Anti-CD203c antibody conjugates and uses thereof

Abstract

Provided herein are anti-CD203c antibodies and antigen-binding portions linked to c- Kit inhibitors. Such antibody-drug conjugates can be used for specific targeting of mast cells and / or basophils in proliferative and inflammatory disorders.

Description

ANTLCD203C ANTIBODY CONJUGATES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 595,425, filed November 2, 2023, and U.S. Provisional Patent Application No. 63 / 578,246, filed August 23, 2023, the contents of each of which are incorporated by reference herein in their entirety for all purposes.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (UHFL_003_02WO_SeqList_ST26.xml; Size: 146,432 bytes; and Date of Creation: August 19, 2024) are herein incorporated by reference in their entirety.TECHNICAL FIELD

[0003] The disclosure is related to antibody-drug conjugates and uses thereof for treatment of proliferative and inflammatory disorders.BACKGROUND

[0004] Mast cells are central drivers of pathology in many chronic diseases, including systemic mastocytosis and mast cell leukemia, which are driven by aberrant mast cell proliferation. There is currently a paucity of mast cell-selective drugs, which leaves a significant unmet medical need for patients suffering from mast cell driven disease. Many existing small molecule approaches target the mast cell survival receptor c-Kit. While this approach has demonstrated some clinical benefit, systemic inhibition of c- Kit can lead to side effects such as on-target dose-limiting bone marrow suppression, alterations in taste sensation, and changes in hair and skin color. These unwanted effects of systemic c-Kit inhibition are observed because the receptor is expressed on a wide variety of cells, including melanocytes, olfactory and taste receptor cells, some myeloid cells and, importantly, hematopoietic stem cells in the bone marrow. For each of these cell classes, c-Kit signaling may be an important survival signal. Kinase inhibitors with broader activities beyond c-Kit have suffered from even greater off-target systemic toxicity and can also show limited therapeutic benefit due to sub-optimal pharmacokinetics. Drug designs that enable more specific, top-down targeting of mast cells and basophils would have the potential to control aberrant activation and / orproliferation of those cells while protecting cell populations that are not diseasemediating. There remains a significant unmet medical need to develop therapies with more effective and longer duration benefit that combine potency and specificity of targeting with minimal side effects.SUMMARY

[0005] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor.

[0006] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the antibody or antigen-binding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:(a) the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;(b) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;(c) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 18; a HCDR2 comprising SEQ ID NO: 19; and a HCDR3 comprising SEQ ID NO: 20; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 22; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 23;(d) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 26; and a HCDR3 comprising SEQ ID NO: 27; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 29; a LCDR2 comprising SEQ ID NO: 30; and a LCDR3 comprising SEQ ID NO: 31;(e) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 34; and a HCDR3 comprising SEQ ID NO: 35; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 37; a LCDR2 comprising SEQ ID NO: 38; and a LCDR3 comprising SEQ ID NO: 39;(f) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;(g) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;(h) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 57; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 59; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 60;(i) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 66; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(j) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(k) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 76; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 77;(l) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 79; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 81; a LCDR2 comprising SEQ ID NO: 82; and a LCDR3 comprising SEQ ID NO: 83;(m) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 85; a HCDR2 comprising SEQ ID NO: 86; and a HCDR3 comprising SEQ ID NO: 87;and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 89; a LCDR2 comprising SEQ ID NO: 90; and a LCDR3 comprising SEQ ID NO: 91;(n) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;(o) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO: 103; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;(p) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;(q) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;(r) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 121; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 123; and a LCDR3 comprising SEQ ID NO: 124;(s) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;(t) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ IDNO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;(u) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 139; a HCDR2 comprising SEQ ID NO: 140; and a HCDR3 comprising SEQ ID NO: 141; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 143;(v) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 145; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 147; a LCDR2 comprising SEQ ID NO: 148; and a LCDR3 comprising SEQ ID NO: 149; or(w) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.

[0007] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein:(a) the VH domain amino acid sequence comprises SEQ ID NO: 1, and the VL domain amino acid sequence comprises SEQ ID NO: 5;(b) the VH domain amino acid sequence comprises SEQ ID NO: 9, and the VL domain amino acid sequence comprises SEQ ID NO: 13;(c) the VH domain amino acid sequence comprises SEQ ID NO: 17, and the VL domain amino acid sequence comprises SEQ ID NO: 21;(d) the VH domain amino acid sequence comprises SEQ ID NO: 24, and the VL domain amino acid sequence comprises SEQ ID NO: 28;(e) the VH domain amino acid sequence comprises SEQ ID NO: 32, and the VL domain amino acid sequence comprises SEQ ID NO: 36;(f) the VH domain amino acid sequence comprises SEQ ID NO: 40, and the VL domain amino acid sequence comprises SEQ ID NO: 44;(g) the VH domain amino acid sequence comprises SEQ ID NO: 47, and the VL domain amino acid sequence comprises SEQ ID NO: 51;(h) the VH domain amino acid sequence comprises SEQ ID NO: 55, and the VL domain amino acid sequence comprises SEQ ID NO: 58;(i) the VH domain amino acid sequence comprises SEQ ID NO: 61, and the VL domain amino acid sequence comprises SEQ ID NO: 65;(j) the VH domain amino acid sequence comprises SEQ ID NO: 68, and the VL domain amino acid sequence comprises SEQ ID NO: 70;(k) the VH domain amino acid sequence comprises SEQ ID NO: 72, and the VL domain amino acid sequence comprises SEQ ID NO: 75;(l) the VH domain amino acid sequence comprises SEQ ID NO: 78, and the VL domain amino acid sequence comprises SEQ ID NO: 80;(m) the VH domain amino acid sequence comprises SEQ ID NO: 84, and the VL domain amino acid sequence comprises SEQ ID NO: 88;(n) the VH domain amino acid sequence comprises SEQ ID NO: 92, and the VL domain amino acid sequence comprises SEQ ID NO: 96;(o) the VH domain amino acid sequence comprises SEQ ID NO: 100, and the VL domain amino acid sequence comprises SEQ ID NO: 104;(p) the VH domain amino acid sequence comprises SEQ ID NO: 108, and the VL domain amino acid sequence comprises SEQ ID NO: 112;(q) the VH domain amino acid sequence comprises SEQ ID NO: 116, and the VL domain amino acid sequence comprises SEQ ID NO: 117;(r) the VH domain amino acid sequence comprises SEQ ID NO: 120, and the VL domain amino acid sequence comprises SEQ ID NO: 122;(s) the VH domain amino acid sequence comprises SEQ ID NO: 125, and the VL domain amino acid sequence comprises SEQ ID NO: 129;(t) the VH domain amino acid sequence comprises SEQ ID NO: 133, and the VL domain amino acid sequence comprises SEQ ID NO: 134;(u) the VH domain amino acid sequence comprises SEQ ID NO: 138, and the VL domain amino acid sequence comprises SEQ ID NO: 142;(v) the VH domain amino acid sequence comprises SEQ ID NO: 144, and the VL domain amino acid sequence comprises SEQ ID NO: 146; or(w) the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

[0008] In some embodiments of the antibody-drug conjugates provided herein, the antibody or antigen-binding portion is human, humanized, or chimeric.

[0009] In some embodiments, the antibody or antigen-binding portion comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY. In some embodiments, the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2.

[0010] In some embodiments, immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is a wildtype human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgGl constant region, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A or a wild-type human IgG2 constant region, wherein numbering is according to the EU index as in Kabat. In some embodiments, the immunoglobulin constant region comprises the amino acid sequence of any one of SEQ ID NOs: 155-160.

[0011] In some embodiments, the antibody or antigen-binding portion is an Fab, an Fab', an F(ab')2, an Fv, an scFv, a maxibody, a minibody, a diabody, a triabody, a tetrabody, or a bis-scFv.

[0012] In some embodiments, the antibody or antigen-binding portion is tetrameric, tetravalent, or multispecific.

[0013] In some embodiments, the c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib. In some embodiments, the c-Kit inhibitor is dasatinib.

[0014] In some embodiments, the c-Kit inhibitor is linked to the antibody or antigenbinding portion via a peptide linker. In some embodiments, the peptide linker comprises two amino acid residues independently selected from glycine, alanine, phenylalanine, lysine, arginine, valine, and citrulline. In some embodiments, the peptide linker is an alanine-alanine (Ala-Ala) linker. In some embodiments, the peptide linker is L-Ala-L-Ala, D-Ala-D-Ala, D-Ala-L-Ala, or L-Ala-D-Ala. In some embodiments, the peptide linker is L-Ala-L-Ala. In some embodiments, the peptide linker is L-Ala-L-Ala; and wherein the antibody-drug conjugate comprises a PEG spacer.

[0015] In some embodiments, the antibody or antigen-binding portion comprises the c- Kit inhibitor and a linker comprising the structure:(a)

[0016] In some embodiments, the c-Kit inhibitor is linked to the antibody or antigenbinding portion via an acid cleavable linker, a glutathione (GSH) cleavable linker, a Fe(II) cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, a phosphatase cleavable linker, a sulfatase cleavable linker, a photo-responsive cleavable linker, or a bioorthogonal cleavable linker. In some embodiments, the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a hydrazone linker. In some embodiments, the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a non-cleavable linker.

[0017] In some embodiments, the antibody-drug further comprises a PEG spacer.

[0018] In some embodiments, the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 2 to about 5. In some embodiments, the DAR is about 4.

[0019] Provided herein is a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein, and a pharmaceutically acceptable carrier.

[0020] Provided herein is a method for treating a proliferative disorder in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate or a pharmaceutical composition provided herein. Provided herein is amethod for ameliorating a symptom of a proliferative disorder in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate or a pharmaceutical composition provided herein. In some embodiments, the proliferative disorder is a mast cell proliferative disorder. In some embodiments, the proliferative disorder is c-Kit-reliant. In some embodiments, the proliferative disorder is systemic mastocytosis, mast cell leukemia, or basophil leukemia.

