Human CD33 antibody and glucocorticoid conjugates

Abstract

The present disclosure provides human CD33 antibodies and antibody glucocorticoid receptor agonist conjugates and methods of using the conjugates in the treatment of bone marrow cell related diseases.

Description

Technical Field

[0001] The present disclosure provides a human CD33 antibody and an antibody glucocorticoid receptor agonist conjugate, a method of using the conjugate to treat a bone marrow cell-related disease, a method of preparing the conjugate, and a pharmaceutical composition comprising the human CD33 antibody glucocorticoid receptor conjugate. Background Art

[0002] Anti-human CD33 antibodies and conjugates have been described previously. Many of these antibodies are myeloid cell depleting antibodies that modulate CD33-mediated responses. For example, lintuzumab is a humanized antibody that reportedly depletes myeloid cells and modulates CD33-mediated cytokine responses via ADCC and CDC activity, and was discontinued as a monotherapy for AML due to lack of efficacy; talin-vadatuximab, as a lintuzumab conjugate, was discontinued due to safety issues, and lintuzumab Ac225 is currently in Phase I / II for AML. (Perl, A., Hematology Am Soc Hematol Educ Program., Issue 1: Pages 54-65, 2017; Bothell, Wash. (BUSINESS WIRE), June 19, 2017; Abedin, S., Blood, Volume 136 Supplement 1: Pages 9-10, 2020). Other such anti-human CD33 antibody therapeutics include BI 836858, which did not show clinical activity in a Phase I AML study but is currently being evaluated in combination with other compounds in the treatment of acute myeloid leukemia (AML); IMGN779, which is currently in Phase I trials for AML; and gemtuzumab ozogamicin, which is currently the only anti-human CD33 antibody conjugate approved for the treatment of AML. (Vasu S et al., Haematologica., Vol. 107, No. 3: pp. 770-773, 2022; Mol. Cancer Ther., Vol. 17, No. 6: pp. 1271-1279, 2018). AL003 is a humanized CD33 antibody that reportedly modulates CD33-mediated responses and is in Phase II trials for the treatment of Alzheimer's disease. (Alector, GlobeNewswire, November 10, 2021).

[0003] WO2017 / 210471 discloses certain glucocorticoid receptor agonists (GC) and immunoconjugates thereof that can be used to treat inflammatory diseases. WO2018 / 089373 discloses novel steroids, protein conjugates thereof, and methods for treating diseases, disorders, and conditions, the methods comprising administering steroids and conjugates. To date, no human CD33 GC conjugates have been 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 consume bone marrow cells ("non-depleting anti-human CD33 antibodies"), are internalized into bone marrow cells, do not modulate CD33-mediated responses (e.g., cytokine expression, inflammatory responses), and do not significantly degrade CD33 ("non-degrading anti-human CD33 antibodies"). The present disclosure also provides compositions comprising such anti-human CD33 antibodies and methods of using such anti-human CD33 antibodies and compositions. Such anti-human CD33 antibodies can be conjugated with therapeutic agents (e.g., inflammatory agents, glucocorticoids, cytotoxic agents, siRNA, saRNA, peptides, small molecules, antibodies, and binding fragments thereof) for targeted delivery of the therapeutic agent to bone marrow cells expressing human CD33 to treat bone marrow cell-related diseases. In addition, certain anti-human CD33 antibodies of the present disclosure do not significantly affect the availability of CD33 receptors on the cell surface, and thus can provide repeated CD33-targeted delivery of therapeutic agents to bone marrow cells. The present disclosure provides certain novel fully human CD33 antibody glucocorticoid (GC) conjugates, wherein the antibody binds to human CD33. The present disclosure also provides compositions comprising novel anti-human CD33 antibody GC conjugates and methods of using such anti-human CD33 antibody GC conjugates and compositions thereof. The present disclosure also provides certain novel anti-human CD33 GC conjugates, which can be used to treat rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, atopic dermatitis, psoriasis, inflammatory bowel disease, multiple sclerosis, Sjögren's syndrome, fibrotic diseases (such as scleroderma) and macrophage activation syndrome. Thus, certain 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 a desired binding affinity and / or association and dissociation rates, 2) are internalized into bone marrow cells after binding to CD33, 3) do not deplete bone marrow 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 CD33 cell surface receptors on bone marrow cells, 7) modulate glucocorticoids in vitro The invention relates to an agent that has the following properties: 1) a cytokine response mediated by a glucocorticoid receptor agonist (e.g., inhibition of IL-6 and TNFα), 2) a target-specific glucocorticoid receptor agonist-mediated response in vivo (e.g., inhibition of target site-specific tissue inflammation and gene expression, such as FKBP5), 3) a low immunogenicity risk, 4) an inhibition of plasmacytoid dendritic cell differentiation and IFNα production in vitro, and / or 5) good developability properties, such as acceptable stability, solubility, viscosity, low aggregation, hydrophobicity, and / or good pharmacokinetic properties to facilitate development, manufacturing and / or formulation.

[0005] Thus, in some embodiments, antibodies that bind to human CD33 ("anti-human CD33 antibodies") are provided, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDR1 comprises SEQ ID NO: 4, the LCDR2 comprises SEQ ID NO: 5, and the LCDR3 comprises 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, wherein the antibody comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDR1 comprises SEQ ID NO: 13, the LCDR2 comprises SEQ ID NO: 5, and the LCDR3 comprises SEQ ID NO: 6. In some embodiments, an anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 14. In some embodiments, an anti-human CD33 antibody comprises a HC comprising SEQ ID NO: 9 and a LC comprising SEQ ID NO: 15.

[0007] In some embodiments, an antibody that binds to human CD33 is provided, wherein the antibody comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDR1 comprises SEQ ID NO: 26, the LCDR2 comprises SEQ ID NO: 5, and the LCDR3 comprises 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 other embodiments, the anti-human CD33 antibody comprises a HC comprising SEQ ID NO: 9 and a LC comprising SEQ ID NO: 10 or 15.

[0008] In some embodiments, an antibody that binds to human CD33 is provided, wherein the antibody comprises a VH and a VL, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein 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, and the LCDR3 comprises SEQ ID NO: 6. In some embodiments, an anti-human CD33 antibody comprises a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20. In some embodiments, an anti-human CD33 antibody comprises a HC comprising SEQ ID NO: 21 and a 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 internalizing antibody. In other embodiments, the anti-human CD33 antibody has a human IgG1 or human IgG4 isotype.

[0010] In other embodiments, the anti-human CD33 antibody has a human IgG1 Fc region comprising a modification of L234A, L235A and P329A (EU numbering) (also referred to as IgG1AAA), which reduces or eliminates binding to Fcγ and C1q receptors. In such embodiments, the anti-human CD33 antibody with an IgG1AAA modified Fc region has reduced or eliminated Fc effector function activity, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (all residues are numbered according to EU numbering). Such antibodies are referred to as "IgG1-effector null" antibodies. In such embodiments, the anti-human CD33 antibody with an IgG1AAA modified Fc region does not consume CD33-expressing cells (e.g., bone marrow cells). In such embodiments, the anti-human CD33 antibody is a non-degrading antibody. In other embodiments, when compared to IgG1, the anti-human CD33 antibody with an IgG1AAA skeleton significantly reduces and / or eliminates the degradation of the CD33 receptor. In such embodiments, the anti-human CD33 antibody is a non-degrading antibody.

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

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

[0013] In some embodiments, antibody fragments (e.g., Fab or scFv) that bind to human CD33 are provided, wherein the antibody fragments comprise VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein 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, and the LCDR3 comprises SEQ ID NO: 6. In some embodiments, an 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 comprising an engineered cysteine ​​residue for generating an antibody conjugate compound (also referred to as a bioconjugate) (see WO 2018 / 232088 A1). More particularly, in such embodiments, the anti-human CD33 antibody comprises a cysteine ​​at amino acid residue 124 (EU numbering) or a cysteine ​​at amino acid residue 378 (EU numbering); or a cysteine ​​at amino acid residue 124 (EU numbering) and a cysteine ​​at amino acid residue 378 (EU numbering).

[0015] In some embodiments, the present disclosure provides nucleic acids encoding HC or LC, or VH or VL of novel antibodies that bind to anti-human CD33, or vectors comprising such nucleic acids.

[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, nucleic acids encoding heavy or light chains of antibodies that bind to anti-human CD33 are provided. In some embodiments, nucleic acids comprising sequences encoding SEQ ID NO: 9, 10, 15, 21, or 22 are provided. In some embodiments, nucleic acids comprising sequences encoding heavy chains of antibodies comprising SEQ ID NO: 9 or 21 are provided. For example, the nucleic acid may comprise a sequence of SEQ ID NO: 11 or 23. In some embodiments, nucleic acids comprising sequences encoding light chains of antibodies comprising SEQ ID NO: 10, 15, or 22 are provided. For example, the nucleic acid may comprise a sequence of SEQ ID NO: 12, 16, or 24.

[0018] In some embodiments of the present disclosure, nucleic acids encoding VH or VL of anti-human CD33 antibodies are provided. In some embodiments, nucleic acids comprising sequences encoding SEQ ID NO: 7, 8, 14, 19 or 20 are provided. In some embodiments, nucleic acids comprising sequences encoding antibody VH comprising SEQ ID NO: 7 or 19 are provided. In some embodiments, nucleic acids comprising sequences encoding antibody VL comprising SEQ ID NO: 8, 14 or 20 are provided.

