 (s, 1H), 5.61 (s, 1H), 5.16 - 5.12 (m, 3H), 4.98 - 4.96 (m, 1H), 4.81 (d, J = 3.1 Hz, 1H), 4.49 - 4.36 (m, 6H), 3.61 (t, J = 7.3 Hz, 2H), 2.41 (t, J = 7.3 Hz, 2H), 2.30 - 2.29 (m, 4H), 2.17 - 2.15 (m, 2H), 1.88 - 1.77 (m, 4H), 1.69 - 1.61 (m, 1H), 1.40 (s, 3H), 1.31 (d, J = 7.2 Hz, 3H), 1.18 (d, J = 7.2 Hz, 3H), and 0.93 - 0.87 (m, 6H).

Example

[0163] Example 1. Generation of the exemplified anti-human CD33 antibody GC conjugate Example 1a: Generation and manipulation of the anti-human CD33 antibody Antibody Generation: The anti-human CD33 antibodies described herein were discovered from a phage display Fab library using solution panning against the extracellular domain of human CD33 with an Fc tag (hCD33-Fc). Briefly, the library was first panned against human IgG Fc to remove Fc binders. Human CD33-specific binders were enriched after 3 rounds of panning and identified by single-point phage ELISA screening against his-tagged hCD33. After conversion to the IgG format and purification, antibodies that bind to CD33-expressing myeloid cells were confirmed by fluorescence-activated cell sorting (FACS) assays using human and cynomolgus monkey PBMCs, respectively.

[0164] Antibodies generated from the above process were screened for internalization activity in CD33-expressing myeloid cells. Antibodies were labeled with pHrodo™ Red, incubated with human PBMCs, and analyzed by FACS. The pHrodo-labeled antibody generates a red fluorescence signal in a low pH environment after being internalized into lysosomes.

[0165] Antibodies were selected for specific binding to both human and cynomolgus monkey CD33 on myeloid cells and high internalization efficiency.

[0166] Engineering of Anti-Human CD33 Antibodies: Critical engineering including antibody sequence germlining, affinity maturation, amino acid modification, and developability studies was performed to generate the anti-human CD33 antibodies described herein. The engineering overcame the challenges of achieving and balancing the desired affinity for human and cynomolgus monkey CD33, improving the functional characteristics of the antibody (e.g., reducing receptor degradation and cell depletion properties), and improving the immunogenic risk profile, viscosity, and developability for subcutaneous administration.

[0167] The VH and Vκ sequences of the anti-human CD33 antibodies described herein are highly homologous to the human germline. Four framework residues were identified in the light chain framework region of the parental antibody (A12, S18, S22, and A40 (Kabat numbering)) and converted to germline residues without affecting the specificity and affinity for human CD33, resulting in an antibody (Ab1) with a high percentage of human germline identity, potentially reducing immunogenic risk, and thus providing an improved development profile.

[0168] Ab1 was further engineered as a Fab using phage display platform technology (Anal Biochem. 1998 256(2):169-77). The amino acid residue substitution at HCDR3 E95T (Kabat numbering) was found to be important in improving the affinity for human CD33, reducing the interaction with an Ig-binding chromatography column, and improving the viscosity of the antibody at high concentrations. Additionally, HCDR3 E95T and LCDR1 D28P residue substitutions were identified and engineered to improve the isoelectric point of the anti-human CD33 antibody GC conjugate.

[0169] Ab1 showed a significant difference in affinity for human CD33 (3.4E-09) and cynomolgus monkey CD33 (2.41E-05) (see Table 4). An affinity difference of more than 10-fold can make preclinical toxicity studies and data interpretation difficult. Using Ab1 as a template, a targeted mutagenesis library was designed to screen for mutations that improve affinity for cynomolgus monkey CD33 using antibody structural information of the cynomolgus monkey CD33 extracellular domain (ECD), CDR mutagenesis scanning, and computer modeling. Four amino acid residues in LCDR1 (D28, V29, F30, and R31 (Kabat numbering)) were identified as important for increasing cynomolgus monkey CD33 binding while maintaining a desirable binding affinity for human CD33. The combination of the HCDR3 E95T amino acid residue substitution identified above and the four LCDR1 amino acid residue substitutions significantly improved the binding affinity for cynomolgus monkey CD33 (see Table 4), and significantly minimized the affinity difference between human CD33 and cynomolgus monkey CD33 while maintaining a desirable binding affinity for human CD33. The combination of these engineered residues resulted in the generation of Ab2 and Ab3 (Tables 2a, 2b, and 3).

[0170] Framework selection: Amino acid residue substitutions L234A, L235A, and P329A (EU numbering) were incorporated into the IgG1 Fc region (IgG1AAA), reducing the binding of the antibody to Fcγ receptors and C1q, and as a result, reducing effector function activities such as ADCC and CDC. Furthermore, the IgG1AAA framework significantly reduced the degradation of the CD33 receptor by the CD33 antibody, as shown below.

[0171] The antibody further contains amino acid residue substitutions S124C and A378C (EU numbering) in the IgG1 HC constant region for specific conjugation to a therapeutic agent (e.g., glucocorticoid).

[0172] The amino acid sequence of human CD33 ECD is provided by SEQ ID NO: 28, and the amino acid sequence of cynomolgus CD33 ECD is provided by SEQ ID NO: 29.