[0021] Provided herein is a method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate or a pharmaceutical composition provided herein. Provided herein is a method for ameliorating a symptom of an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate or a pharmaceutical composition provided herein. In some embodiments, the inflammatory disorder is a mast cell inflammatory disorder. In some embodiments, the inflammatory disorder is c-Kit-reliant. In some embodiments, the inflammatory disorder is a chronic inflammatory disorder. In some embodiments, the inflammatory disorder is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0022] Provided herein is a use of an antibody-drug conjugate or a pharmaceutical composition provided herein in the preparation of a medicament.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows a schematic of an anti-CD203c-c-Kit inhibitor conjugate mechanism of action. Anti-CD203c conjugates are internalized on CD203c+granulocytes and trafficked to the lysosome where they are proteolytically processed, releasing free c-Kit inhibitor which potently inhibits c-Kit receptor activity, leading to leading to the suppression of c-Kit related granulocyte activation and degranulation, and / or cell death via apoptosis.

[0024] FIG. 2 shows a schematic of the targeting modality disclosed herein. A granulocyte (or CD203c) targeting antibody is conjugated via a linker to dasatinib, a dasatinib derivative, or another c-Kit inhibitor.

[0025] FIG. 3 shows results from a direct binding ELISA of anti-CD203c antibodies against human and rhesus CD203c. Purified anti-CD203c IgG-null antibodies weretitrated (in nM) in a direct binding ELISA against purified recombinant human and rhesus CD203c proteins.

[0026] FIG. 4A - FIG. 4B show results from an analysis of binding of anti-CD203c antibodies to cell-surface expressed CD203c on stimulated KU812 cells. Purified anti- CD203c IgG-null antibodies were titrated (in nM) in a flow-based assay against the basophilic cell line KU812 which expresses CD203c.

[0027] FIG. 5A - FIG. 5B show results from an analysis of internalization of anti- CD203c antibodies by TF1 (CD2O3c10) and KU812 (CD203chigh) cells. Purified anti- CD203c antibodies were tested for their ability to internalize using Fab-ZAP-mediated killing of cells as an indirect measure of internalization capacity. Internalization efficiency was dependent on level of CD203c expression.

[0028] FIG. 6A - FIG. 6B show results from biophysical characterization of anti- CD203c antibodies. Antibody aggregation propensity was measured using an affinitycapture self-interaction nanoparticle spectroscopy (AC-SINS) assay (FIG. 6A). The clinical antibodies alirocumab and bococizumab were used as negative and positive controls, respectively. In addition, antibody specificity was measured by direct ELISA against double-stranded DNA and insulin using bococizumab and briakinumab as positive controls (non-specific) and bevacizumab and ustekinumab as negative controls (antigen-specific) (FIG. 6B).

[0029] FIG. 7A - FIG. 7C show results from an analysis of internalization of 2.23- AF594 conjugate on KU812 cells. Internalization of fluorescently conjugated antibody 2.23 in KU812 cells was visualized using the Amnis® ImageStream® imaging flow cytometer platform (FIG. 7A). Internalization rate was measured at 4°C and 37°C (FIG. 7B), in addition to co-localization with LAMP2 as a marker of lysosomal uptake (FIG. 7C).

[0030] FIG. 8A - FIG. 8B show structures of the BTP-Glu(AA-cKit-inhibitor 1)- PEG(24u) (FIG. 8A) and BTP-Glu(AA-cKit-inhibitor 2)-PEG(24u) linker-payloads (FIG. 8B)

[0031] FIG. 9 shows synthesis of the BTP-Glu(AA-cKit-inhibitor l)-PEG(24u) linkerpayload. The linker-payload was prepared by adding a mixture of c-Kit inhibitor 1.2HC1 salt and BTP-Bl-Glu(OH)-PEG(24u) to HATU reagent prepared in DMF and N-MethylMorpholine (NMM).

[0032] FIG. 10 shows a RPLC (reversed-phase liquid chromatography) chromatogram of the BTP-Glu(AA-c-Kit inhibitor l)-PEG(24u) linker-payload. Detection was performed at 214 nm and 254 nm.

[0033] FIG. 11 shows a LC-MS ((liquid chromatography-mass spectrometry) spectrum of the BTP-Glu(AA-c-Kit inhibitor l)-PEG(24u) linker-payload. The purified linker-payload was diluted to 0.05 mg / mL and 10 pl was analyzed by LC-MS using a flow rate of 0.4 mL / min. The expected mass was confirmed via doubly and triply charged species.

[0034] FIG. 12 shows synthesis of the BTP-Glu(AA-cKit-inhibitor 2)-PEG(24u) linker-payload. The linker-payload was prepared by adding a mixture of c-Kit inhibitor 2.2HC1 salt and BTP-Bl-Glu(OH)-PEG(24u) to HATU reagent prepared in DMF and N-MethylMorpholine (NMM).

[0035] FIG. 13 shows a RPLC chromatogram of the BTP-Glu(AA-c-Kit inhibitor 2)- PEG(24u) linker-payload. Detection was performed at 214 nm and 254 nm.

[0036] FIG. 14 shows a LC-MS spectrum of the BTP-Glu(AA-c-Kit inhibitor 2)- PEG(24u) linker-payload. The purified linker-payload was diluted to 0.05 mg / mL and 10 pl was analyzed by LC-MS using a flow rate of 0.4 mL / min. The expected mass was confirmed via doubly and triply charged species.

[0037] FIG. 15 shows a deconvoluted LC-MS spectrum for 2.23-BTP c-Kit inhibitor1 ADC (1 / 2 mAb analysis).

[0038] FIG. 16A - FIG. 16B show results from analytical characterization of 2.23- BTP c-Kit inhibitor 1 ADC. FIG. 16A shows a hydrophobic interaction chromatogram at 280 nm comparing naked 2.23 antibody (2.23-IgG) and 2.23-ADC. FIG. 16A shows a size exclusion chromatogram at 280 nm for the 2.23-ADC compared to PBS buffer alone.

[0039] FIG. 17 shows a deconvoluted LC-MS spectrum for 2.23-BTP c-Kit inhibitor2 ADC (1 / 2 mAb analysis).

[0040] FIG. 18A - FIG. 18B show results from analytical characterization of 2.23- BTP c-Kit inhibitor 2 ADC. FIG. 18A shows a hydrophobic interaction chromatogram at 280 nm comparing naked 2.23 antibody (2.23-IgG) and 2.23-ADC. FIG. 18B shows a size exclusion chromatogram at 280 nm for the 2.23-ADC compared to PBS buffer alone.

[0041] FIG. 19 shows an analytical summary for 2.23-c-Kitl-ADC.

[0042] FIG. 20 shows an analytical summary for 2.23-c-Kit2-ADC.

[0043] FIG. 21A - FIG. 21B show results from an assessment of ADC potency in cell killing and c-Kit receptor inhibition assays. In FIG. 21A, 2.23-cKitl-ADC, 2.23-cKit2- ADC, 2.23-IgG and free payload were assessed in a KU812 cell killing assay using CellTiterGLO to measure viability. In FIG. 21B, potency of inhibition of 2.23-cKitl- ADC, 2.23-cKit2-ADC, 2.23-IgG and free payload were compared in a c-Kit receptor phosphorylation assay.

[0044] FIG. 22 shows results from a functional characterization of 2.23-cKit2-ADC. Potency of 2.23-cKit2-ADC, 2.23-IgG and free payload were compared in a KU812 cell killing assay. Both cell viability and cytotoxicity were measured at 24, 48 and 72h using CellTiterGLO and CellToxGreen, respectively.

[0045] FIG. 23 shows results from an analysis of specificity of 2.23-cKit2-ADC. Specificity of 2.23-cKit2-ADC targeting was demonstrated by comparing potency against a relevant isotype control-cKit2-ADC conjugate in KU812 cells after 72h incubation.

[0046] FIG. 24A - FIG. 24B show results from an analysis of selectivity of 2.23-cKit2- ADC cell killing. The effects of 2.23-cKit2-ADC, 2.23-IgG, isotype control-cKit2- ADC and free pay load treatment were measured in RKO cells. FIG. 24 A shows that RKO cells do not express CD203c or c-Kit. FIG. 24B shows that 2.23-cKit-ADC does not affect RKO cell viability.

[0047] FIG. 25A - FIG. 25B show results from an analysis of selectivity of 2.23-cKit2- ADC cell killing. The effects of 2.23-cKit2-ADC, 2.23-IgG, isotype control-cKit2- ADC and free payload treatment were measured in RXF393 cells. FIG. 25A shows that RXF393 cells express low levels of CD203c but not c-Kit. FIG. 25B shows that 2.23- cKit-ADC does not affect RXF393 cell viability.

[0048] FIG. 26A - FIG. 26B show results from an analysis of selectivity of 2.23-cKit2- ADC cell killing. The effects of 2.23-cKit2-ADC, 2.23-IgG, isotype control-cKit2- ADC and free payload treatment were measured in HepG2 cells. FIG. 26A shows that HepG2 cells express CD203c but not c-Kit. FIG. 26B shows that 2.23-cKit-ADC does not affect HepG2 cell viability.

[0049] FIG. 27A - FIG. 27D show linker variants for cKit2 payload testing in KU812 cells and results from viability and cytotoxicity studies. “dAla” refers to “delta-Ala”, a structure where one alanine in the linker has been removed. “dAlaAla” refers to a structure where both alanines in the linker have been removed.

[0050] FIG. 28A - FIG. 28B show structures and results from viability and cytotoxicity studies in KU812 cells testing anlotinib as a potential payload for a 2.23 ADC.

[0051] FIG. 29A - FIG. 29B show structures and results from viability and cytotoxicity studies in KU812 cells testing imatinib as a potential payload for a 2.23 ADC.

[0052] FIG. 30A - FIG. 30B show structures and results from viability and cytotoxicity studies in KU812 cells testing crenolanib as a potential payload for a 2.23 ADC.

[0053] FIG. 31A - FIG. 31B show structures and results from viability and cytotoxicity studies in KU812 cells testing ponatinib as a potential payload for a 2.23 ADC.

[0054] FIG. 32A - FIG. 32B show structures and results from viability and cytotoxicity studies in KU812 cells testing avapritinib as a potential payload for a 2.23 ADC.