[0019] Some embodiments of the present disclosure provide vectors comprising a nucleic acid sequence encoding an antibody heavy chain or light chain. For example, such vectors may 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 vectors may 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 is a vector 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 is a composition comprising a first vector containing a nucleic acid sequence encoding an antibody heavy chain and a second vector containing a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a first vector containing 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 containing a nucleic acid sequence encoding SEQ ID NO: 9 and a second vector containing a nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO: 9 and a second vector containing a nucleic acid sequence encoding SEQ ID NO: 15. In some embodiments, the composition comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO: 21 and a second vector containing 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] Nucleic acids of the present disclosure can be expressed in host cells, for example, after the nucleic acids have been operably connected to expression control sequences. Expression control sequences capable of expressing nucleic acids operably connected thereto are well known in the art. Expression vectors may include sequences encoding one or more signal peptides that promote secretion of polypeptides from host cells. Expression vectors containing nucleic acids of interest (for example, nucleic acids encoding heavy or light chains of antibodies) can be transferred to host cells by well-known methods (for example, stable or transient transfection, transformation, transduction or infection). In addition, the expression vector may include one or more selection markers, such as tetracycline, neomycin and dihydrofolate reductase, to help detect 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 stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing 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 expressing the HC and LC polypeptides of an antibody disclosed herein. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing the HC polypeptide of an antibody described herein and a second vector expressing the LC polypeptide of an antibody described herein. Such host cells (e.g., mammalian host cells) can express antibodies that bind to anti-human CD33 as described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0026] In some embodiments, a cell (eg, 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 (eg, 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, the cell (e.g., 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, the cell (e.g., 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, the cell (e.g., 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, the cell (e.g., 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 also provides methods for producing antibodies that bind to human CD33 as described herein by culturing the above-described host cells, such as mammalian host cells, under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium. The culture medium in which the antibody has been secreted can be purified by conventional techniques. Various protein purification methods can be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0030] The present disclosure also provides antibodies or antigen-binding fragments thereof produced by any of the methods described herein.

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

[0032] Considering that the anti-human CD33 antibodies described herein do not consume bone marrow cells, they provide advantages over bone marrow cell-depleting antibodies in treating bone marrow cell-related immune diseases, such as avoiding complications such as problematic concurrent immunodeficiency, long-term immunosuppression, etc. caused by bone marrow cell consumption, and enhancing / inducing the immunomodulatory function of bone marrow cells expressing CD33. In addition, as shown below, the anti-human CD33 antibodies described herein are internalized into bone marrow cells. Therefore, the anti-human CD33 antibodies described herein can be conjugated with a therapeutic agent for targeted delivery of the therapeutic agent to human bone marrow cells to induce immunomodulation or other therapeutic effects through the therapeutic agent to treat bone marrow cell-related diseases. In addition, the anti-human CD33 antibodies described herein do not significantly degrade cell surface or intracellular CD33, nor do they significantly affect the availability of CD33 receptors on the cell surface, and therefore, the anti-human CD33 antibodies described herein can allow repeated CD33-targeted delivery of therapeutic agents to bone marrow cells to treat bone marrow cell-related diseases.

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

[0034] Thus, in one embodiment, the invention provides a conjugate of formula I:

[0035]

[0036] wherein Ab is an antibody that binds to human CD33 ("anti-human CD33 antibody"), wherein

[0037] for:

[0038]

[0039] And n is 1 to 5.

[0040] In another embodiment, the present invention provides a conjugate of formula I:

[0041]

[0042] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein the VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0043] The HCDR1 comprises SEQ ID NO: 1;

[0044] The HCDR2 comprises SEQ ID NO: 2;

[0045] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0046] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0047] The LCDR2 comprises SEQ ID NO:5; and

[0048] The LCDR3 comprises SEQ ID NO: 6;

[0049] in

[0050] for:

[0051]

[0052] And n is 1 to 5.

[0053] In another embodiment, the present invention provides a conjugate of formula I:

[0054]

[0055] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein 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:

[0056] The HCDR1 comprises SEQ ID NO: 1;

[0057] The HCDR2 comprises SEQ ID NO: 2;

[0058] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0059] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0060] The LCDR2 comprises SEQ ID NO:5; and

[0061] The LCDR3 comprises SEQ ID NO: 6;

[0062] in

[0063] for:

[0064]

[0065] And n is 1 to 5.

[0066] In another embodiment, the present invention provides a conjugate of formula I:

[0067]

[0068] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein 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:

[0069] The HCDR1 comprises SEQ ID NO: 1;

[0070] The HCDR2 comprises SEQ ID NO: 2;

[0071] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0072] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0073] The LCDR2 comprises SEQ ID NO:5; and

[0074] The LCDR3 comprises SEQ ID NO: 6;

[0075] in

[0076] for:

[0077]

[0078]

[0079] And n is 1 to 5.

[0080] In another embodiment, the present invention provides a conjugate of formula I:

[0081]

[0082] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein 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:

[0083] The HCDR1 comprises SEQ ID NO: 1;

[0084] The HCDR2 comprises SEQ ID NO: 2;

[0085] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0086] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0087] The LCDR2 comprises SEQ ID NO:5; and

[0088] The LCDR3 comprises SEQ ID NO: 6;

[0089] in

[0090] for:

[0091]

[0092]

[0093] And n is 1 to 5.

[0094] In another embodiment, the present invention provides a conjugate of formula I:

[0095]

[0096] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein 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:

[0097] The HCDR1 comprises SEQ ID NO: 1;

[0098] The HCDR2 comprises SEQ ID NO: 2;

[0099] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0100] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0101] The LCDR2 comprises SEQ ID NO:5; and

[0102] The LCDR3 comprises SEQ ID NO: 6;

[0103] in

[0104] for:

[0105]

[0106]

[0107] And n is 1 to 5.

[0108] In another embodiment, the present disclosure provides a conjugate of Formula I:

[0109]

[0110] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein 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:

[0111] The HCDR1 comprises SEQ ID NO: 1;

[0112] The HCDR2 comprises SEQ ID NO: 2;

[0113] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0114] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0115] The LCDR2 comprises SEQ ID NO:5; and

[0116] The LCDR3 comprises SEQ ID NO: 6;

[0117] in

[0118] for:

[0119]

[0120] And n is 1 to 5.

[0121] In another embodiment, the present disclosure provides a conjugate of Formula I:

[0122]

[0123] Wherein Ab is an anti-human CD33 antibody, wherein Ab comprises VH and VL, wherein 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:

[0124] The HCDR1 comprises SEQ ID NO: 1;

[0125] The HCDR2 comprises SEQ ID NO: 2;

[0126] The HCDR3 comprises SEQ ID NO: 3 or 17;

[0127] The LCDR1 comprises SEQ ID NO: 4, 13, 18 or 26;

[0128] The LCDR2 comprises SEQ ID NO:5; and

[0129] The LCDR3 comprises SEQ ID NO: 6;

[0130] in

[0131] for:

[0132]

[0133] And n is 1 to 5.

[0134] In one embodiment, n is 2-5.

[0135] In one embodiment, n is 3-5.

[0136] In one embodiment, n is 3-4.

[0137] In one embodiment, n is 4.

[0138] In one embodiment, n is 3.

[0139] In one embodiment, n is 2.

[0140] In some embodiments, Ab in the conjugate of Formula 1 comprises VH and VL, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDR1 comprises SEQ ID NO: 4, the LCDR2 comprises SEQ ID NO: 5, and the 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, wherein Ab2 comprises a HC comprising SEQ ID NO: 9 and a LC comprising SEQ ID NO: 10.

[0141] In some embodiments, Ab in the conjugate of Formula 1 comprises VH and VL, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDR1 comprises SEQ ID NO: 13, the LCDR2 comprises SEQ ID NO: 5, and the 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, wherein Ab3 comprises a HC comprising SEQ ID NO: 9 and a LC comprising SEQ ID NO: 15.

[0142] In some embodiments, Ab in the conjugate of Formula 1 comprises VH and VL, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein 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, and the LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab comprises a VH comprising SEQ ID NO: 19 and a VL comprising SEQ ID NO: 20. In some embodiments, Ab is Abl, wherein Abl comprises a HC comprising SEQ ID NO: 21 and a LC comprising SEQ ID NO: 22.

[0143] As used herein, "GC" in the formula:

[0144]

[0145] refers to a suitable glucocorticoid receptor agonist payload, wherein GC is one of the following Formulae IIa, IIb or IIc:

[0146]

[0147]

[0148] As used herein, the "L" in the formula

[0149]

[0150] Refers to a suitable linker group that connects Ab to GC. Suitable linkers known to those of ordinary skill in the art include, for example, cleavable and non-cleavable linkers. More specifically, a suitable linker "L" is one of the following formulas IIIa to IIIf:

[0151]

[0152]

[0153] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IV:

[0154]

[0155] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVa:

[0156]

[0157] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVb:

[0158]

[0159] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVc:

[0160]

[0161] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVd:

[0162]

[0163] In one embodiment, the present disclosure provides a conjugate of Formula V:

[0164]

[0165] In another embodiment, the present disclosure provides a conjugate of Formula Va:

[0166]

[0167] 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.

[0168] Due to their key role in regulating immune responses, dysregulation of myeloid cells is associated with a variety of myeloid cell-related immune diseases, including autoimmune / inflammatory diseases, which are caused by abnormal activation of myeloid cells and lymphocytes (increased proinflammatory cytokines). Examples of myeloid cell-related immune diseases include rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome, and fibrotic diseases such as scleroderma.

[0169] In one embodiment, the present disclosure provides a method for treating a bone marrow cell-related disease in a subject in need thereof, 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 as disclosed herein, e.g., a conjugate of Formula I, or a pharmaceutically acceptable salt thereof. In certain embodiments, the bone marrow cell-related disease is an immune disease, a neurodegenerative disease, or a cancer. In certain embodiments, the bone marrow cell-related disease is an immune disease, such as rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile spontaneous arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome, or a fibrotic disease such as scleroderma. In one embodiment, the present disclosure also provides a method for treating rheumatoid arthritis in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure also provides a method for treating systemic lupus erythematosus in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating lupus nephritis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating cutaneous lupus in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating giant cell arteritis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating polymyalgia in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating psoriatic arthritis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating atopic dermatitis in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure also provides a method for treating psoriasis in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure also provides a method for treating ulcerative colitis in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof.In one embodiment, the present disclosure also provides a method for treating Crohn's disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating dermatomyositis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating juvenile spontaneous arthritis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating multiple sclerosis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating Sjögren's syndrome in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the present disclosure also provides a method for treating macrophage activation syndrome in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the disclosure also provides a method for treating a fibrotic disease such as scleroderma in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, bone marrow cell-related diseases are neurodegenerative diseases (e.g., Alzheimer's disease). In one embodiment, the disclosure also provides a method for treating Alzheimer's disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, bone marrow cell-related diseases are bone marrow cell-related cancers (e.g., AML). In one embodiment, the disclosure also provides a method for treating an AML in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject.