[0173]

Table 2

[0174]

Table 3

[0175]

Table 4

[0176] Example 1b: Generation of anti-human CD33 Ab2-GC conjugate (when n = 4)

[0177]

Chemical formula

[0178] The exemplified anti-human CD33 antibody Ab2 (see Tables 2 and 3) was first ultra-reduced at 37 °C for 2 hours or at 21 °C for over 16 hours in the presence of a 40-fold molar excess of dithiothreitol (DTT). This first reduction step was used to remove various capping groups containing cysteine and glutathione that bind to engineered cysteines at positions 124 and 378 of the heavy chain during expression. After the reduction step, the sample was purified through a desalting resin to remove unbound caps and the reducing agent. The subsequent 2-hour oxidation step was carried out at room temperature (about 21 °C) in the presence of a 10-fold molar excess of dehydroascorbic acid (DHAA) to reform the native interchain disulfides between the light and heavy chains and the hinge disulfide pairs. After the 2-hour oxidation step, 4 - 8 molar equivalents of glucocorticoid receptor agonist payload-linker (“GC-L”), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2’,1’:4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide prepared in Preparation 8, was added using a 10 mM stock solution dissolved in DMSO. The sample was then incubated at room temperature for 30 - 60 minutes to allow efficient conjugation of GC-L to the engineered cysteines. Subsequently, a subsequent finishing step such as size exclusion chromatography (SEC) or tangential flow filtration (TFF) was used to buffer exchange the sample into an appropriate formulation buffer and remove DMSO and any excess linker-payload.

[0179] Drug to antibody ratio (DAR) evaluation: To evaluate the average number of linker-payloads present on the final conjugate, two analytical methods were used: 1) reverse phase (RP) HPLC and 2) time of flight (TOF) mass spectrometry. Both methods required an initial sample reduction step that included adding dithiothreitol (DTT) to a final concentration of approximately 10 mM and subsequently incubating at 42 °C for 5 minutes.

[0180] Reverse phase HPLC method: 10 - 30 μg of the reduced anti-human CD33 antibody Ab2 GC conjugate sample was injected onto a Phenyl 5PW, 4.6 mm × 7.5 cm, 10 μm column (Tosh Parts No. 0008043). Buffer A was composed of 0.1% trifluoroacetic acid (TFA) in water, while buffer B was composed of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column was equilibrated in 20% buffer B prior to sample injection, followed by a gradient of 28% - 40% buffer B over approximately 8.5 column volumes. The average DAR was determined by calculating the contribution from each individual DAR species from the percentage of the fraction multiplied by the number of DARs per contributing species. Since this value is based on a partially reduced sample and represents only half of the molecule, it was then multiplied by 2 to account for the intact antibody GC conjugate. The DAR calculation for the exemplary anti-human CD33 Ab2 GC conjugate of Example 1b is provided in Table 4.

[0181] [Table 5]

[0182] Time-of-Flight Mass Spectrometry Method: 8 μg of the partially reduced sample was injected onto a Poroshell 300sb-C3 2.1×12.5 mm, 5 μm column (Agilent part No. 821075-924). Buffer A was composed of 0.1% trifluoroacetic acid (TFA) in water, while Buffer B was composed of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column was equilibrated in 0% B buffer before sample injection, followed by a gradient of 10% B to 80% B over approximately 28 column volumes. The average DAR was determined by calculating the contribution from each individual DAR species from the percentage of the fraction multiplied by the number of DARs per contributing species. Since this value is based on the partially reduced sample and represents only half of the molecule, it was then multiplied by 2 to account for the intact antibody GC conjugate. The DAR calculation for the anti-human CD33 Ab2 GC conjugate of Example 1b is provided in Table 5.

[0183]

Table 6

[0184] Example 1c. Generation of anti-human CD33 Ab2 GC conjugate (where n = 3)

[0185]

Chemical formula

[0186] The conjugate of Example 1c was prepared in a manner similar to the procedure described in Example 1b, using a 4:1 GC-L:Ab2 molar ratio and an incubation at approximately 21°C for 20 minutes to yield a final DAR of approximately 3.

[0187] Example 1d. Thiosuccinimide hydrolysis: The thiosuccinimide ring of the compound of conjugated formula Ia can be hydrolyzed under conditions well-known in the art as shown below (see, for example, WO 2017 / 210471, paragraph 001226) to obtain the ring-opened product of formula Ib.

[0188]

Chem.

[0189] In addition, the above thiosuccinimide ring of conjugated formula Ia can undergo at least partial in vivo hydrolysis under standard or well-known formulation conditions to provide the ring-opened product of formula Ib.

[0190] Example 2. Binding activity Example 2a. Binding affinity: The affinity and binding kinetics of the exemplified anti-human CD33 antibodies against human and cynomolgus CD33 ECD 18-232-His proteins were determined by surface plasmon resonance using a Biacore 8K (GE Healthcare). Briefly, according to the Instrument Handbook, the antibody was captured on a Biacore protein A chip, and subsequently CD33 ECD was flowed at a 2-fold serial dilution in PBS-P20-BSA (0.005% surfactant P20, 0.1 mg / mL BSA) from 200 nM to 0.782 nM. All measurements were performed at 37 °C. A multi-cycle kinetic setting was used where each analyte concentration was run in separate cycles with surface regeneration after each sample injection. Regeneration was optimized to maintain consistent surface properties for each cycle. Each cycle started with a 3-minute injection of 0.1 μg / mL antibody at a flow rate of 10 μl / min, followed by a 3-minute injection of antigen at a flow rate of 50 μl / min and a 15-minute dissociation phase in PBS-P20-BSA. Then, the chip surface was regenerated using three 30-second injections at a flow rate of 50 μl / min of pH 1.5, 10 mM glycine buffer. The data was fitted to a 1:1 binding model to derive k a and k d and calculate K D ​

[0191] The results in Table 3 show that the exemplified anti-human CD33 Ab2 GC conjugate of Example 1b maintained a binding affinity similar to that of the unconjugated anti-human CD33 Ab2 for human and cynomolgus CD33. Furthermore, Ab1, Ab2, and Ab3 bound to human CD33 with desirable affinities, and Ab1 and Ab2 had improved binding to cynomolgus monkeys.