[0055] FIG. 33A - FIG. 33B show structures and results from viability and cytotoxicity studies in KU812 cells testing elenestinib as a potential payload for a 2.23 ADC.

[0056] FIG. 34A - FIG. 34B show structures and results from viability and cytotoxicity studies in KU812 cells testing bezuclastinib as a potential payload for a 2.23 ADC.

[0057] FIG. 35A - FIG. 35B show structures and results from viability and cytotoxicity studies in KU812 cells testing midostaurin as a potential payload for a 2.23 ADC.

[0058] FIG. 36A - FIG. 36B show results from a pharmacokinetic analysis in rats of naked 2.23 vs 2.23-cKit 2 ADC.

[0059] FIG. 37 shows results from an in-vivo non-tumor bearing tolerability study of 2.23-cKit2 ADC using female NOD SCID gamma (NSG) mice.

[0060] FIG. 38A - FIG. 38B show results from an in-vivo efficacy study of 2.23-cKit2 ADC in a KU812 tumor model in NSG mice. Results are reported in both linear (FIG. 38A) and log (FIG. 38B) scales.

[0061] FIG 39 shows results from a dose-dependent in-vivo efficacy study of 2.23- cKit2 ADC in a KU812 tumor model in NSG mice.

[0062] FIG. 40 shows results from a multiple dose pharmacokinetics study of 2.23- cKit2 ADC in cynomolgus monkeys.

[0063] FIG. 41A - FIG. 411 show results from blood parameters measured in cynomolgus monkey after treatment with 2.23-cKit2 ADC. This panel included White Blood cells (FIG. 41A), Red blood cells (FIG. 41B), Hemoglobin (FIG. 41C), Platelets (FIG. 41D), Neutrophils (FIG. 41E), Lymphocytes (FIG. 41F), Monocytes (FIG. 41G), Eosinophils (FIG. 41H) and Basophils (FIG. 411).

[0064] FIG. 42A - FIG. 42D show results from a direct filter-binding radiometric kinase activity assay measuring the potency of cKit2 free payload in inhibiting tyrosine kinases including Bruton’s Tyrosine Kinase (BTK; FIG. 42A), wild-type c-Kit (FIG. 42B) and variant c-Kit (D816V; FIG. 42C). Calculated IC50 values are summarized in FIG. 42D

[0065] FIG. 43 shows results from a KINOMEscan competition assay (Eurofins) used to assess the selectivity of the cKit2 free payload compared to other benchmark tyrosine kinase inhibitors.DETAILED DESCRIPTION

[0066] The disclosure provides an anti-CD203c antibody or an antigen -binding portion thereof linked to a c-Kit inhibitor and medical uses thereof. It has been shown that both IgE-dependent and IgE-independent activation of mast cells leads to a robust upregulation of cell surface CD203c. CD203c is specific to the mast cell / basophil lineage and is not expressed on other hematopoietic stem cell precursors, unlike c-Kit. It has been shown that CD203c differentiates erythroid and granulocyte lineages. Therefore, mast cell / basophil targeting via CD203c provides the benefit of avoiding myelosuppression. Anti-CD203c antibodies and antigen-binding portions conjugated to small molecule c-Kit inhibitors have the potential to deliver greatly increased potency and specificity over prior approaches, thus improving the therapeutic index for mast cell proliferative disease. Antibody-drug conjugates (ADCs) provided herein are advantageous because they do not lead to systemic inhibition of c-Kit and adverse effects associated with such inhibition.ANTIBODIES AND ANTIGEN-BINDING PORTIONS

[0067] ADCs provided herein comprise anti-CD203c antibodies or antigen-binding portions. Anti-CD203c antibodies and antigen-binding portions that may be components of ADCs are described in more detail below.

[0068] Antibodies and antigen-binding portions disclosed herein specifically bind human CD203c. In some embodiments, antibodies and antigen-binding portions may cross-react with CD203c from species other than human, for example, cynomolgus monkey (Macaca fascicularis) CD203c. In some embodiments, an antibody may be specific for only human CD203c and may exhibit no non-human cross-reactivity.

[0069] The term “antibody” broadly refers to an immunoglobulin (Ig) molecule, generally, comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential target binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0070] In a full-length antibody, each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH domain) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable domain (abbreviated herein as VL domain) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH domain and VL domain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0071] The term “Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof. The numbering of the residues in the Fc region is according to the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. An Fc region can be present in dimer or monomeric form. The Fc region binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0072] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY) and class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2) or subclass. IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen combining domains, each composed of a VH) and a VL. Generally, IgA antibodies are composed of two monomers, each monomer composed of two heavy chains and two light chains (as for IgG, IgD, and IgE antibodies); in this way the IgA molecule has four antigen binding domains, each again composed of a VH and a VL. Certain IgA antibodies are monomeric in that they are composed of two heavy chains and two light chains. Secreted IgM antibodies are generally composed of five monomers, each monomer composed of two heavy chains and two light chains (as for IgG and IgE antibodies). Thus, the IgM molecule has ten antigen binding domains, each again composed of a VH and a VL. A cell surface form of IgM has a two heavy chain / two light chain structure similar to IgG, IgD and IgE antibodies.

[0073] The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD203c). It has been shown that the antigen-binding function of an antibody can be performed by portions or fragments of a full-length antibody. Examples of binding portions encompassed within the term “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb (domain antibody) fragment (Ward et al., (1989) Nature 341 :544-546; WO 90 / 05144 Al, each herein incorporated by reference in its entirety), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). The disclosure also encompasses a Fab' fragment. Fab' fragments can be formed by the reduction of F(ab')2 fragments. Fab' is derived from F(ab')2; therefore, it may contain a small portion of Fc. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Suchsingle chain antibodies are also intended to be encompassed within the term “antigenbinding portion” of an antibody. In some embodiments, scFv molecules may be incorporated into a fusion protein. In some embodiments, provided herein is a single chain camelid antibody. In some embodiments, provided herein is a shark heavy chain antibody (V-NAR). See, English et al. (2020) Antibody Therapeutics, 3(1): 1-9. Examples of antigen-binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.). In some embodiments, provided herein is a single domain antibody. In general, the term “antibody” when used herein encompasses an “antibody portion”. An antibody portion generally retains the antigen-binding properties of a full-length antibody.

[0074] Antibodies and antibody portions provided herein may be in multispecific (e.g., bispecific or trispecific) formats. Such multispecific molecules specifically bind to two or more different molecular targets or epitopes. In some embodiments, an antibody or antigen-binding portion provided herein is tetrameric, tetravalent or multispecific. In some embodiments, an antibody or an antigen-binding portion is a bispecific molecule that binds specifically to a first antigen and a second antigen, wherein the first antigen is CD203c, and the second antigen is not CD203c. In some embodiments, an antibody or an antigen-binding portion is a diabody. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123). In some embodiments, an antibody or an antigen-binding portion is a triabody, a tetrabody, a bis-scFv or a tandem scFv. In some embodiments, an antibody or an antigen-binding portion is a dual affinity re-targeting protein.

[0075] In some embodiments, an anti-CD203c antibody or antigen-binding portion disclosed herein is a Fab, a Fab', a F(ab')2, a Fv, a scFv, a maxibody, a minibody, a diabody, a triabody, a tetrabody, or a bis-scFv.

[0076] As used herein, the terms “immunological binding” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule (e.g., antibody or antigen-binding portion thereof) and an antigen for which the immunoglobulin is specific. The strength, or affinity, of immunological binding interactions can be expressed in terms of thedissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koir) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See, Malmqvist, Nature 361 : 186-187 (1993)). The ratio of Koir / Kon enables the cancellation of all parameters not related to affinity and is equal to the dissociation constant Kd. (See, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody or antigen-binding portion provided herein is said to specifically bind CD203c when the equilibrium binding constant (Kd) is <10 pM, preferably < 10 nM, more preferably < 10 nM, and most preferably < 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. In some embodiments, the Kd of an antibody is measured by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system. In some embodiments, the Kd of an antibody is measured by a platform based on bio-layer interferometry technology, such as the Octet system (Creative Biolabs, Shirley, NY).

[0077] In some embodiments, an anti-CD203c antibody or antigen-binding portion provided herein is monovalent or bivalent and comprises a single or double chain. Functionally, the binding affinity of an antibody or antigen-binding portion may be within the range of 10'5M to 10'12M. For example, the binding affinity of an antibody or antigen-binding portion is from 10'6M to 10'12M, from 10'7M to 10'12M, from 10’8M to IO’12M, from 10’9M to 10'12M, from 10’5M to 10-11M, from 10-6M to 10'11M, from 10'7M to 10'11M, from 10'8M to 10'11M, from 10'9M to 10'11M, from 10'10M to 10’11M, from 10’5M to IO’10M, from IO’6M to IO’10M, from IO’7M to IO’10M, from 10'8M to 10'10M, from 10'9M to 10'10M, from 10'5M to 10'9M, from 10'6M to 10'9M, from 10'7M to 10'9M, from 10'8M to 10'9M, from 10'5M to 10'8M, from 10’6M to 1 O’8M, from 1 O’7M to 1 O’8M, from 10’5M to 1 O’7M, from 1 O’6M to 1 O’7M or from 10'5M to 10'6M.

[0078] In some embodiments, an anti-CD203c antibody or antigen-binding portion is monoclonal. The term “monoclonal antibody” (Mab) refers to an antibody, or antigen-binding portion thereof, that is derived from a single copy or clone, including for example any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Preferably, a monoclonal antibody exists in a homogeneous or substantially homogeneous population.

[0079] In some embodiments, an anti-CD203c antibody or antigen-binding portion is human, humanized, or chimeric.