[0170] In one embodiment, the present disclosure also provides a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent as disclosed herein, such as a conjugate of Formula I, or a pharmaceutically acceptable salt thereof, for use in treatment. In one embodiment, the present disclosure provides a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent as disclosed herein, such as a conjugate of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of bone marrow cell-related diseases. In certain embodiments, the bone marrow cell-related disease is an immune disease, a neurodegenerative disease, or a cancer. In certain embodiments, the bone marrow cell-related disease is an immune disease, such as rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile spontaneous arthritis, multiple sclerosis, Sjögren'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 the treatment of rheumatoid arthritis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating systemic lupus erythematosus. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating lupus nephritis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating cutaneous lupus. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating giant cell arteritis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating polymyalgia. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating psoriatic arthritis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating atopic dermatitis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating psoriasis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating ulcerative colitis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating Crohn's disease. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating dermatomyositis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating juvenile idiopathic arthritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating multiple sclerosis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating Sjögren's syndrome.In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating slow macrophage activation syndrome. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating fibrotic diseases such as scleroderma. In some embodiments, myeloid cell-related diseases are neurodegenerative diseases (e.g., Alzheimer's disease). In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating Alzheimer's disease. In some embodiments, myeloid cell-related diseases are myeloid cell-related cancers (e.g., AML). In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating AML.

[0171] In one embodiment, the present disclosure also provides a conjugate comprising an anti-human CD33 antibody conjugated to a therapeutic agent as disclosed herein, such as a conjugate of Formula I, or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for treating a bone marrow cell-related disease. In certain embodiments, the bone marrow cell-related disease is an immune disease, a neurodegenerative disease, or a cancer. In certain embodiments, the bone marrow cell-related disease is an immune disease, such as rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile spontaneous arthritis, multiple sclerosis, Sjögren'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 manufacture of a medicament for treating rheumatoid arthritis. In one embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for treating 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 treating 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 treating 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 treating 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 treating polymyalgia. 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 treating 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 treating 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 treating 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 treating 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 treating 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 treating 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 treating 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 treating 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 treating 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 drug 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 drug 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 drug for the treatment of fibrotic diseases such as scleroderma. In some embodiments, myeloid cell-related diseases are neurodegenerative diseases (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 drug for the treatment of Alzheimer's disease. In some embodiments, myeloid cell-related diseases are myeloid cell-related cancers (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 drug for the treatment of AML.

[0172] The present disclosure provides a method for producing 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 producing a conjugate, the method comprising conjugating a compound of the present disclosure with an anti-human CD33 antibody or an antigen-binding fragment thereof.

[0173] The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IV with an anti-human CD33 antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVa with an anti-human CD33 antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVb with an anti-human CD33 antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVc with an anti-human CD33 antibody. The present disclosure provides a method for producing a conjugate, the method comprising conjugating a compound of formula IVd with an anti-human CD33 antibody.

[0174] In some embodiments, the conjugate produced is a conjugate of Formula I.

[0175] The present disclosure provides a method for producing a conjugate, the method comprising the steps of:

[0176] (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;

[0177] (b) oxidizing the reduced anti-human CD33 antibody with an oxidizing agent to produce an oxidized anti-human CD33 antibody; and

[0178] (c) contacting the oxidized anti-human CD33 antibody with a compound of the present disclosure to produce the conjugate.

[0179] The present disclosure provides a method for producing a conjugate, the method comprising the steps of:

[0180] (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;

[0181] (b) oxidizing the reduced anti-human CD33 antibody with an oxidizing agent to produce an oxidized anti-human CD33 antibody; and

[0182] (c) contacting the oxidized anti-human CD33 antibody with a compound of the formula

[0183]

[0184] to produce the conjugate.

[0185] 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.

[0186] In one embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of an anti-human CD33 antibody conjugated to a therapeutic agent as described herein, or a pharmaceutically acceptable salt thereof, or an antibody, nucleic acid or vector as described herein and one or more pharmaceutically acceptable carriers, diluents or excipients. In one embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients. In one embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of formula I and one or more pharmaceutically acceptable carriers, diluents or excipients. In one embodiment, the present disclosure also provides a method for preparing a pharmaceutical composition, the method 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 includes novel intermediates and methods for synthesizing conjugates of formula I. DETAILED DESCRIPTION

[0187] Unless otherwise indicated, the term "CD33" or "CD33 receptor" as used herein 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), which belongs to the immunoglobulin superfamily sialic acid-binding Ig-like lectin (SIGLEC) family. The term also includes naturally occurring CD33 variants, such as splice variants or allelic variants. The amino acid sequence of human CD33 is known in the art, such as the NCBI reference sequence XP_011525833.1 (SEQ ID NO: 25). The amino acid sequence of cynomolgus monkey CD33 is known in the art, such as the 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.

[0188] As used herein, the term "myeloid cell-related disease" refers to a disease associated with CD33-expressing myeloid cells. Such myeloid cell-related diseases may, for example, include immune diseases, neurodegenerative diseases, or myeloid cell-related cancers. Myeloid cell-related immune diseases may be, for example, rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile spontaneous arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome, or fibrotic diseases such as scleroderma. Myeloid cell-related neurodegenerative diseases may be, for example, Alzheimer's disease. Myeloid cell-related cancers may be, for example, acute myeloid leukemia (AML).

[0189] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. These antibodies may belong to 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" includes at least a portion of an antibody that retains the ability to interact with an antigen, such as, for example, Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, or linear antibodies, which may, for example, be fused to Fc regions or IgG heavy chain constant regions.

[0190] An exemplary antibody is an immunoglobulin G (IgG) type antibody, which is composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked by interchain 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 primarily responsible for antigen recognition. The carboxyl terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. 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 contains the Fc region and 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. IgG isotypes 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 ed., Bethesda, MD: U. S. Pat. of Health and Human Services, Public Health Service, National Institutes of Health (1991). The terms EU index number or EU number are used interchangeably herein.

[0191] VH and VL regions can be further subdivided into hypervariable regions known as complementary determining regions (CDRs), which are interspersed with more conservative regions known as framework regions (FRs). CDRs are exposed to the surface of the protein and are important regions of the antigen-binding specificity of antibodies. Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from amino-terminal to carboxyl-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this article, 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". These CDRs contain most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs can be performed 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, 274, 937-951 (1998)), and the like. Biology, 406, 228-256 (2011)) or IMGT (International Immunogenetics Database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212). The North CDR definitions are used for exemplary anti-human CD33 antibodies as described herein.

[0192] Exemplary embodiments of the antibodies of the present disclosure also include antibody fragments or antigen-binding fragments that comprise 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 fragment, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragment, or linear antibodies, which can be fused, for example, to an Fc region or an IgG heavy chain constant region.

[0193] As used herein, the term "Fc region" refers to the region of an antibody that includes the CH2 and CH3 domains of an antibody heavy chain. Optionally, the Fc region may include a portion or the entire hinge region of an antibody heavy chain. Biological activities such as effector functions may be attributed to the Fc region, which vary with 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 receptors), and B cell activation.

[0194] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. "Fcγ receptor" or "FcγR" is an FcR that binds to an IgG antibody, and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., Vol. 9: 457-492 (1991); 25 Capel et al., Immunomethods, Vol. 4: 25-34 (1994); and deHaas et al., J. Lab. Clin. Med., Vol. 126: 330-341 (1995).

[0195] 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., bone marrow cells) in a subject after treatment compared to the number of bone marrow cells before treatment. Bone marrow cell number and viability can be measured using well-known assays such as trypan blue staining and Vi-cell counters. Non-depleting antibodies generally do not induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis of bone marrow cells.

[0196] As used herein, the term "receptor availability" refers to CD33 receptor expression on the cell surface. CD33 cell surface receptor expression may refer to de novo expression of CD33 receptor on the cell surface and / or CD33 receptor recycled to the cell surface after internalization.

[0197] 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. For example, a non-degrading CD33 antibody as used herein does not significantly affect receptor availability.

[0198] As used herein, the terms "modulate," "modulate," "modulate," and the like refer to changing or altering a measurable value, and include changing or altering such a measurable value upward (ie, up-regulating) or downward (ie, down-regulating).

[0199] As used herein, unless otherwise indicated, the term "binding" refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule that brings the two proteins or molecules into proximity as determined by common methods known in the art.

[0200] The term "therapeutic agent" as used herein refers to a therapeutic composition, such as an anti-inflammatory agent, a glucocorticoid, a cytotoxic agent, siRNA, saRNA, a peptide, an oligonucleotide, a small molecule, a nanoparticle, a lipid nanoparticle, an exosome, an antibody or a fragment thereof, or a combination thereof, which can be conjugated to an anti-human CD33 antibody as disclosed herein to form a conjugate (e.g., an antibody drug conjugate). In some embodiments, such conjugates induce immunomodulatory or other therapeutic effects for the treatment of myeloid cell-related diseases by specifically targeting the CD33 receptor on myeloid cells with an anti-human CD33 antibody as disclosed herein and then delivering the therapeutic agent to the myeloid cells.

[0201] In addition, the therapeutic agent can be conjugated to an anti-human CD33 antibody as disclosed herein in a variety of ways and at different locations or portions of the antibody such that when conjugated, such linkage 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. The terms "linked" and "conjugated" used interchangeably herein refer to the association, attachment, connection, covalent attachment or linkage or otherwise combination of a first molecule or compound (e.g., an antibody or fragment thereof) and a second molecule or compound (e.g., a therapeutic agent as described herein).

[0202] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, including molecules containing single-stranded and / or double-stranded nucleotides, such as DNA, cDNA and RNA molecules, incorporating natural, modified and / or analog nucleotides. The polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or synthesis reactions.

[0203] 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, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, or more. As described herein, the drug moiety can be conjugated to the polypeptide at one or more sites in the polypeptide. In certain embodiments, the conjugate has an average drug to antibody ratio (DAR) (molar ratio) in the range of 2 to 5, or 3 to 5, or 3 to 4. In certain embodiments, the conjugate has an average DAR of 3 to 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.

[0204] As used herein, those skilled in the art will also understand that the conjugate of Formula I may also be referred to as an anti-human CD33 antibody glucocorticoid conjugate ("anti-human CD33 Ab GC conjugate").

[0205] The anti-human CD33 conjugates of the present disclosure, such as anti-human CD33 antibody GC conjugates, can be formulated as pharmaceutical compositions that are administered by any route that makes the conjugate bioavailable, including, for example, intravenous or subcutaneous administration. Such pharmaceutical compositions can be prepared using techniques and methods known in the art (see, for example, Remington: The Science and Practice of Pharmacy, edited by A. Adejare, 23rd edition, published in 2020, Elsevier Science).