[0192] [Table 7]

[0193] Example 2b. Cell surface binding potency: The binding potencies of the exemplified anti-human CD33 antibodies and the anti-human CD33 Ab2 GC conjugate of Example 1b to human monocytes were tested in a fluorescence-activated cell sorting (FACS) assay. Human PBMCs were isolated from human blood samples by standard Ficoll-Paque™ plus (GE HEALTHCARE) density gradient centrifugation. Freshly isolated cell PBMCs were 2×10 6Resuspend in cells / mL, let stand at room temperature for 15 minutes, then seed into a round-bottom 96-well plate (COSTAR®) at 100 μL / well, and wash with FACS buffer (PBS containing 2% fetal bovine serum from Corning®). Add each of the exemplified anti-human CD33 antibody and the respective control IgG antibody conjugated to Alexa Fluor® 647 to the wells according to the manufacturer's protocol (Thermo Fisher Scientific), dilute 4-fold and make duplicates. Then, add an equal volume of 2× antibody cocktail containing PE-Cy5 anti-human CD14 antibody (clone M5E2), Per-CP anti-human CD45 antibody (clone H130), FITC anti-human CD11b (clone ICRF44), and Alexa Fluor-647 anti-human CD66b (G10F5) to the wells. Incubate the cells at 4°C for 30 minutes, wash twice with FACS buffer, and resuspend in FACS buffer with a final volume of 100 μl. Add Viability dye, Live / Dead Yellow (Thermo Fisher Scientific, L34968), and analyze the sample via a flow cytometer (LSRFortessa™ X-20, BD BIOSCIENCES). Perform data analysis using FlowJo software and statistical analysis using GraphPad Prism9. The data represent the mean fluorescence intensity (MFI) of CD33-expressing cells from monocytes or neutrophils.

[0194] The results in FIGS. 1A-1B show the exemplified anti-human CD33 Ab2 GC conjugate and Ab2 of Example 1b that bound to human CD33 expressed on the monocyte cell surface with a desirable binding potency. FIG. 1B shows the binding of Ab3 to the monocyte cell surface.

[0195] Example 3: Functional Activity Example 3a. Antibody internalization: The exemplified anti-human CD33 antibody was labeled with pHrodo™ Red, an amine-reactive dye (Thermo Fisher, P36014). The pH-sensitive dye is non-fluorescent extracellularly but emits bright fluorescence in acidic, low-pH lysosomes. This property enables visualization and quantification of antibody internalization by flow cytometry. Human PBMCs (500K / well) were incubated on ice for 30 minutes in FACS buffer to arrest baseline internalization. Cells were stained on ice for 1 hour with the pHrodo-labeled antibody (10 μg / mL) in FACS buffer and then transferred to 37 °C for 2 hours. The cells were then stained with the remaining panel of surface detection markers: CD45 mAb (clone HI30), CD11b (clone ICRF44), CD14 (clone M5E2).

[0196] The results in Table 4 show that greater than about 80% of the exemplified anti-human CD33 Ab2 GC conjugate and Ab2 of Example 1b were internalized into primary human monocytic cells within about 2 hours from binding to cell surface CD33. In comparison, only about 50% of gemtuzumab (hIgG4PAA) was internalized into primary human monocytic cells within about 2 hours of binding to cell surface CD33. The faster and / or increased rate of internalization of the exemplified anti-human CD33 Ab2 GC conjugate and Ab2 of Example 1b into monocytic cells suggests that Ab2 can effectively deliver the therapeutic agent conjugated thereto to CD33-expressing cells.

[0197] [Table 8]

[0198] Example 3b. CD33 surface receptor availability after internalization: CD33 cell surface receptor availability in monocytes after internalization upon binding to the exemplified anti-human CD33 antibody was evaluated. Briefly, human monocytes isolated from PBMCs were plated at 2 × 10 in 96-well plates 4Cells were seeded in wells and incubated on ice for 60 minutes to prevent internalization. An exemplary anti-human CD33 antibody (30 μg / mL) was added to the cells and allowed to bind on ice for 30 minutes. Then 100 μL of culture medium was added to the cells and the plates were placed in an incubator at 37 °C at 3.5, 24, 48, 72, and 96 hour time points. Baseline samples were stained immediately to represent the surface binding of the exemplified anti-human CD33 antibody prior to internalization. The cells were Fc-blocked for 20 minutes and then stained on ice for 30 minutes with Alexa Fluor-647 labeled anti-human CD33 exemplified antibody, Alexa Fluor-700 anti-human CD45 (HI30), and Brilliant Violet-605 anti-human CD11b (ICRF44). The cells were analyzed by flow cytometry.

[0199] The results in Table 5 show that the cell surface CD33 availability on monocytes increases from about 1.1% to a maximum of about 31.9% from baseline over a period of about 3.5 hours to about 96 hours after internalization. The surface CD33 can be from de novo CD33 expression and / or internalized CD33 recycled to the cell surface. These results indicate that the regenerated CD33 on human monocytes can potentially bind to additional anti-human CD33 antibodies for repeated delivery of CD33 antibodies or therapeutics conjugated to CD33 antibodies to CD33-expressing cells.

[0200]

Table 9

[0201] Example 3c. CD33 Receptor Degradation: The effect of the exemplified anti-human CD33 Ab2 on intracellular CD33 degradation was evaluated by protein immunoblot assay. Human PBMCs were isolated from human blood samples by standard Ficoll-Paque™ plus (GE HEALTHCARE) density gradient centrifugation. Freshly isolated PBMCs were seeded in 24-well cell culture plates at 1×10 7Cells were seeded at 5 mL / well. Cells were treated overnight in a cell incubator at 37 °C using anti-human CD33 Ab2 and hIgG1 (effector null) negative control. This step enabled determination of the degradation of internalized CD33 receptor. Cells were collected the next day and lysed with RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor.