[0080] In some embodiments, an anti-CD203c antibody or antigen-binding portion is humanized. The term “humanized” is intended to refer to an antibody, or an antigenbinding portion thereof, that has been engineered to comprise one or more human framework regions in the variable domain together with non-human (e.g., mouse, rat, or hamster) CDRs of the heavy and / or light chain. In some embodiments, a humanized antibody comprises sequences that are entirely human except for the CDRs. An anti- CD203c antibody molecule or antigen-binding portion thereof may comprise one or more human variable domain framework scaffolds into which the CDRs have been inserted. In some embodiments, the VH domain, the VL domain, or both the VH domain and the VL domain of an anti-CD203c antibody or antigen-binding portion provided herein comprise one or more human framework region amino acid sequences. Examples of humanized antibodies and suitable techniques for their generation are provided in Hwang et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321 :522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239: 1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. 5,225,539; U.S. 5,530,101; U.S. 5,585,089; U.S. 5,693,761; U.S. 5,693,762; U.S. 6,180,370; and WO 90 / 07861, each of which is incorporated herein by reference in its entirety. When choosing FR to flank CDRs, for example when humanizing or optimizing an antibody, FRs from antibodies that contain CDR sequences in the same canonical class are preferred.

[0081] In some embodiments, an anti-CD203c antibody or antigen-binding portion is chimeric. The term “chimeric” is intended to refer to an antibody molecule, or an antigen-binding portion thereof, in which the variable domain sequences are derived from one species and at least one constant region sequence is derived from another species. For example, one or all the variable domains of the light chain(s) and / or one or all the variable domains of the heavy chain(s) of a mouse antibody (e.g., a mouse monoclonal antibody) may each be joined to a human constant region, such as, without limitation an IgGl or an IgG4 human constant region. Examples of chimeric antibodiesand suitable techniques for their generation are provided in U.S. 4,816,567; U.S. 4,975,369; and U.S. 4,816,397, each of which is incorporated herein by reference in its entirety.

[0082] In some embodiments, an anti-CD203c antibody or antigen-binding portion is human (e.g., fully human). A human antibody comprises human CDR sequences, human framework sequences and human constant region sequences.

[0083] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof that comprises one or more amino acid sequences of antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23. The combinations of VH domain, VL domain, and CDR sequences forming these antibodies are provided in Tables 1-23. In some embodiments, the VH domain sequence and / or the VL domain sequence comprises a signal sequence (also known as a signal peptide) at the amino-terminus.Table 1. Amino acid sequences of anti-CD203c antibody 2.1CDR sequences are underlined in variable region sequences.Table 2. Amino acid sequences of anti-CD203c antibody 2.2CDR sequences are underlined in variable region sequences.Table 3. Amino acid sequences of anti-CD203c antibody 2.3CDR sequences are underlined in variable region sequences.Table 4. Amino acid sequences of anti-CD203c antibody 2.4CDR sequences are underlined in variable region sequences.Table 5. Amino acid sequences of anti-CD203c antibody 2.5CDR sequences are underlined in variable region sequences.Table 6. Amino acid sequences of anti-CD203c antibody 2.6CDR sequences are underlined in variable region sequences.Table 7. Amino acid sequences of anti-CD203c antibody 2.7CDR sequences are underlined in variable region sequences.Table 8. Amino acid sequences of anti-CD203c antibody 2.8CDR sequences are underlined in variable region sequences.Table 9. Amino acid sequences of anti-CD203c antibody 2.9CDR sequences are underlined in variable region sequences.Table 10. Amino acid sequences of anti-CD203c antibody 2.10CDR sequences are underlined in variable region sequences.Table 11. Amino acid sequences of anti-CD203c antibody 2.11CDR sequences are underlined in variable region sequences.Table 12. Amino acid sequences of anti-CD203c antibody 2.12CDR sequences are underlined in variable region sequences.Table 13. Amino acid sequences of anti-CD203c antibody 2.13CDR sequences are underlined in variable region sequences.Table 14. Amino acid sequences of anti-CD203c antibody 2.14CDR sequences are underlined in variable region sequences.Table 15. Amino acid sequences of anti-CD203c antibody 2.15CDR sequences are underlined in variable region sequences.Table 16. Amino acid sequences of anti-CD203c antibody 2.16CDR sequences are underlined in variable region sequences.Table 17. Amino acid sequences of anti-CD203c antibody 2.17CDR sequences are underlined in variable region sequences.Table 18. Amino acid sequences of anti-CD203c antibody 2.18CDR sequences are underlined in variable region sequences.Table 19. Amino acid sequences of anti-CD203c antibody 2.19CDR sequences are underlined in variable region sequences.Table 20. Amino acid sequences of anti-CD203c antibody 2.20CDR sequences are underlined in variable region sequences.Table 21. Amino acid sequences of anti-CD203c antibody 2.21CDR sequences are underlined in variable region sequences.Table 22. Amino acid sequences of anti-CD203c antibody 2.22CDR sequences are underlined in variable region sequences.Table 23. Amino acid sequences of anti-CD203c antibody 2.23CDR sequences are underlined in variable region sequences.

[0084] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion cross-competes for binding to CD203c with antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23, or an antibody that comprises one or more amino acid sequences of antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23 (see Tables 1-23).

[0085] The terms “cross-compete”, “cross-competition”, “cross-block”, “crossblocked” and “cross-blocking” are used interchangeably herein to mean the ability of an antibody or an antigen-binding portion thereof to interfere with the binding directly or indirectly through allosteric modulation of the anti-CD203c antibodies of the disclosure to the target CD203c (e.g., human CD203c). The extent to which an antibody or portion thereof is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block or cross-compete, can be determined using competition binding assays. One example of a binding competition assay is Homogeneous Time Resolved Fluorescence (HTRF). One particularly suitable quantitative cross-competition assay uses a FACS- or an Alphascreen-based approach to measure competition between the labelled (e.g., His-tagged, biotinylated or radioactive labelled) antibody or portion thereof and the other antibody or portion thereof in terms of their binding to the target. In general, a cross-competing antibody or portion thereof is, for example, one which will bind to the target in the crosscompetition assay such that, during the assay and in the presence of a second antibody or portion thereof, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g. in a FACS-based competition assay) of the maximum theoretical displacement (e.g. displacement by cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked) by the potentially cross-blocking antibody or fragment thereof that is present in a givenamount. In some embodiments, cross-competing antibodies or portions thereof have a recorded displacement that is between 10% and 100%, or between 50% and 100%.

[0086] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein:(a) the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;(b) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;(c) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 18; a HCDR2 comprising SEQ ID NO: 19; and a HCDR3 comprising SEQ ID NO: 20; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 22; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 23;(d) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 26; and a HCDR3 comprising SEQ ID NO: 27; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 29; a LCDR2 comprising SEQ ID NO: 30; and a LCDR3 comprising SEQ ID NO: 31;(e) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 34; and a HCDR3 comprising SEQ ID NO: 35; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 37; a LCDR2 comprising SEQ ID NO: 38; and a LCDR3 comprising SEQ ID NO: 39;(f) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;(g) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;(h) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 57; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 59; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 60;(i) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 66; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(j) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(k) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 76; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 77;(l) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 79; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 81; a LCDR2 comprising SEQ ID NO: 82; and a LCDR3 comprising SEQ ID NO: 83;(m) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 85; a HCDR2 comprising SEQ ID NO: 86; and a HCDR3 comprising SEQ ID NO: 87; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 89; a LCDR2 comprising SEQ ID NO: 90; and a LCDR3 comprising SEQ ID NO: 91;(n) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;(o) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO:103; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;(p) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;(q) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;(r) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 121; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 123; and a LCDR3 comprising SEQ ID NO: 124;(s) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;(t) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;(u) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 139; a HCDR2 comprising SEQ ID NO: 140; and a HCDR3 comprising SEQ ID NO: 141; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 143;(v) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO:145; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 147; a LCDR2 comprising SEQ ID NO: 148; and a LCDR3 comprising SEQ ID NO: 149; or(w) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.

[0087] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein:(a) the VH domain amino acid sequence comprises SEQ ID NO: 1, and the VL domain amino acid sequence comprises SEQ ID NO: 5;(b) the VH domain amino acid sequence comprises SEQ ID NO: 9, and the VL domain amino acid sequence comprises SEQ ID NO: 13;(c) the VH domain amino acid sequence comprises SEQ ID NO: 17, and the VL domain amino acid sequence comprises SEQ ID NO: 21;(d) the VH domain amino acid sequence comprises SEQ ID NO: 24, and the VL domain amino acid sequence comprises SEQ ID NO: 28;(e) the VH domain amino acid sequence comprises SEQ ID NO: 32, and the VL domain amino acid sequence comprises SEQ ID NO: 36;(f) the VH domain amino acid sequence comprises SEQ ID NO: 40, and the VL domain amino acid sequence comprises SEQ ID NO: 44;(g) the VH domain amino acid sequence comprises SEQ ID NO: 47, and the VL domain amino acid sequence comprises SEQ ID NO: 51;(h) the VH domain amino acid sequence comprises SEQ ID NO: 55, and the VL domain amino acid sequence comprises SEQ ID NO: 58;(i) the VH domain amino acid sequence comprises SEQ ID NO: 61, and the VL domain amino acid sequence comprises SEQ ID NO: 65;(j) the VH domain amino acid sequence comprises SEQ ID NO: 68, and the VL domain amino acid sequence comprises SEQ ID NO: 70;(k) the VH domain amino acid sequence comprises SEQ ID NO: 72, and the VL domain amino acid sequence comprises SEQ ID NO: 75;(l) the VH domain amino acid sequence comprises SEQ ID NO: 78, and the VL domain amino acid sequence comprises SEQ ID NO: 80;(m) the VH domain amino acid sequence comprises SEQ ID NO: 84, and the VL domain amino acid sequence comprises SEQ ID NO: 88;(n) the VH domain amino acid sequence comprises SEQ ID NO: 92, and the VL domain amino acid sequence comprises SEQ ID NO: 96;(o) the VH domain amino acid sequence comprises SEQ ID NO: 100, and the VL domain amino acid sequence comprises SEQ ID NO: 104;(p) the VH domain amino acid sequence comprises SEQ ID NO: 108, and the VL domain amino acid sequence comprises SEQ ID NO: 112;(q) the VH domain amino acid sequence comprises SEQ ID NO: 116, and the VL domain amino acid sequence comprises SEQ ID NO: 117;(r) the VH domain amino acid sequence comprises SEQ ID NO: 120, and the VL domain amino acid sequence comprises SEQ ID NO: 122;(s) the VH domain amino acid sequence comprises SEQ ID NO: 125, and the VL domain amino acid sequence comprises SEQ ID NO: 129;(t) the VH domain amino acid sequence comprises SEQ ID NO: 133, and the VL domain amino acid sequence comprises SEQ ID NO: 134;(u) the VH domain amino acid sequence comprises SEQ ID NO: 138, and the VL domain amino acid sequence comprises SEQ ID NO: 142;(v) the VH domain amino acid sequence comprises SEQ ID NO: 144, and the VL domain amino acid sequence comprises SEQ ID NO: 146; or(w) the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

[0088] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein: the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83. Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein: the VH domain amino acidsequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

[0089] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain sequence, the VL domain sequence, or both the VH domain and the VL domain sequences comprise an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the VH domain sequence, the VL domain sequence, or both the VH domain and the VL domain sequences of antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23.