[0206] As used herein, the terms "treating", "treatment" or "to treat" include inhibiting, slowing down, stopping, controlling, delaying or reversing the progression or severity of existing symptoms or disorders, or improving existing symptoms or disorders, but do not necessarily mean completely eliminating existing symptoms or disorders. Treatment includes administering proteins or nucleic acids or vectors or compositions to treat symptoms or disorders in patients (especially humans).

[0207] As used herein, the term "inhibits" or "inhibiting" refers to, for example, reducing, lowering, slowing, decreasing, stopping, destroying, eliminating, antagonizing or blocking a biological response or activity, but does not necessarily mean complete elimination of a biological response.

[0208] As used herein, the term "subject" refers to mammals, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Preferably, the subject is a human.

[0209] As used herein, the term "effective amount" refers to the amount or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt, which provides the desired effect in the subject receiving diagnosis or treatment after single or multiple administration to the subject. As used herein, the term "effective amount" further refers to the amount or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt, which will induce the desired biological or medical response of the subject, for example, reducing or inhibiting protein activity, or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing diseases, etc. In a non-limiting embodiment, the term "effective amount" refers to the necessary amount (in terms of dosage and for time period and for administration mode) or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt when administered to a subject, which can effectively at least partially alleviate, inhibit, prevent and / or improve the condition, disorder or disease to achieve the desired therapeutic result. The effective amount is also the amount in which any toxic or harmful effect or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt of the present invention is exceeded by the beneficial effect.

[0210] The effective amount can be determined by one skilled in the art by using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for a patient, the attending diagnostician will consider many factors, including, but not limited to: the species of the patient; the size, age, and general health of the patient; the specific disease or disorder involved; the extent or severity of the disease or disorder; the response of the individual patient; the specific conjugate being administered; the mode of administration; the bioavailability characteristics of the administered preparation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances.

[0211] 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, for example, Berge, SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).

[0212] Table 1: Abbreviations and definitions

[0213]

[0214]

[0215] The conjugates or salts thereof of the present invention can be easily prepared by a variety of procedures known to those of ordinary skill in the art, some of which are described in the preparation and in the following examples. It is recognized by those of ordinary skill in the art that the specific synthetic steps of each route described can be combined in different ways, or combined with steps from different schemes, to prepare the conjugates or salts thereof of the present invention. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration and crystallization. Unless otherwise stated, all substituents are as previously defined. Reagents and starting materials are readily available to those of ordinary skill in the art. The following preparations, examples and determinations further illustrate the present invention, but should not be construed as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0216] Figure 1A to Figure 1B Anti-human CD33 Ab2 and the Ab2 GC conjugate of Example 1b (1A) and Ab3 (1B) are shown to bind to cell surface CD33 on human monocytes in a fluorescence activated cell sorting (FACS) assay.

[0217] FIG. 2A to FIG. 2B It is shown that Ab2 binding to CD33 expressing cells does not degrade cell surface or intracellular CD33 (2B) relative to the hIgG1 negative control (2A).

[0218] FIG. 3A to FIG. 3D It was shown that treatment with the anti-human CD33 Ab2 GC conjugate of Example 1b modulated IL-6 (3A) and TNFα (3B) cytokine expression in human PBMCs, and that this response was modulated by GC but not Ab2 (3A-3B) or Ab3 (3C-3D).

[0219] FIG. 4A to FIG. 4C It was shown that in subsequent oxazolone challenges (Challenge 1-4A, Challenge 2-4B, and Challenge 3-4C), treatment with the anti-human CD33 Ab2 GC conjugate of Example 1b significantly modulated (inhibited) oxazolone-induced skin inflammation in a dose-dependent manner, and that the inhibition was driven by GC rather than Ab2.

[0220] Preparation 1

[0221] 6-Bromo-2-fluoro-3-methoxybenzaldehyde

[0222]

[0223] Two reactions were carried out in parallel. At -78 ° C, in 30 minutes, LDA (2M, 730 mL) was slowly added to a solution of 4-bromo-2-fluoro-1-methoxybenzene (250 g, 1.2 mol) in THF (1500 mL). After another 30 minutes, at -78 ° C, in 30 minutes, DMF (140 mL, 1.8 mol) was slowly added. After 1 hour, the two reactions were combined, and the mixture was diluted with citric acid aqueous solution (2000 mL) and extracted with EtOAc (1500 mL × 2). The combined organic layer was washed with saturated NaCl aqueous solution (1000 mL) and concentrated under reduced pressure to obtain a residue. The residue was ground with petroleum ether (1000 mL) at room temperature within 12 hours to obtain the title compound (382 g, 67% yield). ES / MS m / z 233.9 (M+H).

[0224] Preparation 2

[0225] 2-Fluoro-3-methoxy-6-methylbenzaldehyde

[0226]

[0227] 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)Cl 2 (12 g, 0.02 mol) and Cs 2 CO 3 (340 g, 1.1 mol) was added to a mixture of 1,4-dioxane (600 mL) and water (120 mL). The mixture was stirred at 120°C. After 12 hours, the three reactions were combined and the mixture was washed with saturated NH 4 The product was diluted with Cl aqueous solution (1000 mL) and extracted with MTBE (1500 mL×2). The combined organic layers were washed with saturated NaCl aqueous solution (1000 mL) and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography, eluted with 40:1 petroleum ether:EtOAc to give the title compound (180 g, 59%). ES / MS m / z 169.3 (M+H).

[0228] Preparation 3

[0229] 2-Fluoro-3-hydroxy-6-methylbenzaldehyde

[0230]

[0231] 2-Fluoro-3-methoxy-6-methylbenzaldehyde (175 g, 1.0 mol) was added to DCM (1050 mL). BBr was added at 0 °C. 3 (200 mL, 2.1 mol) was slowly added to the solution. The reaction was stirred at room temperature. After 1 hour, saturated NaHCO 3 The mixture was diluted with aqueous solution (1000 mL) until pH = 7-8, and then extracted with MTBE (1500 mL x 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).

[0232] Preparation 4

[0233] tert-Butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate

[0234]

[0235] 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 was diluted with water (600 mL) and extracted with EtOAc (800 mL×2). The combined organic layers were washed with saturated aqueous NaCl solution (800 mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography, eluted with 50:1 petroleum ether: EtOAc to obtain a crude product. The crude product was ground 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 + ).

[0236] Preparation 5

[0237] (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

[0238]

[0239] Perchloric acid (70% aqueous solution, 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]phenanthrene-3-one (4.4 g, 12 mmol, also known as "16α-hydroxyprednisolone") 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 warmed to room temperature. After 1 hour, DMF (10 mL) was added to the suspension at room temperature. After 18 hours, the reaction was heated with saturated NaHCO 3 The aqueous solution was quenched and extracted with 9:1 DCM:isopropanol. The organic layers were combined and purified by MgSO 4 Dry, filter, and concentrate under reduced pressure to give a residue. The residue was purified by reverse phase chromatography using 1:1 NH 4 HCO 3 Aqueous solution (10 mM + 5% MeOH):ACN eluted to give the title compound, peak 1 (1.72 g, 25%). ES / MS m / z 618.6 (M+H). 1 HNMR (400.13 MHz, DMSO-d 6 )δ0.93-0.87(m,6H),1.40(s,3H),1.71-1.60(m,1H),1.89-1.76(m,4H),2.18-2.12(m,2H),2.2 9(s,4H),4.23-4.17(m,1H),4.32-4.30(m,1H),4.50-4.43(m,1H),4.81(d,J=3.2Hz,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=7.7Hz,1H),7.12(t,J=8.5Hz,1H),7.33-7.30(m,1H).

[0240] Preparation 6

[0241] (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 (hereinafter referred to as Also known as "GC1")

[0242]

[0243] The residue from Preparation 5 was purified by reverse phase chromatography using 1:1 NH 4 HCO 3 Aqueous solution (10 mM + 5% MeOH):ACN eluted to give the title compound, peak 2 (1.24 g, 18%). ES / MS m / z 618.6 (M+H). 1 HNMR (400.13 MHz, d 6 -DMSO)δ0.88(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=5.2Hz,1H),4.12-4.00(m,1H),4.37-4.30(m,2H),4.79(d,J=3.1Hz,1H),5.00-4 .93(m,2H),5.10-5.06(m,3H),5.31(d,J=6.7Hz,1H),5.95(s,1H),6.18(dd,J=1.8,10.1Hz,1H),6.34(s,1H),6.53-6 .48(m,2H),6.58(s,1H),6.87(d,J=8.5Hz,1H),6.99(t,J=7.7Hz,1H),7.09(t,J=8.5Hz,1H),7.33(d,J=10.1Hz,1H).

[0244] Preparation 7

[0245] (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine

[0246]

[0247] 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 give 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).

[0248] Preparation 8

[0249] 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-(2 ...

[0250]

[0251] To (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 To a solution of [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) was added 2,6-lutidine (11 mL, 97 mmol) followed by HATU (17 g, 43 mmol). 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 with three portions of water, one portion of saturated aqueous NaCl solution, and then washed with Na 2 SO 4 Dry (MeOH added to aid dissolution), filter and evaporate to give the crude product. The crude product was purified by silica gel chromatography using a gradient of 1% to 10% MeOH in DCM to give the title compound (24 g, 68%). ES / MS m / z 911.4 (M+H). 1H NMR (400.13MHz, DMSO): δ9.88(s,1H),8.20(d,J=7.1Hz,1H),8.11(d,J=7.2Hz,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=8.4Hz,1H),6.34(s,1H),6.18(dd,J=1.8,10.0Hz,1H),5.95(s,1H),5.76(s,1H),5. 31(d,J=6.8Hz,1H),5.13-5.04(m,3H),4.78(d,J=3.1Hz,1H),4.41-4.30(m,4H),4.10-4.00(m,1H),3.61(t,J=7.3Hz,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=7.2Hz,3H),1.19-1.11(m,5H),0.88(s,3H).