[0202] Quantification was performed by bicinchoninic acid (BCA) assay and normalized for loading in protein immunoblot assay. CD33 levels were detected using a non-competing anti-CD33 antibody (AbCam clone EPR4423).

[0203] The results in FIGS. 2A-2B showed that the exemplified anti-human CD33 Ab2 did not degrade CD33 in terms of binding and internalization, and the CD33 levels were equivalent to the isotype control.

[0204] Example 3d. Pro-inflammatory cytokine regulation and immune cell depletion: The exemplified anti-human CD33 Ab2 GC conjugate and anti-human CD33 Ab2 of Example 1b were evaluated for their effects on pro-inflammatory cytokine regulation in human PBMC. PBMC were used to mimic in vivo conditions. Briefly, the exemplified anti-human CD33 Ab2 or the anti-human CD33 Ab2 GC conjugate of Example 1b was incubated with human PBMC at 2×10 6 cells / well in a 96-well plate in a 37 °C incubator for 1 hour. Subsequently, the cells were stimulated with LPS (Sigma catalog L2880) at 100 pg / mL in a 37 °C incubator for 24 hours. The culture supernatant was collected and the indicated cytokines (Proinflammatory Panel II (human) 4-Plex kit, MSD, K15053D-2) were measured.

[0205] The results in FIGS. 3A-3D and Table 6a show that the anti-human CD33 Ab2 GC conjugate of Example 1b significantly regulated (inhibited) the pro-inflammatory cytokine IL-6 and TNFα responses, similar to that of GC1 alone. The unconjugated anti-human CD33 Ab2 did not regulate the CD33-mediated expression of the pro-inflammatory cytokines IL-6 and TNFα, indicating that the cytokine responses observed with the exemplified anti-human CD33 Ab2 GC conjugate were regulated by glucocorticoids.

[0206] Table 6b shows that treatment with the anti-human CD33 Ab2 GC conjugate and anti-human CD33 Ab2 of Example 1b did not induce PBMC immune cell depletion compared to the isotype control.

[0207]

Table 10

[0208]

Table 11

[0209] Example 3e. Plasmacytoid dendritic cell assay: The ability of the exemplified anti-human CD33 Ab2 GC conjugate to regulate human plasmacytoid dendritic cells (pDC) was evaluated in a pDC differentiation assay. pDC, which are known to express CD33, are primary cells that produce type I interferon associated with the pathogenesis of lupus. Briefly, human PBMC were cultured in IL-3-supplemented culture medium with stimulation by CpG (immunostimulatory DNA containing unmethylated cytosine-phosphate-guanosine) in the presence of anti-human CD33 Ab2 or the anti-human CD33 Ab2 GC conjugate of Example 1b or GC1 alone. Differentiation of pDC was measured by flow cytometry. The functional effect of pDC was measured by type I IFN (IFNα) production.

[0210] The results in Table 6c show that the anti-human CD33 Ab2 GC conjugate of Example 1b significantly regulated (inhibited) the differentiation of pDCs and inhibited cytokine IFNα secretion, similar to that of GC1 alone. The unconjugated anti-human CD33 Ab2 had no effect on pDC differentiation and IFNα secretion, indicating that the inhibition of cytokine response observed with the exemplified anti-human CD33 Ab2 GC conjugate was regulated by glucocorticoids. Thus, the results demonstrate that the anti-human CD33 Ab2 GC conjugate of Example 1b specifically targets the functional activity of pDCs.

[0211] [Table 12]

[0212] Example 4. Effector function activity Antibody-dependent cell-mediated cytotoxicity (ADCC): The in vitro ADCC assay of the exemplified antibodies was evaluated using a reporter gene-based ADCC assay. CHO cells expressing human CD33 and human CD20 were used as target cell lines, and Jurkat cells (Eli Lilly and Company) expressing functional FcγRIIIa (V158)-NFAT-Luc were used as effector cell lines. All test antibodies and cells were diluted in assay medium containing RPMI-1640 (without phenol red) supplemented with 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 2 mM L-glutamine, 500 U / mL penicillin-streptomycin, and 0.1% w / v BSA. The test antibodies were first diluted to a 3-fold concentration of 3.3 μg / mL and then serially diluted 7-fold at a ratio of 1:4. 50 μL / well of each antibody was aliquoted in duplicate into a white opaque-bottom 96-well plate (Costar, #3917). A CD20 antibody was used as a positive control. Then, the CHO target cells were 5×10 in 50 μL aliquots 4Added to the plate in cells / wells and incubated at 37°C for 1 hour. Next, Jurkat V158 cells were added to the wells at 150,000 cells / well in 50 μL aliquots, incubated at 37°C for 4 hours, and then 100 μL / well of One-Glo luciferase substrate (Promega, #E8130) was added. The contents of the plate were mixed using a plate shaker at low speed, incubated at room temperature for 5 minutes, and the luminescence signal was read using a BioTek microplate reader (BioTek Instruments) with an integration of 0.2 cps. The data was analyzed using GraphPad Prism9, and the relative luminescence units (RLU) for each antibody concentration were plotted in a scatter format of antibody concentration vs. RLU.

[0213] The exemplified anti-human CD33 antibody having an effector null IgG1AAA backbone did not induce ADCC activity (results not shown), suggesting that the exemplified antibodies have a low potential to deplete CD33-expressing cells via ADCC-mediated killing.