[0090] Provided herein is an anti-CD203c antibody or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH and VL domain sequences are those of antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23, with 1, 2 or 3 conservative amino acid substitutions in the VH domain sequence, the VL domain sequence, or both the VH domain and the VL domain sequences. In some embodiments, conservative amino acid substitutions are made only in the FR sequences and not in the CDR sequences of an antibody or antigen-binding portion.

[0091] The present disclosure encompasses modifications to the amino acid sequence of the antibody molecule or antigen-binding portion thereof as defined herein. For example, the disclosure includes antibody molecules and corresponding antigenbinding portions thereof comprising functionally equivalent variable domains and CDRs which do not significantly affect their properties as well as variants which have enhanced or decreased activity and / or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with the desired binding affinity to CD203c. Insertions which include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues, are envisaged. Examples of terminal insertions include an antibody molecule with an N-terminal methionyl residue or the antibody molecule fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

[0092] In some embodiments, the anti-CD203c antibody or antigen-binding portion provided herein may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. The antibody or antigen-binding portion may be mutated to alter such post-translational modifications, for example by adding, removing or replacing one or more amino acid residues to form or remove a glycosylation site.

[0093] In some embodiments, the anti-CD203c antibody or antigen-binding portion provided herein may be modified for example by amino acid substitution to remove potential proteolytic sites in the antibody or portion.

[0094] In some embodiments, an anti-CD203c antibody or an antigen-binding portion thereof comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY. In some embodiments, the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2. In some embodiments, the immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises one or more mutations to increase FcyR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP) activity, and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region comprises one or more mutations to reduce or prevent FcyR binding, ADCC activity, ADCP activity and / or CDC activity. In some embodiments, the immunoglobulin constant region is a wild-type human IgGl constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgGl constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S or a human IgG4 constant region comprising the amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat. In some embodiments, a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to the EU index as in Kabat (Ward et cd.. 1995 Therap. Immunol. 2:77-94).

[0095] In some embodiments, an anti-CD203c antibody or an antigen-binding portion thereof may comprise an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.

[0096] In some embodiments, an anti-CD203c antibody may comprise an immunoglobulin constant region comprising any one of the amino acid sequences in Table 30. The Fc region sequences in Table 30 begin at the CHI domain. In some embodiments, an anti-CD203c antibody may comprise an immunoglobulin constant region comprising an amino acid sequence of an Fc region of human IgG4 or human IgGl. In some embodiments, an anti-CD203c antibody may comprise an immunoglobulin constant region comprising an amino acid sequence of an Fc region of human IgG4, human IgG4(S228P), human IgG2, human IgGl, human IgGl effector null. For example, the human IgG4(S228P) Fc region comprises the following substitution compared to the wild-type human IgG4 Fc region: S228P. For example, the human IgGl effector null Fc region comprises the following substitutions compared to the wild-type human IgGl Fc region: L234A, L235A and G237A. In some embodiments, an anti-CD203c antibody may comprise an immunoglobulin constant region comprising the amino acid sequence of any one of SEQ ID NOS: 155-160. In some embodiments, an anti-CD203c antibody may comprise the six CDR amino acid sequences of any one of the clones in Tables 1-23 and any one of the Fc region amino acid sequences in Table 24. In some embodiments, an anti-CD203c antibody may comprise an immunoglobulin heavy chain constant region comprising any one of the Fc region amino acid sequences in Table 24 and an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.Table 24. Exemplary immunoglobulin constant region amino acid sequencesNUCLEIC ACID MOLECULES, VECTORS, HOST CELLSAND METHODS OF PRODUCING ANTIBODIES

[0097] Provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding an amino acid sequence of an anti-CD203c antibody or anti- CD203c antigen-binding portion described herein (or an amino acid sequence of a (i) VH domain, (ii) a VL domain, or (iii) both a VH domain and a VL domain of an antibody or antigen-binding portion). Further provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding (i) a heavy chain, (ii) a light chain, or (iii) both a heavy chain and a light chain of an anti-CD203c antibody or anti- CD203c antigen-binding portion described herein. In some embodiments, a nucleicacid molecule encoding a VH domain, a VL domain, a heavy chain or a light chain comprises a signal sequence. In some embodiments, a nucleic acid molecule encoding a VH domain, a VL domain, a heavy chain or a light chain does not comprise a signal sequence.

[0098] Also provided herein is an expression vector comprising a nucleic acid molecule described herein. In certain vectors, a nucleic acid molecule is operatively linked to one or more regulatory sequences suitable for expression of the nucleic acid segment in a host cell. In some cases, an expression vector comprises sequences that mediate replication and comprises one or more selectable markers. As used herein, “vector” means a construct that is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0099] Provided herein is a recombinant host cell comprising an expression vector or a nucleic acid molecule disclosed herein. A “host cell” includes an individual cell, a cell line or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. An expression vector can be transfected into a host cell by standard techniques. Non-limiting examples include electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. In some embodiments, a recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both a VH domain and a VL domain of an anti-CD203c antibody or an antigen-binding portion thereof. In some embodiments, a recombinant host cell comprises (i) a first vector or a first nucleic acid molecule encoding a VH domain of an anti-CD203c antibody or an antigen-binding portion thereof and (ii) a second vector or a second nucleic acid molecule encoding a VL domain of an anti-CD203c antibody or an antigen-binding portion thereof.

[0100] Antibody molecules of the invention, or antigen-binding portion thereof, can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies, computationaltechnologies or combinations of such technologies or other technologies readily known in the art.

[0101] Further provided herein is a method for producing an anti-CD203c antibody or an antigen-binding portion thereof, the method comprising: culturing a recombinant host cell comprising an expression vector described herein under conditions whereby the nucleic acid segment is expressed, thereby producing the anti-CD203c antibody or antigen-binding portion. The antibody or antigen-binding portion may then be isolated from the host cell or culture. Anti-CD203c antibodies and antigen-binding portions thereof can be produced by any of a variety of methods known to those skilled in the art. In certain embodiments, anti-CD203c antibodies and antigen-binding portions thereof can be produced recombinantly. For example, nucleic acid sequences encoding one or more of SEQ ID NOS: 1-154, or portions thereof, may be introduced into a bacterial cell (e.g., E. coli, B. subtilis) or a eukaryotic cell (e.g., a yeast such as S. cerevisiae, or a mammalian cell such as a CHO cell line, various Cos cell lines, a HeLa cell, a HEK293 cell, various myeloma cell lines, or a transformed B-cell or hybridoma), or into an in vitro translation system, and the translated polypeptide may be isolated. In some embodiments, antibody light chain proteins and heavy chain proteins are produced in a cell with a signal sequence that is removed upon production of a mature anti-CD203c antibody or antigen-binding portion thereof.

[0102] Those skilled in the art will be able to determine whether an antibody or antigenbinding portion comprising a given polypeptide sequence binds to CD203c protein without undue experimentation using standard methodologies, for example, Western blots, ELISA, and the like.C-KIT INHIBITORS, LINKERS, AND ADCs

[0103] ADCs provided herein comprise small molecule c-Kit inhibitors. A c-Kit inhibitor may be referred to as a “payload” in this disclosure. c-Kit inhibitors that may be components of ADCs are described in more detail below. In some embodiments, a c-Kit inhibitor inhibits multiple tyrosine kinases, including c-Kit.

[0104] In some embodiments, a c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib.

[0105] In some embodiments, a c-Kit inhibitor is dasatinib. Dasatinib is described, for example, in US 6,596,746. Polymorphic forms of dasatinib are described in, for example, US 11,059,813.

[0106] For all c-Kit inhibitors provided herein, the c-Kit inhibitor can include all stereoisomeric forms, mixtures of stereoisomeric forms, diastereomeric forms, and pharmaceutically acceptable salts thereof. The term “pharmaceutically acceptable salts” refers to derivatives of the disclosed inhibitors wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present disclosure include the non-toxic salts of the parent compound formed, e.g., from nontoxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.

[0107] In some embodiments, ADCs provided herein comprise a c-Kit inhibitor linked to an anti-CD203c antibody or antigen-binding portion via a linker.

[0108] A linker is a bifunctional or multifunctional moiety that can be used to link one or more c-Kit inhibitor drug moi eties to an anti-CD203c antibody or antigen-binding portion to form an ADC. In some embodiments, an ADC can be prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody or antigen-binding portion. For example, in some embodiments, a cysteine thiol of an antibody or antigen-binding portion can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC. In some embodiments, the linker is attached to an antibody or antigen-binding portion at an available amino nitrogen atom (z.e., a primary or secondary amine, rather than an amide) or a hydroxylic oxygen atom, or to an available sulfhydryl, such as on a cysteine.

[0109] In some embodiments, a linker is divalent, meaning it can link only one drug molecules to an antibody, or it can be trivalent (able to link two drug molecules to an antibody), or it can be polyvalent. Trivalent, tetravalent, and polyvalent linkers can be used to increase the loading of drug on an antibody, increasing the drug-to-antibody ratio (DAR) without requiring additional sites on the antibody for attaching linkers. Such linkers are known in the art, see e.g., US 6,638,499; US 10,214,560.