[0252] Preparation 9

[0253] 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-dimethoxy 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

[0254]

[0255] In a manner analogous to the procedure described in 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). 1HNMR (500.11MHz, DMSO): δ9.88(s,1H),8.23-8.20(m,1H),8.11(d,J=7.2Hz,1H),7.69(s,1H),7.59(d,J=8.0Hz,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=1.7,10.1Hz,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=3.1Hz,1H),4.49-4.36(m,6H),3.61(t,J=7.3Hz,2H),2.41(t,J=7.3Hz,2H),2.30-2.29(m,4H),2.1 7-2.15(m,2H),1.88-1.77(m,4H),1.69-1.61(m,1H),1.40(s,3H),1.31(d,J=7.2Hz,3H),1.18(d,J=7.2Hz,3H),0.93-0.87(m,6H).

[0256] Example

[0257] Example 1. Generation of an exemplary anti-human CD33 antibody GC conjugate

[0258] Example 1a: Generation and engineering of anti-human CD33 antibodies

[0259] Antibody Generation: Anti-human CD33 antibodies as described herein were discovered from a phage display Fab library using solution panning against the Fc-tagged extracellular domain of human CD33 (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-spot phage ELISA screening against his-tagged hCD33. After conversion to IgG format and purification, antibodies binding to CD33-expressing bone marrow cells were confirmed by fluorescence activated cell sorting (FACS) assay using human and cynomolgus monkey PBMCs, respectively.

[0260] The antibodies generated by the above method were screened for internalization activity on CD33 expressing bone marrow cells. TM pHrodo-labeled antibodies generate red fluorescent signals in a low pH environment after being internalized into lysosomes.

[0261] Antibodies were selected that specifically bound to both human and cynomolgus CD33 on bone marrow cells and had efficient internalization.

[0262] Engineering of anti-human CD33 antibodies: Significant engineering including cross-species alignment of antibody sequences, affinity maturation, amino acid modifications, and developability studies were performed to generate anti-human CD33 antibodies as described herein. This engineering overcame the challenges of achieving and balancing the desired affinity for human and cynomolgus CD33, and improved the functional characteristics of the antibodies (e.g., reduced receptor degradation and cell-depleting properties), improved immunogenicity risk profile, viscosity, and developability for subcutaneous administration.

[0263] The VH and Vκ sequences of the anti-human CD33 antibodies described herein are highly homologous to human germline. Four framework residues (A12, S18, S22 and A40 (Kabat numbering)) were identified in the light chain framework region of the parent antibody 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, which potentially reduces the risk of immunogenicity, thereby providing improved developability properties.

[0264] Ab1 was further engineered into Fab using phage expression platform technology (Anal Biochem. 1998, Vol. 256, No. 2: pp. 169-177). It was found that amino acid residue substitution at HCDR3 E95T (Kabat numbering) was critical in improving affinity for human CD33, reducing interaction with Ig-coupled chromatography columns, and improving the viscosity of the antibody at high concentrations. In addition, HCDR3 E95T and LCDR1 D28P residue substitutions were identified and engineered to improve the isoelectric point of the anti-human CD33 antibody GC conjugate.

[0265] Ab1 exhibits significant differences in affinity for human CD33 (3.4E-09) and cynomolgus CD33 (2.41E-05) (see Table 4). Affinity differences of more than 10-fold can make preclinical toxicology studies and data interpretation challenging. Using Ab1 as a template, a targeted mutagenesis library was designed to screen for mutations that improve affinity for cynomolgus CD33 using antibody structural information, CDR mutation scanning, and computational modeling of the cynomolgus CD33 extracellular domain (ECD). Four amino acid residues in LCDR1 (D28, V29, F30, and R31 (Kabat numbering)) were identified as being critical for improving cynomolgus CD33 binding while maintaining the desired binding affinity for human CD33. The combination of the HCDR3 E95T amino acid residue substitution and the four LCDR1 amino acid residue substitutions identified above significantly improved the binding affinity to cynomolgus monkey CD33 (see Table 4), and significantly minimized the affinity difference between human and cynomolgus monkey CD33 while maintaining the desired binding affinity to human CD33. The combination of these engineered residues enabled the generation of Ab2 and Ab3 (Tables 2a, 2b, and 3).

[0266] Choice of backbone: Incorporation of the antibody amino acid residue substitutions L234A, L235A and P329A (EU numbering) into the IgG1 Fc region (IgG1AAA) attenuates binding of the antibody to Fcγ receptors and C1q, and thus attenuates effector function activities such as ADCC and CDC. In addition, as shown below, the IgG1AAA backbone significantly reduces CD33 receptor degradation by CD33 antibodies.

[0267] The antibody also contains amino acid residue substitutions S124C and A378C (EU numbering) in the IgG1 HC constant region for specific conjugation to therapeutic agents (eg, glucocorticoids).

[0268] The amino acid sequence of human CD33 ECD is provided as SEQ ID NO:28, and the amino acid sequence of cynomolgus monkey CD33 ECD is provided as SEQ ID NO:29.

[0269] Table 2a: CDR amino acid sequences of exemplary anti-human CD33 antibodies

[0270]

[0271] Table 2b: CDR amino acid consensus sequences of exemplary anti-human CD33 antibodies

[0272]

[0273] Table 3: Amino acid sequences of exemplary anti-human CD33 antibodies

[0274] VH V L HC LC Ab1 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 Ab2 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:10 Ab3 SEQ ID NO:7 SEQ ID NO:14 SEQ ID NO:9 SEQ ID NO:15

[0275] Example 1b : Anti-human CD33 Ab2 GC The formation of conjugates, where n is 4

[0276]

[0277] wherein n is 4; and

[0278] Ab is Ab2.

[0279] Exemplary anti-human CD33 antibody Ab2 (see Tables 2 and 3) was first reduced in the presence of a 40-fold molar excess of dithiothreitol (DTT) at 37°C for 2 hours or at about 21°C for >16 hours. This initial reduction step was used to remove various capping groups, including cysteine ​​and glutathione, which were bound to the engineered cysteines at positions 124 and 378 of the heavy chain during expression. After the reduction step, the sample was purified by a desalting column to remove unbound caps as well as the reducing agent. A subsequent 2-hour oxidation step was performed at room temperature (about 21°C) in the presence of a 10-fold molar excess of dehydroascorbic acid (DHAA) to reform the native interchain disulfide and hinge disulfide pairs between the light and heavy chains. After the 2 h oxidation step, 4-8 molar equivalents of the 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 prepared in Preparation 8, were added using a 10 mM stock solution in DMSO. ,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. The sample is then incubated at room temperature for 30-60 minutes to allow efficient conjugation of GC-L with engineered cysteine. A subsequent polishing step (such as size exclusion chromatography (SEC) or tangential flow filtration (TFF)) is then used to buffer exchange the sample into a suitable formulation buffer and remove DMSO and any excess linker-payload.

[0280] Drug to Antibody Ratio (DAR) Assessment: To evaluate the average number of linkers-payloads present on the final conjugates, two analytical methods were used: 1) reverse phase (RP) HPLC and 2) time of flight (TOF) mass spectrometry. Both methods require an initial sample reduction step, which includes the addition of dithiothreitol (DTT) to a final concentration of approximately 10 mM, followed by incubation at 42°C for 5 minutes.

[0281] Reverse Phase HPLC Method: 10 μg to 30 μg of 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 Part # 0008043). A buffer consisted of 0.1% trifluoroacetic acid (TFA) in water, while B buffer consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). Prior to sample injection, the column was equilibrated in 20% B buffer, followed by elution with a gradient of 28% B to 40% B over approximately 8.5 column volumes. The average DAR was determined by calculating the contribution of each individual DAR species by multiplying the fractional percentage by the DAR number of each contributing species. Since this value is based on a partially reduced sample and represents only half of the molecule, this number is multiplied by 2 to account for the intact antibody GC conjugate. The DAR calculations for the exemplary anti-human CD33 Ab2 GC conjugate of Example 1b are provided in Table 4.

[0282] Table 4: Percentage of fractions of each DAR species from partially reduced samples for exemplary anti-human CD33 Ab2 Quantification of average DAR of GC conjugates

[0283]

[0284] 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 #821075-924). Buffer A consisted of 0.1% trifluoroacetic acid (TFA) in water, while buffer B consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). Prior to sample injection, the column was equilibrated in 0% B buffer, followed by elution with a gradient of 10% B to 80% B over approximately 28 column volumes. The average DAR was determined by calculating the contribution of each individual DAR species by multiplying the fractional percentage by the DAR number of each contributing species. Since this value is based on a partially reduced sample and represents only half of the molecule, this number is multiplied by 2 to account for the intact antibody GC conjugate. The DAR calculations for the anti-human CD33 Ab2 GC conjugate of Example 1b are provided in Table 5.

[0285] Table 5: Analysis of exemplary anti-human IgG using fractional percentages based on total ion counts from time-of-flight mass spectrometry. Quantification of average DAR of CD33 Ab2 GC conjugates

[0286]

[0287]

[0288] Example 1c. Generation of anti-human CD33 Ab2 GC conjugate, where n is 3

[0289]

[0290] wherein n is 3; and

[0291] Ab is Ab2.

[0292] 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, incubated at about 21°C for 20 minutes, ultimately yielding a DAR of about 3.

[0293] Example 1d. Sulfosuccinimide hydrolysis: The sulfosuccinimide ring of the conjugate of Formula Ia can be Hydrolysis under well-known conditions, as shown below (see, e.g., WO 2017 / 210471, paragraph 001226) to provide an open-ring compound of formula Ib product.

[0294]

[0295] Furthermore, the above-mentioned thiosuccinimide ring of the conjugate of Formula Ia can be at least partially hydrolyzed in vivo and under standard or well-known formulation conditions to provide the ring-opened product of Formula Ib.

[0296] Example 2. Binding activity

[0297] Example 2a. Binding affinity: Affinity and binding kinetics of exemplary anti-human CD33 antibodies to human and cynomolgus CD33 ECD 18-232-His proteins were determined by surface plasmon resonance using Biacore 8K (GE Healthcare). Briefly, antibodies were captured on a Biacore protein A chip according to the instrument manual, and CD33 ECD was subsequently diluted 2-fold serially (200 nM to 0.782 nM) in PBS-P20-BSA (0.005% surfactant P20, 0.1 mg / mL BSA) and flowed over the chip. All measurements were performed at 37°C. A multi-cycle kinetic setup was used, which ran each analyte concentration in a separate cycle and regenerated the surface after each sample injection. Regeneration was optimized to maintain consistent surface properties between cycles. At the beginning of each cycle, 0.1 μg / mL of antibody was injected at a flow rate of 10 μL / min for 3 minutes, followed by injection of antigen at a flow rate of 50 μL / min for 3 minutes and dissociation in PBS-P20-BSA for 15 minutes. The chip was then regenerated by injecting 10 mM glycine buffer, pH 1.5, at a flow rate of 50 μL / min for 30 seconds, repeated three times. The data were fitted to a 1:1 binding model to obtain ka and k d , and calculate K D .