[0214] Example 5. Biophysical properties The biophysical properties of the exemplified anti-human CD33 antibodies were evaluated for developability.

[0215] Aggregation from cell culture: The exemplified anti-human CD33 antibodies were transiently expressed in CHO cells. The antibody titers and the percentage of high molecular weight (%HMW) species after protein A affinity chromatography purification are shown in Table 7, indicating that Ab1, Ab2, and Ab3 have low aggregation and provide a favorable developability profile.

[0216] Viscosity: The exemplified anti-human CD33 antibodies Ab1, Ab2, Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b were concentrated to about 125 mg / mL in a 5 mM histidine matrix at pH 6. Viscosity for each was measured at 15 °C using a VROC® initium (RheoSense), and the average of nine replicate measurements was used. The results in Table 7 show that the exemplified anti-human CD33 antibodies Ab1, Ab2, Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b have good viscosity profiles with respect to developability.

[0217] Thermal stability: Differential Scanning Calorimetry (DSC) was used to evaluate the stability of the exemplified anti-human CD33 antibodies Ab1, Ab2, Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b against thermal denaturation. The results in Table 7 show that the exemplified anti-human CD33 antibodies Ab1, Ab2, Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b have acceptable thermal melting temperatures in PBS, pH 7.2 with respect to developability.

[0218] Aggregation upon temperature stress: The solution stability over time of the exemplified anti-human CD33 antibodies Ab2, Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b was evaluated at about 100 mg / mL in a common 5 mM histidine pH 6.0 buffer. Concentrated samples were incubated at 5 °C and 35 °C for a period of 4 weeks each. After incubation, samples were analyzed for the percentage of high molecular weight (%HMW) species using size exclusion chromatography (SEC). The results in Table 7 show that the exemplified anti-human CD33 antibodies Ab2, Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b had good stability under thermal stress.

[0219] [Table 13] * ND = not determined

[0220] Example 6. Immunogenicity Evaluation MS Serum Protein Binding: Off-target binding of the exemplified anti-human CD33 antibodies to serum proteins was evaluated. The exemplified anti-human CD33 antibodies Ab1, Ab2, and Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b were coated onto Maxisorp microplates. The plates were blocked, human serum was added to the wells, and the plates were incubated overnight. The bound proteins were eluted, reduced, alkylated, and digested. The peptides were analyzed by mass spectrometer. Identification of peptides and proteins was generated by an in-house proteomics pipeline using a search algorithm with trypsin enzyme and a human database supplemented with the sequence of the test antibody. Ions were quantified by in-house proteomics tools (Chrom-Alignment, Metaconsense and Quant) and analyzed in JMP using one-way ANOVA / comparison means / all pairs, Tukey HSD. Proteins with >30% of ions having a P value <0.05 and FC>2 (compared to isotype control) were considered enriched in the study.

[0221] The results in Table 8 show that the exemplified anti-human CD33 antibodies Ab2 and Ab3, and the anti-human CD33 Ab2 GC conjugate of Example 1b have significantly reduced association with serum proteins when compared to Ab1, and thus potentially have a lower immunogenic risk than Ab1.

[0222] [Table 14]

[0223] Example 7: In Vivo Characterization Preclinical contact hypersensitivity model: The exemplified anti-human CD33 Ab2 GC conjugate and anti-human CD33 antibody Ab2 of Example 1b were evaluated in vivo using a humanized mouse model of contact hypersensitivity (HuNOG-EXL). Ab1 was similarly evaluated in another study. Administration of oxazolone (an inducer of allergic contact dermatitis) to HuNOG-EXL induces a local pro-inflammatory cytokine response and dermatitis. This enables investigation of the anti-inflammatory and immunomodulatory effects of the exemplified molecules in vivo.

[0224] Briefly, huNOG-EXL mice were transplanted with human CD34+ hematopoietic stem cells isolated from human umbilical cord blood at 6 weeks of age. Twenty to twenty-four weeks after stem cell administration, the mice were evaluated for human CD45 engraftment (blood > 25%) and subjected to an oxazolone-induced contact hypersensitivity protocol. On day 0, the mice were grouped by body weight (N = 7 - 8 / group) and administered subcutaneously with the anti-human CD33 Ab2 GC conjugate of Example 1b at 10, 1, and 0.1 mg / kg, or 10 mg / kg of anti-human CD33 Ab1 or Ab2, or an unconjugated isotype control. Twenty-four hours after administration (day 1), the mice were anesthetized with 5% isoflurane, their abdomens were shaved, and sensitized with 100 μL of 3% oxazolone in ethanol (applied to the shaved area). The mice were re-administered as above on days 4, 11, and 16, and 48 hours after each administration (day 6 - challenge 1, day 13 - challenge 2, and day 18 - challenge 3), 2% oxazolone in ethanol was loaded onto both ears (10 μL / side / ear). In the GC1 alone group, mice were administered 3 mg / kg SC 1 hour before sensitization and before each challenge. At the final challenge, the mice were euthanized, and ear tissue and gastrocnemius muscle were collected for analysis of target-selective and non-target-selective tissue expression of GC regulatory genes.

[0225] The results in Table 9a and Figures 4A - 4C show that the anti - human CD33 Ab2 GC conjugate of Example 1b significantly suppressed oxazolone - induced skin inflammation in vivo at all three doses. The results in Table 9a further show that the anti - human CD33 Ab2 GC conjugate of Example 1b suppressed oxazolone - induced skin inflammation by 57% at 10 mg / kg and 34% at 1 mg / kg at dose 3, compared to the unconjugated Ab2 at 10 mg / kg. In another study, the effect of Ab1 also showed no statistical difference from the isotype control (data not shown).