[0110] In some embodiments, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting examples of such reactive functionalities include maleimide, haloacetamides, a- haloacetyl, pyridyl disulfide, activated esters such as succinimide esters, N-hydroxysuccinimide, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method described in Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.[OHl] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Nonlimiting examples of such electrophilic groups include aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting examples of such reactive functionalities include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0112] In some embodiments, a linker may comprise one or more linker components, including but not limited to, a stretcher unit, a peptidomimetic unit, a peptide unit, and a spacer unit. In some embodiments, an ADC provided herein comprises a polyethylene glycol (PEG) spacer. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alaninephenylalanine (“ala-phe”), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N- maleimidomethyl) cyclohexane- 1 carboxylate (“MCC”).

[0113] In some embodiments, a linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting examples of cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); US 5,208,020). Acid-labile linkers are linkers cleavable at acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid- labile linkers. In some embodiments, photolabile linkers are linkers that are useful at the body surface and in body cavities that are accessible to light. Photolabile linkers may also be cleaved by infrared light that can penetrate tissue.

[0114] In some embodiments, a linker is an acid cleavable linker. Cleavage of these linkers is induced by low pH of the tumor acidic microenvironment or lysosomes. Nonlimiting examples include hydrazone, carbonate, and silyl ether linkers.

[0115] In some embodiments, a linker is a glutathione (GSH) cleavable linker. Cleavage of these linkers is induced by high level of GSH in the cytoplasm. Nonlimiting examples include a disulfide linker.

[0116] In some embodiments, a linker is a Fe(II) cleavable linker. Cleavage of these linkers is induced by elevating levels of ferrous iron. Nonlimiting examples include a 1, 2, 4-tri oxolane linker.

[0117] In some embodiments, a linker is a cathepsin cleavable linker. Cleavage of these linkers is induced by cathepsin in lysosomes. Nonlimiting examples include dipeptide, triglycyl (CX), and cyclobutane- 1,1 -dicarboxamide (cBu)-Cit linkers.

[0118] In some embodiments, a linker is a glycosidase cleavable linker. Cleavage of these linkers is induced by P-glucuronidase or P-galactosidase in lysosomes. Nonlimiting examples include P-glucuronide and P-galactoside linkers.

[0119] In some embodiments, a linker is a phosphatase cleavable linker. Cleavage of these linkers is induced by phosphatase and pyrophosphates in lysosomes. Nonlimiting examples include a pyrophosphate linker.

[0120] In some embodiments, a linker is a sulfatase cleavable linker. Cleavage of these linkers is induced by sulfatase in lysosomes. Nonlimiting examples include an aryl sulfate linker.

[0121] In some embodiments, a linker is a photo-responsive cleavable linker. Cleavage of these linkers is induced by irradiation with near-infrared (NIR) light (X = 650-900 nm); irradiation with UV light (X = 365 nm); or irradiation with NIR light (X = 650-900 nm) and intramolecular addition reaction with nearby amine. Nonlimiting examples include heptamethine cyanine fluorophore, O-nitrobenzyl, and PC4AP linkers.

[0122] In some embodiments, a linker is a bioorthogonal cleavable linker. Cleavage of these linkers is induced by the bioorthogonal cleavage pair: Cu(I)-BTTAA / dsProc. Nonlimiting examples include a dsProc linker.

[0123] In some embodiments, a linker may be a “non-cleavable linker”. Non-cleavable linkers are any chemical moiety capable of linking a drug to an antibody in a stable, covalent manner that does not fall off under the categories listed above for cleavable linkers. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage. Furthermore, non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, photolabile-cleaving agent, a peptidase, an esterase, or a chemical orphysiological compound that cleaves a disulfide bond, at conditions under which the drug or the antibody does not lose its activity. In some embodiments, the active part of an ADC with a non-cleavable linker is composed of an amino acid appendage, a linker and a cytotoxic payload. Nonlimiting examples include 2-(maleimidom ethyl)- 1,3- dioxane (MD) linkers, Mal-PAB linkers, and PEG linkers with intermediates of alkyne, triazole and piperazine.

[0124] Additional examples of linkers are described in US 7,498,298.

[0125] In some embodiments, a linker is a peptide linker. In some embodiments, a peptide linker comprises two amino acid residues independently selected from glycine (Gly), alanine (Ala), phenylalanine (Phe), lysine (Lys), arginine (Arg), valine (Vai), and citrulline (Cit). In some embodiments, a peptide linker comprises at least one unnatural amino acid.

[0126] In some embodiments, the peptide linker is an alanine-alanine (Ala-Ala) linker. In some embodiments, the peptide linker is L-Ala-L-Ala, D-Ala-D-Ala, D-Ala-L-Ala, or L-Ala-D-Ala.

[0127] In some embodiments, the peptide linker is a Vai-Ala linker or a Val-Cit linker.

[0128] Linker-payload intermediate compounds may be synthesized, purified, and isolated by methods provided in the Examples, or other methods known in the art. In some embodiments, bis-tolylsulfonyl-propanoyl benzamide (BTP) is used to synthesize a linker-payload. Purification and isolation methods are known in the art and include precipitation, crystallization, filtration, centrifugation, ultrafiltration, and various chromatographic techniques. Chromatography can include, for example, reverse-phase and normal phase; size exclusion; ion exchange; high, medium and low pressure liquid chromatography methods and apparatus; small scale analytical; simulated moving bed (SMB) and preparative thin or thick layer chromatography, as well as techniques of small scale thin layer and flash chromatography. In some embodiments, the completed reaction mixture may be evaporated to dryness followed by re-dissolution in a polar aprotic solvent. The solution may be filtered and then precipitated by combining the solution with a nonpolar antisolvent such as, for instance, hexane or cyclohexane. The precipitate may then be collected by filtration, optionally washed, and then dried.

[0129] In some embodiments, an antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer. In some embodiments, an antibody-drug conjugate comprises dasatinib (or a dasatinib derivative), a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0130] Provided herein is the molecule referred to as “(BTP)-Glu-(AA-c-kit inhibitor l)-PEG(24u) linker-payload” in the Examples and Drawings. Provided herein is the molecule referred to as “BTP-Glu-(AA-c-kit inhibitor 2)-PEG(24u) linker-payload” in the Examples and Drawings.

[0131] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a c-Kit inhibitor, wherein the antibody-drug conjugate comprises the c-Kit inhibitor and a linker comprising the structure:(a)

[0132] Antibody conjugation to linker-payload intermediate compounds may be performed by methods provided in the Examples, or other methods known in the art.

[0133] In some embodiments, site-specific conjugation technology through antibody engineering is used for antibody conjugation to the linker. In some embodiments, chemical structural modification of the linker-antibody attachment is used for antibody conjugation to the linker.

[0134] In some embodiments, the c-Kit inhibitor is linked to the immunoglobulin constant region of the anti-CD203c antibody or antigen-binding portion.

[0135] In some embodiments, conjugation is performed via maleimide structure conjugation, bis(vinylsulfonyl)piperazine conjugation, A -methyl -A - phenylvinylsulfonamide conjugation, or Pt(II)-based conjugation.

[0136] An ADC composition generally comprises a mixture of antibody-drug conjugate compounds, with varied numbers of drug molecules conjugated per antibody. In some embodiments, the average drug-to-antibody ratio (DAR) in the mixture of antibody-drug conjugate compounds is about 2 to about 5. In some embodiments, the average DAR is about 4. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine residues. The average DAR in preparations of antibody-drug conjugates from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.

[0137] Following conjugation, ADCs may be purified and separated from unconjugated reactants and / or conjugate aggregates by purification methods known in the art such as, for example and not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, and combinations thereof.

[0138] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.

[0139] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprisingSEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and aLCDR3 comprising SEQ ID NO: 83.

[0140] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and aLCDR3 comprising SEQ ID NO: 83; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0141] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor; wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

[0142] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

[0143] Provided herein is an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153; and wherein the antibodydrug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0144] Provided herein is the molecule referred to as “2.23-cKitl-ADC” in the Examples and Drawings. Provided herein is the molecule referred to as “2.23-cKit2- ADC” in the Examples and Drawings.PHARMACEUTICAL COMPOSITIONS

[0145] The ADCs provided herein (also referred to herein as “active compounds”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an ADC, and a pharmaceutically acceptable carrier, diluent or excipient. Such materials should be non-toxic and should not interfere with the efficacy of the ADC. The precise nature of the carrier or other material will depend on the route of administration, which may be by injection, bolus, infusion, or any other suitable route, as discussed below.

[0146] In some embodiments of the pharmaceutical compositions disclosed herein, the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83; and wherein the antibodydrug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0147] In some embodiments of the pharmaceutical compositions disclosed herein, the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0148] As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.” As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutically acceptable carrier, diluent or excipient may be a compound or a combination of compounds that does not provoke secondary reactions and that allows, for example, facilitation of the administration of the ADC, an increase in its lifespan and / or in its efficacy in the body or an increase in its solubility in solution.

[0149] A pharmaceutical composition disclosed herein may be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (z.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0150] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In some cases, the composition is sterile and fluid to the extent that easy syringeabilityexists. In some cases, the composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0151] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0152] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®, or com starch; a lubricantsuch as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0153] For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0154] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0155] The pharmaceutical agents can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0156] In some embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.

[0157] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0158] In some embodiments, the ADC may be provided in a lyophilized form for reconstitution prior to administration. For example, lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0159] The pharmaceutical compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.MEDICAL USES OF ADCs

[0160] Provided herein are methods and uses of ADCs and pharmaceutical compositions disclosed herein for providing a therapeutic benefit to a subject with a proliferative disorder. Provided herein are methods and uses of ADCs and pharmaceutical compositions disclosed herein for providing a therapeutic benefit to a subj ect with an inflammatory disorder. The therapeutic ADCs provided herein combine potency and specificity of targeting with minimal side effects. This is particularly important in the case of indolent systemic mastocytosis, which represents more than 90% of cases, where most patients have a normal life span. In these cases, adverse events associated with treatment often outweigh the burden of chronic disease symptoms, from a quality-of-life perspective.