[0298] The results in Table 3 show that the exemplary anti-human CD33 Ab2 GC conjugate of Example 1b retains similar binding affinity to human and cynomolgus CD33 as unconjugated anti-human CD33 Ab2. In addition, Abl, Ab2, and Ab3 bind to human CD33 with the desired affinity, while Abl and Ab2 have improved binding to cynomolgus monkey.

[0299] Table 3. Anti-human CD33 antibody GC conjugates, Ab1, Ab2 and Ab3 of Example 1b for human and cynomolgus monkey CD33 Binding affinity and kinetics of ECD

[0300]

[0301] Example 2b. Cell surface binding ability: The exemplary anti-human CD33 antibodies and the anti-human CD33 Ab2 GC conjugate of Example 1b were tested for their binding ability to human monocytes in a fluorescence activated cell sorting (FACS) assay. TM Human PBMCs were isolated from human blood samples by density gradient centrifugation using GE Healthcare plus. Freshly isolated PBMCs were centrifuged at 2 × 10 6 The cells were resuspended at 100 μL / well and allowed to stand at room temperature for 15 minutes before being plated into a round-bottom 96-well plate. The cells were neutralized and FACS buffer (PBS containing 2% fetal bovine serum, from ) were washed. 647 conjugated exemplary anti-human CD33 antibodies and corresponding control IgG antibodies were added to the wells and diluted 4-fold in duplicate. Then an equal volume of 2× antibody mixture containing the following substances was added to the wells: PE-Cy5 anti-human CD14 antibody (clone M5E2), Per-CP anti-human CD45 antibody (clone H130), FITC anti-human CD11b (clone ICRF44) and AlexaFluor-647 anti-human CD66b (G10F5). The cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer, and resuspended in a final volume of 100 μL of FACS buffer. Live / Dead Yellow (Thermo Fisher Scientific, L34968) was added to the viability dye and analyzed by flow cytometry (LSRFortessa TMX-20; BD BIOSCIENCES) were used to analyze the samples. Data were analyzed using FlowJo software, and statistical analysis was performed using GraphPad Prism 9. The data represent the mean fluorescence intensity (MFI) of CD33-expressing cells from monocytes or neutrophils.

[0302] Figure 1A to Figure 1B The results in show that the exemplary anti-human CD33 Ab2 GC conjugate of Example 1b and Ab2 bind to human CD33 expressed on the surface of monocytes with the desired binding efficacy. Figure 1B Binding of Ab3 to the surface of monocytes is shown.

[0303] Example 3: Functional Activity

[0304] Example 3a. Antibody internalization: An exemplary anti-human CD33 antibody was internalized using the amine-reactive dye pHrodo TM pHrodo Red (ThermoFisher, P36014) labeled. The pH-sensitive dye is non-fluorescent when extracellular, but emits bright fluorescence in acidic low pH lysosomes. This property allows visualization and identification of antibody internalization by flow cytometry. Human PBMCs (500K / well) were incubated on ice for 30 minutes in FACS buffer to allow baseline internalization to stabilize. The cells were stained with pHrodo-labeled antibodies (10 μg / mL) in FACS buffer on ice for 1 hour and then transferred to 37°C for 2 hours. The cells were then stained with the remaining set of surface detection markers: CD45 mAb (clone HI30); CD11b (clone ICRF44); CD14 (clone M5E2).

[0305] The results in Table 4 show that about >80% of the exemplary anti-human CD33 Ab2 GC conjugate and Ab2 of Example 1b were internalized into primary human monocytes within about 2 hours of binding to cell surface CD33. In contrast, only about 50% of gemtuzumab (hIgG4PAA) was internalized into primary human monocytes within about 2 hours of binding to cell surface CD33. The faster and / or increased internalization rate of the exemplary anti-human CD33 Ab2 GC conjugate and Ab2 of Example 1b into monocytes indicates that Ab2 can effectively deliver the therapeutic agent linked thereto into CD33 expressing cells.

[0306] Table 4. Internalization of exemplary anti-human CD33 Ab2 GC conjugates, Ab2, and gemtuzumab of Example 1b

[0307] Internalization% hIgG1AAA isotype control 2.26±0.693 hIgG4PAA Isotype Control 3.49±1.626 Ab2 86.545±4.589 Ab2GC conjugate 86.55±6.859 Gemtuzumab 50.55±2.899

[0308] Example 3b. CD33 cell surface receptor availability after internalization: Evaluation of CD33 cell surface receptor availability on monocytes that have been internalized after binding to an exemplary anti-human CD33 antibody. Briefly, human monocytes isolated from PBMCs were cultured at 2×10 4 Cells / well were seeded in 96-well plates and incubated on ice for 60 minutes to prevent internalization. Exemplary anti-human CD33 antibodies (30 μg / mL) were added to the cells and combined on ice for 30 minutes. 100 μL of culture medium was then added to the cells, and the plates were placed in an incubator at 37°C for 3.5, 24, 48, 72 and 96 hours. Baseline samples were immediately stained to indicate that the exemplary anti-human CD33 antibody surface was bound before internalization. Cell Fc was blocked for 20 minutes, and then stained on ice for 30 minutes with AlexaFluor-647 labeled anti-human CD33 exemplary antibodies, Alexa Fluor-700 anti-human CD45 (HI30) and Brilliant Violet-605 anti-human CD11b (ICRF44). Cells were analyzed by flow cytometry.

[0309] The results in Table 5 show that cell surface CD33 availability on monocytes increases from a baseline of about 1.1% to as high as about 31.9% over the course of about 3.5 hours to about 96 hours after internalization, respectively. Surface CD33 can be from CD33 de novo expression and / or internalized CD33 that is recycled to the cell surface. These results suggest that regenerated CD33 on human monocytes can potentially bind additional anti-human CD33 antibodies for repeated delivery of CD33 antibodies or therapeutic agents linked to CD33 antibodies to CD33 expressing cells.

[0310] Table 5. CD33 cell surface availability after internalization following treatment with exemplary Ab1

[0311] % of CD33 positive cells on cell surface after internalization Baseline 1.1±0.481 3.5 hours 1.06±0.339 24 hours 1.935±0.884 48 hours 9.195±1.260 72 hours 14.185±1.335 96 hours 31.9±2.183

[0312] Example 3c. CD33 receptor degradation: The effect of exemplary anti-human CD33 Ab2 on intracellular CD33 degradation was evaluated by Western immunoblot analysis. TM Human PBMCs were isolated from human blood samples by density gradient centrifugation using GE Healthcare plus. Freshly isolated PBMCs were centrifuged at 1×10 7 Cells / 5mL / well were seeded in culture medium in 24-well cell culture plates. Cells were treated with anti-human CD33 Ab2 and hIgG1 (effector null) negative control antibody overnight in a 37°C cell culture incubator. This step allows the determination of degradation of internalized CD33 receptors. The next day, cells were collected and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors.

[0313] Cell lysate protein was quantified by bispicolinic acid (BCA) assay and normalized for loading on western blots. Non-competitive anti-CD33 antibody (AbCam clone EPR4423) was used to detect CD33 levels.

[0314] FIG. 2A to FIG. 2B The results in show that the exemplary anti-human CD33 Ab2 does not degrade CD33 after binding and internalization, as CD33 levels are comparable to isotype controls.

[0315] Example 3d. Proinflammatory cytokine modulation and immune cell depletion: The effects of the exemplary anti-human CD33 Ab2 GC conjugate and anti-human CD33 Ab2 of Example 1b on proinflammatory cytokine modulation in human PBMCs were evaluated. PBMCs were used to mimic in vivo conditions. Briefly, the exemplary anti-human CD33 Ab2 or the anti-human CD33 Ab2 GC conjugate of Example 1b was incubated with 2×10 6 Human PBMCs at 10 cells / well were incubated in a 96-well plate at 37°C incubator for 1 hour. The cells were then stimulated with 100 pg / mL of LPS (Sigma, catalog number: L2880) in a 37°C incubator for 24 hours. The culture supernatant was collected to measure the indicated cytokines (Proinflammatory Panel II (human) 4-Plex kit, MSD, K15053D-2).

[0316] FIG. 3A to FIG. 3D The results in Table 6a show that the anti-human CD33 Ab2 GC conjugate of Example 1b significantly modulates (inhibits) the proinflammatory cytokine IL-6 and TNFα responses similar to GC1 alone. Unconjugated anti-human CD33 Ab2 does not modulate CD33-mediated expression of proinflammatory cytokines IL-6 and TNFα, indicating that the cytokine responses observed with the exemplary anti-human CD33 Ab2 GC conjugate are regulated by glucocorticoids.

[0317] Table 6b shows that treatment with the anti-human CD33 Ab2 GC conjugate of Example 1b and anti-human CD33 Ab2 did not induce PBMC immune cell depletion compared to the isotype control.

[0318] Table 6a. Proinflammatory cytokine modulation by exemplary anti-human CD33 Ab2 GC conjugates of Example 1b

[0319] % inhibition of human IL-6 secretion at 200 nM % inhibition of human TNFa secretion at 200 nM Isotype 1.638±1.565 3.214±0.264 Ab2 12.045±1.887 5.978±0.548 Ab2GC conjugate 78.604±0.836 80.469±2.579 GC1 83.908±2.815 85.126±3.278

[0320] Table 6b. Exemplary anti-human CD33 Ab2 GC conjugates and anti-human CD33 Ab2 of Example 1b did not induce immune cells Cell consumption

[0321]

[0322] Live cell count (×10 6 cells / mL)

[0323] Example 3e. Plasmacytoid dendritic cell assay: The ability of exemplary anti-human CD33 Ab2GC conjugates to regulate human plasmacytoid dendritic cells (pDCs) was evaluated in a pDC differentiation assay. It is known that pDCs expressing CD33 are the main cells that produce type 1 interferons associated with the pathogenesis of lupus. Briefly, human PBMCs were cultured in medium supplemented with IL-3 in the presence of anti-human CD33 Ab2 or the anti-human CD33 Ab2 GC conjugate of Example 1b or GC1 alone, and CpG (immunostimulatory DNA containing unmethylated cytosine-phosphate-guanosine) was stimulated. The differentiation of pDCs was measured by flow cytometry. The functional effects of pDCs were measured by type I IFN (IFNα) production.