[0226] The results in Table 9b show that the anti - human CD33 Ab2 GC conjugate of Example 1b regulated target tissue - specific (ear) glucocorticoid receptor agonist - mediated FKBP5 gene expression, but did not regulate non - target tissue (gastrocnemius muscle) FKBP5 gene expression. In contrast, mice treated with systemic high - dose glucocorticoid (GC1) showed glucocorticoid agonist receptor - mediated gene induction in both target and non - target tissues.

[0227] Collectively, these data show that the exemplified anti - human CD33 Ab2 GC conjugate of Example 1b specifically delivered glucocorticoids to target cells and then regulated tissue - specific GC agonist receptor - mediated responses.

[0228] [Table 15] * p < 0.0002 vs isotype; ANOVA Tukey; ^p < 0.005 vs Ab2; ANOVA Tukey

[0229] [Table 16]

[0230] Sequence Listing Ab1 HCDR1 (North) for Sequence Numbers 1 Ab1, Ab2, and Ab3 AASGFTFSSYAMS

[0231] HCDR2 (North) for Sequence Numbers 2 Ab1, Ab2, and Ab3 AISGSGGSTY

[0232] HDCR3 (North) for Sequence Number 17 Ab1 AREYSNYDY

[0233] LCDR1 (North) for Sequence Number 18 Ab1 QASQDVFRYLN

[0234] LCDR2 (North) for Sequence Numbers 5 Ab1, Ab2, and Ab3 YDASNLQT

[0235] LCDR3 (North) for Sequence Numbers 6 Ab1, Ab2, and Ab3 QQYEDLPT

[0236] VH for Sequence Number 19 Ab1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYSNYDYWGQGTLVTVSS

[0237] VL for Sequence Number 20 Ab1 DIQMTQSPSSLSASVGDRVTITCQASQDVFRYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIK

[0238] HC for Sequence Number 21 Ab1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYSNYDYWGQGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0239] LC for SEQ ID NO:22 Ab1 DIQMTQSPSSLSASVGDRVTITCQASQDVFRYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0240] HC DNA for SEQ ID NO:23 Ab1

[0241] LC DNA for Array No. 24 Ab1 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACGTTTTCAGGTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAGGATCTCCCTACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0242] Ab2 HCDR1 (North) for Array No. 1 Ab1, Ab2, and Ab3 AASGFTFSSYAMS

[0243] HCDR2 (North) for Array No. 2 Ab1, Ab2, and Ab3 AISGSGGSTY

[0244] HDCR3 (North) for Array No. 3 Ab2 and Ab3 ARTYSNYDY

[0245] LCDR1 (North) for SEQ ID NO:4 Ab2 QASQPTFNYLN

[0246] LCDR2 (North) for SEQ ID NO:5 Ab1, Ab2, and Ab3 YDASNLQT

[0247] LCDR3 (North) for SEQ ID NO:6 Ab1, Ab2, and Ab3 QQYEDLPT

[0248] VH for SEQ ID NO:7 Ab2 and Ab3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSS

[0249] VL for SEQ ID NO:8 Ab2 DIQMTQSPSSLSASVGDRVTITCQASQPTFNYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIK

[0250] HC for SEQ ID NO:9 Ab2 and Ab3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0251] LC for SEQ ID NO:10 Ab2 DIQMTQSPSSLSASVGDRVTITCQASQPTFNYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0252] HC DNA for SEQ ID NO:11 Ab2 and Ab3

[0253] LC DNA for Array No. 12 Ab2 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGCCGACGTTCAATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAGGATCTCCCTACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0254] Ab3 HCDR1 (North) for Array No. 1 Ab1, Ab2, and Ab3 AASGFTFSSYAMS

[0255] HCDR2 (North) for Array No. 2 Ab1, Ab2, and Ab3 AISGSGGSTY

[0256] HDCR3 (North) for Array No. 3 Ab2 and Ab3 ARTYSNYDY

[0257] LCDR1 (North) for SEQ ID NO: 13 Ab3 QASQPTYIYLN

[0258] LCDR2 (North) for SEQ ID NO: 5 Ab1, Ab2, and Ab3 YDASNLQT

[0259] LCDR3 (North) for SEQ ID NO: 6 Ab1, Ab2, and Ab3 QQYEDLPT

[0260] VH for SEQ ID NO: 7 Ab2 and Ab3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSS

[0261] VL for SEQ ID NO: 14 Ab3 DIQMTQSPSSLSASVGDRVTITCQASQPTYIYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIK

[0262] HC for SEQ ID NO: 9 Ab2 and Ab3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0263] LC for SEQ ID NO:15 Ab3 DIQMTQSPSSLSASVGDRVTITCQASQPTYIYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0264] HC DNA for SEQ ID NO:11 Ab2 and Ab3

[0265] LC DNA for Array No. 16 Ab3 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGCCTACGTATATCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAGGATCTCCCTACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0266] Array No. 25 Human CD33 MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0267] Sequence number 26 LCDR1 consensus sequence QASQPTXaa7Xaa8IYLN In the formula, Xaa7 is phenylalanine or tyrosine, and Xaa8 is asparagine or isoleucine

[0268] Sequence number 27 Cynomolgus CD33 MPLLLLPLLWAGALAMDPRVRLEVQESVTVQEGLCVLVPCTFFHPVPYHTRNSPVHGYWFREGAIVSLDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMEKGSTKYSYKSTQLSVHVTDLTHRPQILIPGALDPDHSKNLTCSVPWACEQGTPPIFSWMSAAPTSLGLRTTHSSVLIITPRPQDHGTNLTCQVKFPGAGVTTERTIQLNVSYASQNPRTDIFLGEGSGRKARKQGVVQGAIGGAGVTVLLALCLCLIFFTVKTHRRKAARTAVGRIDTHPATGPTSSKHQKKSKLHGATETSGCSGTTLTVEMDEELHYASLNFHGMNPSEDTSTEYSEVRTQ