[0161] An ADC or pharmaceutical composition disclosed herein may be used in a method of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., treatment before the onset of a condition in a subject to reduce the risk of the condition occurring in the subject; delay its onset; or reduce its severity after onset). The method of treatment may comprise administering the ADC or pharmaceutical composition to a subject in need thereof.

[0162] Provided herein is a method for treating a proliferative disorder in a subject, the method comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein. Provided herein is a method for ameliorating a symptom of a proliferative disorder in a subject, the method comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein. Provided herein is an ADC or a pharmaceutical composition disclosed herein for use in the treatment of a proliferative disorder.

[0163] In some embodiments, the proliferative disorder is a mast cell proliferative disorder. In some embodiments, the proliferative disorder is c-Kit-reliant. In someembodiments, the proliferative disorder is a mast cell proliferative disorder and is c- Kit-reliant.

[0164] In some embodiments, the proliferative disorder is systemic mastocytosis, mast cell leukemia, or basophil leukemia.

[0165] Provided herein is a method for treating an inflammatory disorder in a subject, the method comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein. Provided herein is a method for ameliorating a symptom of an inflammatory disorder in a subject, the method comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein. Provided herein is an ADC or a pharmaceutical composition disclosed herein for use in the treatment of an inflammatory disorder.

[0166] In some embodiments, the proliferative disorder is a mast cell inflammatory disorder. In some embodiments, the inflammatory disorder is c-Kit-reliant. In some embodiments, the inflammatory disorder is a mast cell inflammatory disorder and is c- Kit-reliant.

[0167] In some embodiments of the methods and uses disclosed herein, the inflammatory disease is a chronic inflammatory disease. In some embodiments of the methods and uses disclosed herein, the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0168] In some embodiments of the methods and uses disclosed herein, the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0169] In some embodiments of the methods and uses disclosed herein, the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VHdomain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.

[0170] As used herein, the term “effective amount” or “therapeutically effective amount” refers to the amount of a pharmaceutical agent, e.g., an ADC or a pharmaceutical composition disclosed herein, which is sufficient to reduce or ameliorate the severity and / or duration of a proliferative disorder or an inflammatory disorder, or one or more symptoms thereof, prevent the advancement of a disease, cause regression of a disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disease, or enhance or improve the prophylactic or therapeutic effect(s) of another related therapy (e.g., prophylactic or therapeutic agent) for a proliferative disorder or an inflammatory disorder.

[0171] The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and / or progression of a disease being treated. Appropriate doses of antibody -based protein molecules are well known in the art (Ledermann J. A. et al., 1991, Int. J. Cancer659-664; Bagshawe K.D. et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of an antibody-based protein molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody-based protein is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody-based protein, and the nature of any detectable label or other molecule attached to the antibody -based protein.

[0172] A typical ADC dose will be in the range 100 pg to 1 g for systemic applications, and 1 pg to 1 mg for intradermal injection. An initial higher loading dose, followed by one or more lower doses, may be administered. A dose for a single treatment of an adult subject may be proportionally adjusted for children and infants. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for a subject may be dependent on the pharmacokinetic and pharmacodynamic properties of the ADC composition, the route of administration and the nature of the condition being treated.

[0173] Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g., about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Treatment may be given before, and / or after surgery, and / or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.

[0174] In some embodiments, an ADC or a pharmaceutical composition disclosed herein may be administered as a subcutaneous injection. Subcutaneous injections may be administered using an auto-injector, for example for long term prophylaxis / treatment.

[0175] In some embodiments, the therapeutic effect of an ADC or a pharmaceutical composition disclosed herein may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of an ADC or a pharmaceutical composition disclosed herein may persist in a subject for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.

[0176] In some embodiments, a subject may be treated with an ADC or a pharmaceutical composition disclosed herein and an additional therapeutic agent or therapy that is used to treat a cancer or a symptom or complication of a proliferative disorder or an inflammatory disorder. The ADC or pharmaceutical composition disclosed herein and the additional therapeutic agent or therapy may be administered simultaneously or sequentially.

[0177] In some embodiments, a subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a guinea pig, a mouse or a rat. In some embodiments, a subject is an adult human. In some embodiments, a subject is a pediatric human.

[0178] Further provided herein is an ADC or a pharmaceutical composition disclosed herein, for use in the treatment of a disease or a disorder.

[0179] Provided herein is an ADC or a pharmaceutical composition disclosed herein, for use as a medicament.DEFINITIONS

[0180] The term “a” or “an” refers to one or more of that entity, i.e., can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0181] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0182] The terms “amino-terminal”, “N-terminus”, “carboxyl-terminal”, and “C- terminus” are used herein to denote positions within polypeptide chains. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl-terminus of the reference sequence but is not necessarily at the carboxyl-terminus of the complete polypeptide.

[0183] CD203c (also known as ENPP3, B10, NPP3, PDNP3, PD-IBETA, and ectonucleotide pyrophosphatase / phosphodiesterase 3) is a type II transmembrane protein that belongs to the ectonucleotide pyrophosphatase / phosphosdiesterase 3 (E- NPP3) family of enzymes involved in hydrolysis of oligonucleotides and nucleoside phosphates. CD203c is specific to the mast cell / basophil lineage of hematopoietic effector cells.

[0184] c-Kit (also known as KIT, Cluster of Differentiation 117 (CD117), PBT, SCFR, KIT proto-oncogene receptor tyrosine kinase) is a transmembrane protein that belongs to the immunoglobulin superfamily and binds to the soluble factor SCF (stem cell factor). c-Kit is a receptor tyrosine kinase type III that is highly expressed by hematopoietic stem cells as well as multiple other cell types, such as mature mast cells.

[0185] As used herein, the term “conservative substitution” refers to replacement of an amino acid with another amino acid which does not significantly deleteriously change the functional activity. A preferred example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919):

[0186] As used herein, the term “HCDR” refers to a heavy chain complementarity determining region. As used herein, the term “LCDR” refers to a light chain complementarity determining region.

[0187] The term “isolated molecule” (where the molecule is, for example, a protein, a polypeptide, a polynucleotide, an antibody or an antigen-binding molecule) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be “isolated” from its naturally associated components. A molecule alsomay be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by several methods known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide. For certain purposes, higher resolution may be provided by using high-performance liquid chromatography (HPLC) or other means known in the art for purification.

[0188] As used herein, the term “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, z.e., the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Percentage identity can be calculated using the alignment program Clustal Omega, available at ebi.ac.uk / Tools / msa / clustalo using default parameters. See, Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega” (2011 October 11) Molecular Systems Biology 7:539. For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.

[0189] As used herein, the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) mean that the severity of the subject's condition is reduced, at least partially improved or stabilized and / or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder.

[0190] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously.

[0191] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portionsof documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term’s definition in the application, the definition that appears in this application controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.NUMBERED EMBODIMENTS

[0192] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[0193] Embodiment 1. An antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor.

[0194] Embodiment 2. The antibody-drug conjugate of embodiment 1, wherein the antibody or antigen-binding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:(a) the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;(b) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;(c) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 18; a HCDR2 comprising SEQ ID NO: 19; and a HCDR3 comprising SEQ ID NO: 20;and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 22; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 23;(d) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 26; and a HCDR3 comprising SEQ ID NO: 27; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 29; a LCDR2 comprising SEQ ID NO: 30; and a LCDR3 comprising SEQ ID NO: 31;(e) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 34; and a HCDR3 comprising SEQ ID NO: 35; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 37; a LCDR2 comprising SEQ ID NO: 38; and a LCDR3 comprising SEQ ID NO: 39;(f) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;(g) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;(h) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 57; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 59; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 60;(i) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 66; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(j) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(k) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 76; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 77;(l) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 79; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 81; a LCDR2 comprising SEQ ID NO: 82; and a LCDR3 comprising SEQ ID NO: 83;(m) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 85; a HCDR2 comprising SEQ ID NO: 86; and a HCDR3 comprising SEQ ID NO: 87; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 89; a LCDR2 comprising SEQ ID NO: 90; and a LCDR3 comprising SEQ ID NO: 91;(n) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;(o) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO: 103; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;(p) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;(q) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;(r) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 121; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 123; and a LCDR3 comprising SEQ ID NO: 124;(s) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ IDNO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;(t) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;(u) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 139; a HCDR2 comprising SEQ ID NO: 140; and a HCDR3 comprising SEQ ID NO: 141; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 143;(v) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 145; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 147; a LCDR2 comprising SEQ ID NO: 148; and a LCDR3 comprising SEQ ID NO: 149; or(w) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.