[0324] The results in Table 6c show that the anti-human CD33 Ab2 GC conjugate of Example 1b significantly regulates (inhibits) the differentiation of pDCs and inhibits the secretion of cytokine IFNα similarly to GC1 alone. Unconjugated anti-human CD33 Ab2 does not affect pDC differentiation and IFNα secretion, indicating that the inhibition of cytokine responses observed with the exemplary anti-human CD33 Ab2 GC conjugate is regulated by glucocorticoids. Therefore, the results demonstrate the functional activity of the anti-human CD33 Ab2 GC conjugate of Example 1b that specifically targets pDCs.

[0325] Table 6c. Inhibition of pDC and IFNα by the exemplary anti-human CD33 Ab2GC conjugates of Example 1b

[0326] % inhibition of pDC differentiation % inhibition of human IFNα secretion Isotype 0.92±0.06 0.83±0.21 Ab2 0.97±0.39 1.25±0.19 Ab2GC conjugate 91.89±6.37 95.62±1.57 GC1 95.50±8.32 97.18±2.95

[0327] Example 4. Effector Function Activity

[0328] Antibody-dependent cellular cytotoxicity (ADCC): In vitro ADCC assays of exemplary antibodies were evaluated using a reporter-based ADCC assay. CHO cells expressing human CD33 and human CD20 were used as target cell lines, and Jurkat cells expressing functional FcγRIIIa (V158)-NFAT-Luc (Eli Lilly) were used as effector cell lines. All test antibodies and cells were diluted in assay medium containing RPMI-1640 (phenol red-free) 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 antibody was first diluted to a 3× concentration of 3.3 μg / mL, and then serially diluted 7 times at a 1:4 ratio. 50 μL / well of each antibody was aliquoted in duplicate in a white opaque bottom 96-well plate (Costar, #3917). The CD20 antibody was used as a positive control. Then CHO target cells were cultured at 5×10 4 100 μ L / well.Incubate 1 hour at 37 ℃ with 50 μ L aliquots of individual cells / well.Next, JurkatV158 cells are added to the wells with 150000 cells / well with 50 μ L aliquots, and incubate 4 hours at 37 ℃, then add the One-Glo luciferase substrate (Promega, #E8130) of 100 μ L / well.Use plate oscillator to mix the contents of the plate at low speed, incubate 5 minutes at room temperature, and read luminescent signal on BioTek microplate reader (BioTekInstruments) using 0.2cps integration.Use GraphPadPrism 9 to analyze data, and the relative luminescent unit (RLU) of every kind of antibody concentration is mapped with the scattering format of antibody concentration to RLU.

[0329] Exemplary anti-human CD33 antibodies with an effector-null IgG1AAA backbone did not induce ADCC activity (results not shown), suggesting that the exemplary antibodies have a low probability of depleting CD33-expressing cells through ADCC-mediated killing.

[0330] Example 5. Biophysical properties

[0331] The biophysical properties of exemplary anti-human CD33 antibodies were evaluated for developability.

[0332] Aggregation from cell culture: Exemplary anti-human CD33 antibodies were transiently expressed in CHO cells. Antibody titers and percentage of high molecular weight species (% HMW) after protein A affinity chromatography purification are shown in Table 7 and indicate that Abl, Ab2 and Ab3 have low aggregation, providing good developability properties.

[0333] Viscosity: Exemplary anti-human CD33 antibodies Abl, Ab2, Ab3 and the anti-human CD33 Ab2 GC conjugate of Example 1b were concentrated to approximately 125 mg / mL in 5 mM histidine matrix at pH 6. The viscosity of each material was measured using an initium (RheoSense) at 15° C. using the average of 9 replicate measurements. The results in Table 7 show that exemplary anti-human CD33 antibodies Abl, Ab2, Ab3 and the anti-human CD33 Ab2 GC conjugate of Example 1b have good viscosity properties for developability.

[0334] Thermal stability: Differential scanning calorimetry (DSC) was used to evaluate the stability of the exemplary anti-human CD33 antibodies Abl, Ab2, Ab3 and the anti-human CD33 Ab2 GC conjugate of Example 1b against thermal denaturation. The results in Table 7 show that the exemplary anti-human CD33 antibodies Abl, Ab2, Ab3 and the anti-human CD33 Ab2 GC conjugate of Example 1b have acceptable thermal melting temperatures for developability in PBS at pH 7.2.

[0335] Aggregation under temperature stress: The solution stability of exemplary anti-human CD33 antibodies Ab2, Ab3 and the anti-human CD33 Ab2 GC conjugate of Example 1b over time was evaluated at about 100 mg / mL in a common 5 mM histidine pH 6.0 buffer. The concentrated samples were incubated at 5°C and 35°C for a period of 4 weeks, respectively. After incubation, the percentage of high molecular weight species (% HMW) in the samples was analyzed by size exclusion chromatography (SEC). The results in Table 7 show that the exemplary anti-human CD33 antibodies Ab2, Ab3 and the anti-human CD33 Ab2 GC conjugate of Example 1b have good stability under thermal stress.

[0336] Table 7. Biophysical properties of exemplary anti-human CD33 antibodies and the Ab2 GC conjugate of Example 1b

[0337] Ab1 Ab2 Ab3 Ab2GC conjugate % HMW species after Protein A chromatography 2.67 1.10 1.39 NA sCHO titer (g / L) 1.7 4.7 4.6 NA Viscosity(cP) 5.5 6.5 7.7 7.3 <![CDATA[T 起始 (℃)]]> 64.4 64.6 57.8 Tm1(℃) 71.8 70.93 64.0 Tm2(℃) 79 77.5 79.68 77.0 HMW% after 4 weeks incubation at 5°C ND* <0.1 <0.1 0.95 HMW% after 4 weeks incubation at 35°C ND* 0.86 1.58 3.08

[0338] *ND = Not Determined

[0339] Example 6. Immunogenicity Assessment

[0340] MS serum protein binding: Evaluation of off-target binding of exemplary anti-human CD33 antibodies to serum proteins. Exemplary anti-human CD33 antibodies Ab1, Ab2 and Ab3 and the anti-human CD33 Ab2GC conjugate of Example 1b were coated onto Maxisorp microplates. The plates were blocked and human serum was added to the wells, and the plates were incubated overnight. The bound proteins were eluted, reduced, alkylated and digested. Peptides were analyzed by mass spectrometry. Peptide and protein identifications were generated by an in-house proteomics pipeline using a search algorithm with trypsin and a human database with additional test antibody sequences. Ions were quantified by in-house proteomics tools (Chrom-Alignment, Metaconsense and Quant) and analyzed in JMP using one-way analysis / mean comparison / all pairs, Tukey HSD. Proteins with ions with >30% P-value <0.05 and FC>2 (compared to isotype control) were considered enriched in the study.

[0341] The results in Table 8 show that exemplary 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 Abl, and thus potentially have a lower immunogenicity risk than Abl.

[0342] Table 8: MS serum protein analysis of exemplary anti-human CD33 antibodies and the anti-human CD33 Ab2 GC conjugate of Example 1b Analysis

[0343]

[0344]

[0345] Example 7: In vivo characterization

[0346] Preclinical contact hypersensitivity model: The exemplary anti-human CD33 Ab2 GC conjugate of Example 1b and the anti-human CD33 antibody Ab2 were evaluated in vivo using a humanized mouse model of contact hypersensitivity (HuNOG-EXL). Ab1 was similarly evaluated in a separate study. Oxazolone, an allergic contact dermatitis inducer, was administered to HuNOG-EXL to induce a local proinflammatory cytokine response and skin inflammation. This allowed the in vivo anti-inflammatory and immunomodulatory effects of the exemplary molecules to be explored.

[0347] Briefly, huNOG-EXL mice were transplanted with human CD34+ hematopoietic stem cells isolated from human umbilical cord blood at 6 weeks of age. 20-24 weeks after stem cell administration, mice were assessed for adequate human CD45 engraftment (>25% in the blood) and subjected to an oxazolone-induced contact hypersensitivity protocol. On day 0, mice were grouped by body weight (N=7-8 / group) and subcutaneously administered with 10, 1, and 0.1 mg / kg of the anti-human CD33 Ab2 GC conjugate of Example 1b, or 10 mg / kg of anti-human CD33 Ab1 or Ab2, or a non-binding isotype control. 24 hours after administration (day 1), mice were anesthetized with 5% isoflurane, the abdomen was shaved, and sensitized with 100 μL of a 3% oxazolone solution in ethanol (applied to the shaved area). Mice were dosed again as described above on days 4, 11, and 16, and challenged bilaterally with 2% oxazolone in ethanol (10 μL / side / ear) 48 hours after each dose (day 6-challenge 1, day 13-challenge 2, and day 18-challenge 3). For the GC1 group alone, mice were dosed subcutaneously at 3 mg / kg before sensitization and 1 hour before each challenge. After the last challenge, mice were euthanized, and ear tissues and gastrocnemius muscles were collected for analysis of target-selective and non-target-selective tissue expression of GC-regulated genes.

[0348] Table 9a and FIG. 4A to FIG. 4C The results in Table 9a show that the anti-human CD33 Ab2 GC conjugate of Example 1b significantly inhibited oxazolone-induced skin inflammation in vivo in all 3 challenges. The results in Table 9a further show that the anti-human CD33 Ab2 GC conjugate of Example 1b inhibited oxazolone-induced skin inflammation by 57% at 10 mg / kg and 34% at 1 mg / kg at challenge 3 compared to 10 mg / kg of unconjugated Ab2. In a separate study, the effect of Ab1 also showed no statistically significant difference from the isotype control (data not shown).

[0349] The results in Table 9b show that the anti-human CD33Ab2GC conjugate of Example 1b regulates target tissue-specific (ear) glucocorticoid receptor agonist-mediated FKBP5 gene expression, but does not regulate non-target tissue (gastrocnemius) 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.

[0350] Overall, these data demonstrate that the exemplary anti-human CD33 Ab2 GC conjugate of Example 1b specifically delivers glucocorticoids to target cells, thereby modulating tissue-specific GC agonist receptor-mediated responses.

[0351] Table 9a. Anti-human CD33 Ab GC conjugates of exemplary anti-human CD33 Ab2 and Example 1b in type IV hypersensitivity human In vivo activity in a HER2-positive mouse model; data from challenge 3.