[0269] Accession No. 28 Human CD33 Extracellular Domain DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVT

[0270] Accession No. 29 Cynomolgus Macaque CD33 Extracellular Domain DPRVRLEVQESVTVQEGLCVLVPCTFFHPVPYHTRNSPVHGYWFREGAIVSLDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMEKGSTKYSYKSTQLSVHVTDLTHRPQILIPGALDPDHSKNLTCSVPWACEQGTPPIFSWMSAAPTSLGLRTTHSSVLIITPRPQDHGTNLTCQVKFPGAGVTTERTIQLNVS

Claims

1. An antibody that binds to human CD33, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, wherein HCDR2 comprises SEQ ID NO: 2, wherein HCDR3 comprises SEQ ID NO: 3, wherein LCDR1 comprises SEQ ID NO: 4, wherein LCDR2 comprises SEQ ID NO: 5, and wherein LCDR3 comprises SEQ ID NO:

6. An antibody.

2. An antibody that binds to human CD33, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, wherein HCDR2 comprises SEQ ID NO: 2, wherein HCDR3 comprises SEQ ID NO: 3, wherein LCDR1 comprises SEQ ID NO: 13, wherein LCDR2 comprises SEQ ID NO: 5, and wherein LCDR3 comprises SEQ ID NO:

6. An antibody.

3. An antibody that binds to human CD33, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, wherein HCDR2 comprises SEQ ID NO: 2, wherein HCDR3 comprises SEQ ID NO: 3, wherein LCDR1 comprises SEQ ID NO: 26, wherein LCDR2 comprises SEQ ID NO: 5, and wherein LCDR3 comprises SEQ ID NO:

6. An antibody.

4. The antibody according to any one of claims 1 to 3, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO: 8 or 14.

5. The antibody according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprises SEQ ID NO: 9, and the LC comprises SEQ ID NO: 10 or 15.

6. An antibody that binds to human CD33, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 17, the LCDR1 comprises SEQ ID NO: 18, the LCDR2 comprises SEQ ID NO: 5, the LCDR3 comprises SEQ ID NO: 6, an antibody.

7. The antibody according to claim 6, wherein the VH comprises SEQ ID NO: 19 and the VL comprises SEQ ID NO:

20.

8. The antibody according to claim 6 or 7, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprises SEQ ID NO: 21, and the LC comprises SEQ ID NO:

22.

9. The antibody comprises a light chain and a heavy chain, and the heavy chain contains a cysteine at amino acid residue 124 (EU numbering), contains a cysteine at amino acid residue 378 (EU numbering), or contains a cysteine at amino acid residue 124 (EU numbering) and a cysteine at amino acid residue 378 (EU numbering), the antibody according to any one of claims 1 to 4 or 6 to 7.

10. The antibody according to any one of claims 1 to 4 or 6 to 7, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), and the HC is of human IgG1 isotype.

11. The antibody according to any one of claims 1 to 10, wherein the antibody is an endogenous antibody.

12. The antibody according to any one of claims 1 to 10, wherein the antibody is a non-depleting antibody.

13. The antibody according to any one of claims 1 to 10, wherein the antibody is a non-degradable antibody.

14. A nucleic acid comprising a sequence encoding SEQ ID NO: 9, 10, 15, 21, or 22.

15. A vector comprising the nucleic acid according to claim 14.

16. The vector according to claim 15, comprising a first nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22.

17. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9 or 21 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 10, 15, or 22.

18. A cell comprising the vector according to claim 15 or 16.

19. The cell according to claim 18, wherein the cell is a mammalian cell.

20. A process for producing an antibody, comprising culturing the cell according to claim 18 or 19 under conditions such that the antibody is expressed, and recovering the antibody expressed from the culture medium.

21. An antibody produced by the process according to claim 20.

22. A conjugate comprising the antibody according to any one of claims 1 to 13 or claim 21 conjugated to a therapeutic agent.

23. The conjugate according to claim 22, wherein the therapeutic agent comprises a cytotoxic agent, siRNA, saRNA, peptide, oligonucleotide, small molecule, nanoparticle, lipid nanoparticle, exosome, antibody or antigen-binding fragment thereof, or a combination thereof.

24. A conjugate of the following formula: 【Chemical 1】 wherein Ab is an antibody that binds to human CD33; wherein the structure: [Chemical Formula 2] is the structure: 【Chemical Formula 3】 and n is from 1 to 5.

25. Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3 or 17, the LCDR1 comprises SEQ ID NO: 4, 13, 18, or 26, the LCDR2 comprises SEQ ID NO: 5, the LCDR3 comprises SEQ ID NO:

6.

26. The structure: 【Chemical Formula 4】 is the structure: [Chemical Formula 5] The conjugate according to claim 24.

27. The structure: 【Chemical Formula 6】 is the structure: 【Chemical Formula 7】 The conjugate according to claim 24.

28. The structure: [Chemical Formula 8] is the structure: 【Chemical Formula 9】 The conjugate according to claim 24 or 26.

29. The structure: 【Chemical Formula 10】 is the structure: 【Chemical 11】 The conjugate according to claim 24 or 27.

30. The structure: 【Chemical 12】 is the structure: 【Chemical 13】 The conjugate according to claim 24 or 26.

31. The structure: 【Chemical 14】 is the structure: 【Chemical Formula 15】 The conjugate according to claim 24 or 26.

32. The structure: 【Chemical 16】 is the structure: 【Chemical 17】 The conjugate according to claim 24 or 27.

33. The structure: 【Chemical 18】 is the structure: 【Chemical Formula 19】 The conjugate according to claim 24 or 27, wherein...