[0352]

[0353] *p < 0.0002 compared to isotype; ANOVA Tukey; ^p < 0.005 compared to Ab2; ANOVA Tukey

[0354] Table 9b. In the humanized mouse model of type IV hypersensitivity, the expression of β-catenin in target tissue (ear) and non-target tissue (gastrocnemius muscle) was Induction of human glucocorticoid response genes by the CD33 Ab2 GC conjugate of Example 1b and Ab2

[0355]

[0356] Sequence Listing

[0357] Ab1

[0358] SEQ ID NO:1 HCDR1 of Ab1, Ab2 and Ab3 (North)

[0359] AASGFTFSSYAMS

[0360] SEQ ID NO:2 HCDR2 of Ab1, Ab2 and Ab3 (North)

[0361] AISGSGGSTY

[0362] SEQ ID NO: 17 HDCR3 of Ab1 (North)

[0363] AREYSNYDY

[0364] SEQ ID NO: 18 LCDR1 of Ab1 (North)

[0365] QASQDVFRYLN

[0366] SEQ ID NO:5 LCDR2 (North) of Ab1, Ab2 and Ab3

[0367] YDASNLQT

[0368] SEQ ID NO:6 LCDR3 of Ab1, Ab2 and Ab3 (North)

[0369] QQYEDLPT

[0370] SEQ ID NO: 19 VH of Ab1

[0371] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYSNYDYWGQGTLVTVSS

[0372] SEQ ID NO: 20 VL of Ab1

[0373] DIQMTQSPSSLSASVGDRVTITCQASQDVFRYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIK

[0374] SEQ ID NO:21 HC of Ab1

[0375] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREESNYDYWGQGTLVTVSSASTKGPCVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0376] SEQ ID NO:22LC of Ab1

[0377] DIQMTQSPSSLSASVGDRVTITCQASQDVFRYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0378] SEQ ID NO: 23 HCDNA of Ab1

[0379]

[0380] SEQ ID NO:24LCDNA of Ab1

[0381] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACGTTTTCAGGTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAGGATCTCCCTACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0382] Ab2

[0383] SEQ ID NO:1 HCDR1 of Ab1, Ab2 and Ab3 (North)

[0384] AASGFTFSSYAMS

[0385] SEQ ID NO:2 HCDR2 of Ab1, Ab2 and Ab3 (North)

[0386] AISGSGGSTY

[0387] SEQ ID NO:3 HDCR3 of Ab2 and Ab3 (North)

[0388] ARTYSNYDY

[0389] SEQ ID NO:4 LCDR1 of Ab2 (North)

[0390] QASQPTFNYLN

[0391] SEQ ID NO:5 LCDR2 (North) of Ab1, Ab2 and Ab3

[0392] YDASNLQT

[0393] SEQ ID NO:6 LCDR3 of Ab1, Ab2 and Ab3 (North)

[0394] QQYEDLPT

[0395] SEQ ID NO: 7 VH of Ab2 and Ab3

[0396] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSS

[0397] SEQ ID NO: 8 VL of Ab2

[0398] DIQMTQSPSSLSASVGDRVTITCQASQPTFNYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIK

[0399] SEQ ID NO: 9 HC of Ab2 and Ab3

[0400] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSSASTKGPCVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0401] SEQ ID NO: 10 LC of Ab2

[0402] DIQMTQSPSSLSASVGDRVTITCQASQPTFNYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0403] SEQ ID NO:11 HCDNA of Ab2 and Ab3

[0404]

[0405] SEQ ID NO: 12LCDNA of Ab2

[0406] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGCCGACGTTCAATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAGGATCTCCCTACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0407] Ab3

[0408] SEQ ID NO:1 HCDR1 of Ab1, Ab2 and Ab3 (North)

[0409] AASGFTFSSYAMS

[0410] SEQ ID NO:2 HCDR2 of Ab1, Ab2 and Ab3 (North)

[0411] AISGSGGSTY

[0412] SEQ ID NO:3 HDCR3 of Ab2 and Ab3 (North)

[0413] ARTYSNYDY

[0414] SEQ ID NO:LCDR1(North) of 13Ab3

[0415] QASQPTYIYLN

[0416] SEQ ID NO:5 LCDR2 (North) of Ab1, Ab2 and Ab3

[0417] YDASNLQT

[0418] SEQ ID NO:6 LCDR3 of Ab1, Ab2 and Ab3 (North)

[0419] QQYEDLPT

[0420] SEQ ID NO: 7 VH of Ab2 and Ab3

[0421] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSS

[0422] SEQ ID NO: 14 VL of Ab3

[0423] DIQMTQSPSSLSASVGDRVTITCQASQPTYIYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIK

[0424] SEQ ID NO: 9 HC of Ab2 and Ab3

[0425] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTYSNYDYWGQGTLVTVSSASTKGPCVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0426] SEQ ID NO: 15 LC of Ab3

[0427] DIQMTQSPSSLSASVGDRVTITCQASQPTYIYLNWYQQKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYEDLPTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0428] SEQ ID NO:11 HCDNA of Ab2 and Ab3

[0429]

[0430] SEQ ID NO: 16LCDNA of Ab3

[0431] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGCCTACGTATATCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAGGATCTCCCTACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0432] SEQ ID NO:25 Human CD33

[0433] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0434] SEQ ID NO:26 LCDR1 consensus sequence

[0435] QASQPTXaa 7 Xaa 8 IYLN

[0436] where Xaa- 7 is phenylalanine or tyrosine, and Xaa 8 is asparagine or isoleucine

[0437] SEQ ID NO:27 Cynomolgus monkey CD33

[0438] MPLLLLPLLWAGALAMDPRVRLEVQESVTVQEGLCVLVPCTFFHPVPYHTRNSPVHGYWFREGAIVSLDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMEKGSTKYSYKSTQLSVHVTDLTHRPQILIPGALDPDHSKNLTCSVPWACEQGTPPIFSWMSAAPTSLGLRTTHSSVLIITPRPQDHGTNLTCQVKFPGAGVTTERTIQLNVSYASQNPRTDIFLGEGSGRKARKQGVVQGAIGGAGVTVLLALCLCLIFFTVKTHRRKAARTAVGRIDTHPATGPTSSKHQKKSKLHGATETSGCSGTTLTVEMDEELHYASLNFHGMNPSEDTSTEYSEVRTQ

[0439] SEQ ID NO: 28 Human CD33 extracellular domain

[0440] DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVT

[0441] SEQ ID NO: 29 Cynomolgus monkey CD33 extracellular domain

[0442] 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), wherein 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: The HCDR1 comprises SEQ ID NO: 1; The HCDR2 comprises SEQ ID NO: 2; The HCDR3 comprises SEQ ID NO: 3; The LCDR1 comprises SEQ ID NO:4; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6.

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), wherein 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: The HCDR1 comprises SEQ ID NO: 1; The HCDR2 comprises SEQ ID NO: 2; The HCDR3 comprises SEQ ID NO: 3; The LCDR1 comprises SEQ ID NO: 13; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6.

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), wherein 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: The HCDR1 comprises SEQ ID NO: 1; The HCDR2 comprises SEQ ID NO: 2; The HCDR3 comprises SEQ ID NO: 3; The LCDR1 comprises SEQ ID NO:26; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6. 4 . The antibody of 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), wherein 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), wherein 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: 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; and The LCDR3 comprises SEQ ID NO:

6.

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

20.

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

22.

9. The antibody according to any one of claims 1 to 4 or 6 to 7, wherein the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises: 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).

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), wherein the HC is of human IgG1 isotype.

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

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

18. A cell comprising the vector according to any one of claims 15 to 16.

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

20. A method of producing an antibody, the method comprising culturing the cell according to claims 18 to 19 under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium.

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

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

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

24. A conjugate of the formula: Wherein Ab is an antibody that binds to human CD33, in for: And n is 1 to 5.

25. The conjugate of claim 24, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein 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: 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; and The LCDR3 comprises SEQ ID NO: 6; 26. The conjugate according to claim 24, wherein for:

27. The conjugate according to claim 24, wherein for:

28. The conjugate according to claim 24 or 26, wherein for:

29. The conjugate according to claim 24 or 27, wherein for:

30. The conjugate according to claim 24 or 26, wherein for:

31. The conjugate according to claim 24 or 26, wherein for:

32. The conjugate according to claim 24 or 27, wherein for:

33. The conjugate according to claim 24 or 27, wherein for:

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

6.

35. The conjugate of claim 34, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO:

8.

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

10.

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

6.

38. The conjugate of claim 37, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO:

14.

39. The conjugate of any one of claims 37 to 38, wherein the Ab comprises a heavy chain (HC) and a light chain (LC), the HC comprising SEQ ID NO: 9, and the LC comprising SEQ ID NO:

15.

40. The conjugate of any one of claims 24 to 33, wherein the Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein 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: 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, and The LCDR3 comprises SEQ ID NO:

6.

41. The conjugate of claim 40, wherein the VH comprises SEQ ID NO: 19 and the VL comprises SEQ ID NO:

20.

42. The conjugate of any one of claims 40 to 41, wherein the Ab comprises a heavy chain (HC) and a light chain (LC), the HC comprising SEQ ID NO: 21, and the LC comprising SEQ ID NO:

22.

43. The conjugate of any one of claims 24 to 35, 37 to 38, 40 to 41, wherein the Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises: 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).

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

45. The conjugate of claim 44, wherein the human IgG1 isotype is effector-null.

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

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

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

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

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

51. The conjugate of 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 bone marrow cell-related disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate according to claims 22 to 51 or a pharmaceutical composition according to claim 52.

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

55. The method of claim 54, wherein the immune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, giant cell arteritis, polymyalgia, 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 of claim 55, wherein the immune disease is rheumatoid arthritis.

57. The conjugate of 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 treating a bone marrow cell related disease.

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, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome or 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 treating a bone marrow cell-related disease.

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, psoriatic arthritis, atopic dermatitis, psoriasis, ulcerative colitis, Crohn's disease, dermatomyositis, juvenile idiopathic arthritis, multiple sclerosis, Sjögren's syndrome, macrophage activation syndrome or fibrotic disease.

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

66. A method of producing a conjugate, the method comprising the steps of: (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; and (c) contacting the oxidized anti-human CD33 antibody with a compound of the formula to produce the conjugate.

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

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