34. wherein said Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), said VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and said VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein said HCDR1 comprises SEQ ID NO: 1, wherein said HCDR2 comprises SEQ ID NO: 2, wherein said HCDR3 comprises SEQ ID NO: 3, wherein said LCDR1 comprises SEQ ID NO: 4, wherein said LCDR2 comprises SEQ ID NO: 5, wherein said LCDR3 comprises SEQ ID NO: 6, and the conjugate according to any one of claims 24 to 33.

35. The conjugate according to claim 34, wherein said VH comprises SEQ ID NO: 7 and said VL comprises SEQ ID NO:

8.

36. The conjugate according to any one of claims 34 to 25, wherein said Ab comprises a heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO:

10.

37. wherein said Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), said VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and said VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein said HCDR1 comprises SEQ ID NO: 1, wherein said HCDR2 comprises SEQ ID NO: 2, wherein said HCDR3 comprises SEQ ID NO: 3, wherein said LCDR1 comprises SEQ ID NO: 13, wherein said LCDR2 comprises SEQ ID NO: 5, wherein said LCDR3 comprises SEQ ID NO: 6, and the conjugate according to any one of claims 24 to 33.

38. The conjugate according to claim 37, wherein said VH comprises SEQ ID NO: 7 and said VL comprises SEQ ID NO:

14.

39. The conjugate according to claim 37 or 38, wherein said Ab comprises a heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO:

15.

40. wherein said Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), said VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and said VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein said HCDR1 comprises SEQ ID NO: 1, wherein said HCDR2 comprises SEQ ID NO: 2, wherein said HCDR3 comprises SEQ ID NO: 17, wherein said LCDR1 comprises SEQ ID NO: 18, wherein said LCDR2 comprises SEQ ID NO: 5, The conjugate according to any one of claims 24 to 33, wherein the LCDR3 contains SEQ ID NO:

6.

41. The conjugate according to claim 40, wherein the VH contains SEQ ID NO: 19 and the VL contains SEQ ID NO:

20.

42. The conjugate according to claim 40 or 41, wherein the Ab contains a heavy chain (HC) containing SEQ ID NO: 21 and a light chain (LC) containing SEQ ID NO:

22.

43. The Ab contains a heavy chain and a light chain, and the heavy chain contains cysteine at amino acid residue 124 (EU numbering), cysteine at amino acid residue 378 (EU numbering), or cysteine at amino acid residue 124 (EU numbering) and cysteine at amino acid residue 378 (EU numbering); the conjugate according to any one of claims 24 to 35, 37 to 38, 40 to 41.

44. The conjugate according to any one of claims 24 to 35, 37 to 38, 40 to 41, wherein the Ab contains a heavy chain (HC) and a light chain (LC), and the HC is of human IgG1 isotype.

45. The conjugate according to claim 44, wherein the human IgG1 isotype is effector null.

46. The conjugate according to any one of claims 24 to 45, wherein n is 2 to 5.

47. The conjugate according to any one of claims 24 to 45, wherein n is 3 to 5.

48. The conjugate according to any one of claims 24 to 45, wherein n is 3 to 4.

49. The conjugate according to any one of claims 24 to 45, wherein n is 4.

50. The conjugate according to any one of claims 24 to 45, wherein n is 3.

51. The conjugate according to any one of claims 24 to 45, wherein n is 2.

52. A pharmaceutical composition comprising the conjugate according to any one of claims 22 to 51 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

53. A method of treating a subject in need of treatment for a bone marrow cell-related disease, the method comprising administering to the subject an effective amount of the conjugate according to any one of claims 22 to 51 or the pharmaceutical composition according to claim 52.

54. The method according to claim 53, wherein the myeloid cell-related disease is an immune disease, a neurodegenerative disease, or cancer.

55. The method according to claim 54, wherein the immune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome, or a fibrotic disease.

56. The method according to claim 55, wherein the immune disease is rheumatoid arthritis.

57. The conjugate according to any one of claims 22 to 51, for use in therapy.

58. The conjugate according to any one of claims 22 to 51, or the pharmaceutical composition according to claim 29, for use in the treatment of myeloid cell-related diseases.

59. The conjugate or pharmaceutical composition for use according to claim 58, wherein the myeloid cell-related disease is an immune disease, a neurodegenerative disease, or cancer.

60. The conjugate or pharmaceutical composition for use according to claim 59, wherein the immune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome, or a fibrotic disease.

61. The conjugate or pharmaceutical composition for use according to claim 60, wherein the immune disease is rheumatoid arthritis.

62. Use of the conjugate according to any one of claims 22 to 51, or the pharmaceutical composition according to claim 52, in the manufacture of a medicament for the treatment of myeloid cell-related diseases.

63. The use according to claim 62, wherein the myeloid cell-related disease is an immune disease, a neurodegenerative disease, or cancer.

64. The use according to claim 64, wherein the immune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia rheumatica, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome, or a fibrotic disease.

65. Use according to claim 65, wherein the immune disease is rheumatoid arthritis.

66. A method for generating a conjugate, comprising: (a) reducing an anti-human CD33 antibody with a reducing agent to produce a reduced anti-human CD33 antibody, wherein the anti-human CD33 antibody comprises one or more engineered cysteine residues; (b) oxidizing the reduced anti-human CD33 antibody with an oxidizing agent to produce an oxidized anti-human CD33 antibody; (c) contacting the oxidized anti-human CD33 antibody with a compound of the formula 【Chemical 20】 to produce the conjugate.

67. The method according to claim 66, wherein the reducing agent is dithiothreitol and the oxidizing agent is dehydroascorbic acid.

Images