[0195] Embodiment 3. The antibody-drug conjugate of embodiment 2, wherein:(a) the VH domain amino acid sequence comprises SEQ ID NO: 1, and the VL domain amino acid sequence comprises SEQ ID NO: 5;(b) the VH domain amino acid sequence comprises SEQ ID NO: 9, and the VL domain amino acid sequence comprises SEQ ID NO: 13;(c) the VH domain amino acid sequence comprises SEQ ID NO: 17, and the VL domain amino acid sequence comprises SEQ ID NO: 21;(d) the VH domain amino acid sequence comprises SEQ ID NO: 24, and the VL domain amino acid sequence comprises SEQ ID NO: 28;(e) the VH domain amino acid sequence comprises SEQ ID NO: 32, and the VL domain amino acid sequence comprises SEQ ID NO: 36;(f) the VH domain amino acid sequence comprises SEQ ID NO: 40, and the VL domain amino acid sequence comprises SEQ ID NO: 44;(g) the VH domain amino acid sequence comprises SEQ ID NO: 47, and the VL domain amino acid sequence comprises SEQ ID NO: 51;(h) the VH domain amino acid sequence comprises SEQ ID NO: 55, and the VL domain amino acid sequence comprises SEQ ID NO: 58;(i) the VH domain amino acid sequence comprises SEQ ID NO: 61, and the VL domain amino acid sequence comprises SEQ ID NO: 65;(j) the VH domain amino acid sequence comprises SEQ ID NO: 68, and the VL domain amino acid sequence comprises SEQ ID NO: 70;(k) the VH domain amino acid sequence comprises SEQ ID NO: 72, and the VL domain amino acid sequence comprises SEQ ID NO: 75;(l) the VH domain amino acid sequence comprises SEQ ID NO: 78, and the VL domain amino acid sequence comprises SEQ ID NO: 80;(m) the VH domain amino acid sequence comprises SEQ ID NO: 84, and the VL domain amino acid sequence comprises SEQ ID NO: 88;(n) the VH domain amino acid sequence comprises SEQ ID NO: 92, and the VL domain amino acid sequence comprises SEQ ID NO: 96;(o) the VH domain amino acid sequence comprises SEQ ID NO: 100, and the VL domain amino acid sequence comprises SEQ ID NO: 104;(p) the VH domain amino acid sequence comprises SEQ ID NO: 108, and the VL domain amino acid sequence comprises SEQ ID NO: 112;(q) the VH domain amino acid sequence comprises SEQ ID NO: 116, and the VL domain amino acid sequence comprises SEQ ID NO: 117;(r) the VH domain amino acid sequence comprises SEQ ID NO: 120, and the VL domain amino acid sequence comprises SEQ ID NO: 122;(s) the VH domain amino acid sequence comprises SEQ ID NO: 125, and the VL domain amino acid sequence comprises SEQ ID NO: 129;(t) the VH domain amino acid sequence comprises SEQ ID NO: 133, and the VL domain amino acid sequence comprises SEQ ID NO: 134;(u) the VH domain amino acid sequence comprises SEQ ID NO: 138, and the VL domain amino acid sequence comprises SEQ ID NO: 142;(v) the VH domain amino acid sequence comprises SEQ ID NO: 144, and the VL domain amino acid sequence comprises SEQ ID NO: 146; or(w) the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

[0196] Embodiment 4. The antibody-drug conjugate of embodiment 1, wherein the antibody or antigen-binding portion is human, humanized, or chimeric.

[0197] Embodiment 5. The antibody-drug conjugate of any one of embodiments 1-4, wherein the antibody or antigen-binding portion comprises an immunoglobulin constant region.

[0198] Embodiment 6. The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

[0199] Embodiment 7. The antibody-drug conjugate of embodiment 6, wherein the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2.

[0200] Embodiment 8. The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region.

[0201] Embodiment 9. The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgGl constant region, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A or a wild-type human IgG2 constant region, wherein numbering is according to the EU index as in Kabat.

[0202] Embodiment 10. The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region comprises the amino acid sequence of any one of SEQ ID NOs: 155-160.

[0203] Embodiment 11. The antibody-drug conjugate of any one of embodiments 1-10, wherein the antibody or antigen-binding portion is an Fab, an Fab', an F(ab')2, an Fv, an scFv, a maxibody, a minibody, a diabody, a triabody, a tetrabody, or a bis-scFv.

[0204] Embodiment 12. The antibody-drug conjugate of any one of embodiments 1-10, wherein the antibody or antigen-binding portion is tetrameric, tetravalent, or multispecific.

[0205] Embodiment 13. The antibody-drug conjugate of any one of embodiments 1-12, wherein the c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib.

[0206] Embodiment 14. The antibody-drug conjugate of any one of embodiments 1-12, wherein the c-Kit inhibitor is dasatinib.

[0207] Embodiment 15. The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a peptide linker.

[0208] Embodiment 16. The antibody-drug conjugate of embodiment 15, wherein the peptide linker comprises two amino acid residues independently selected from glycine, alanine, phenylalanine, lysine, arginine, valine, and citrulline.

[0209] Embodiment 17. The antibody-drug conjugate of embodiment 15, wherein the peptide linker is an alanine-alanine (Ala-Ala) linker.

[0210] Embodiment 18. The antibody-drug conjugate of embodiment 15, wherein the peptide linker is L-Ala-L-Ala, D-Ala-D-Ala, D-Ala-L-Ala, or L-Ala-D-Ala.

[0211] Embodiment 19. The antibody-drug conjugate of embodiment 15, wherein the peptide linker is L-Ala-L-Ala.

[0212] Embodiment 20. The antibody-drug conjugate of any one of embodiments 1-19, wherein the peptide linker is L-Ala-L-Ala; and wherein the antibody-drug conjugate comprises a PEG spacer.

[0213] Embodiment 21. The antibody-drug conjugate of any one of embodiments 1-14, wherein the antibody or antigen-binding portion comprises the c-Kit inhibitor and a linker comprising the structure:(a)

[0214] Embodiment 22. The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via an acid cleavable linker, a glutathione (GSH) cleavable linker, a Fe(II) cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, a phosphatase cleavable linker, a sulfatase cleavable linker, a photo-responsive cleavable linker, or a bioorthogonal cleavable linker.

[0215] Embodiment 23. The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a hydrazone linker.

[0216] Embodiment 24. The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a non-cleavable linker.

[0217] Embodiment 25. The antibody-drug conjugate of any one of embodiments 1-19 and 21-24, further comprising a PEG spacer.

[0218] Embodiment 26. The antibody-drug conjugate of any one of embodiments 1-25, wherein the drug-to-antibody ratio (DAR) is about 2 to about 5.

[0219] Embodiment 27. The antibody-drug conjugate of any one of embodiments 1-26, wherein the DAR is about 4.

[0220] Embodiment 28. A pharmaceutical composition comprising the antibodydrug conjugate of any one of embodiments 1-27, and a pharmaceutically acceptable carrier.

[0221] Embodiment 29. A method for treating a proliferative disorder in a subj ect in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28.

[0222] Embodiment 30. A method for ameliorating a symptom of a proliferative disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28.

[0223] Embodiment 31. The method of embodiment 29 or 30, wherein the proliferative disorder is a mast cell proliferative disorder.

[0224] Embodiment 32. The method of any one of embodiments 29-31, wherein the proliferative disorder is c-Kit-reliant.

[0225] Embodiment 33. The method of any one of embodiments 29-32, wherein the proliferative disorder is systemic mastocytosis, mast cell leukemia, or basophil leukemia.

[0226] Embodiment 34. A method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28.

[0227] Embodiment 35. A method for ameliorating a symptom of an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1- 27 or the pharmaceutical composition of embodiment 28.

[0228] Embodiment 36. The method of embodiment 34 or 35, wherein the inflammatory disorder is a mast cell inflammatory disorder.

[0229] Embodiment 37. The method of any one of embodiments 34-36, wherein the inflammatory disorder is c-Kit-reliant.

[0230] Embodiment 38. The method of any one of embodiments 34-37, wherein the inflammatory disorder is a chronic inflammatory disorder.

[0231] Embodiment 39. The method of any one of embodiments 34-38, wherein the inflammatory disorder is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0232] Embodiment 40. Use of the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28 in the preparation of a medicament.

[0233] The disclosure will be further clarified by the following example, which is intended to be purely exemplary of the disclosure and in no way limiting.EXAMPLE: Generation and testing of anti-CD203c antibodies and antibody-drug conjugatesMethodsIgG expression and purification

[0234] Mammalian codon-optimized synthetic genes encoding the heavy and light chain variable domains of the anti-CD203c antibodies were cloned into mammalian expression vectors comprising effector function null human IgGl (“IgGlnull”; human IgGl containing L234A, L235A, G237A mutations in the lower hinge that abrogate normal immunoglobulin ADCC, ADCP and CDC functions) and human CK domains, respectively. Co-transfection of heavy and light chain containing vectors in a mammalian expression system was performed, followed by protein A-based purification of the IgG, quantification and quality control on denaturing and nondenaturing SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) and analytical SEC (size-exclusion chromatography).Direct binding ELISA for IgG

[0235] Binding and cross-reactivity of the antibodies to the human and rhesus CD203c was initially assessed by binding ELISA. The human CD203c His-tagged recombinant protein and the rhesus monkey CD203c His-tagged recombinant protein were coated to the surface of MaxiSorp™ flat-bottom 96 well plates at a concentration of 1 pg / ml. The purified IgG samples were titrated in 5-fold serial dilutions starting from 100 nM and allowed to bind to the coated antigens. The IgGs were detected using mouse anti-human IgG conjugated to horseradish peroxidase. Binding signals were visualized with 3,3',5,5'-Tetramethylbenzidine Substrate Solution (TMB) and the absorbance measured at 450 nm.Binding to cell-surface CD203c on KU812 cells

[0236] All treatments were performed on cells seeded in T75 flasks at a seeding density of 0.3e6 / ml. Cells were cultured in the presence of 80 ng / ml of SCF and treated with20 ng / ml of IL3 24 hours prior to antibody staining. Following SCF / IL3 treatment, cells were harvested and stained with live dead stain (Zombie UV Biolegend 423108). Cells were then stained with a titration of anti-ENPP3 antibodies or respective isotype control in a 5-fold dilution series starting at 500 nM, and detected with Anti human-FC AF647 antibody (JIR 109-605-098). Cells were also stained with commercial Anti-ENPP3 AF647 (clone NP4D6 BioLegend 324626) as a positive control for ENPP3 expression. Samples were subsequently run on a Yeti analyzer, gating on 10K live cells. Subsequent analysis was performed using FlowJo software and data represented as MFI (median fluorescent intensity).Internalization on TF1 and KU812 cells using Fab-ZAP reagent

[0237] The assay was performed according to manufacturer’ s instructions. KU812 cells were revived and cultured in suspension in RPMI 1640 media supplemented with 10% FBS, 100 units / ml penicillin, 100 pg / ml streptomycin and 80 ng / ml SCF. TF-1 cells were revived and cultured in suspension in RPMI 1640 media supplemented with 10% FBS, 100 units / ml penicillin, 100 pg / ml streptomycin and 2 ng / ml GM-CSF. Targeting antibodies were serially diluted and combined with Fab-ZAP reagent 1 : 1 so that both reagents were at 4x in cell appropriate growth media. Antib ody / Fab -ZAP solutions were incubated for 30 minutes at 37 °C to allow complex formation. Cells were loaded at a final seeding density of 2500 cells / well in appropriate growth media. Fab-ZAP complexes were loaded in an 8-point 4.5-fold serial dilution at final assay concentrations of 0.00013 to 5 nM. Final Fab-ZAP concentrations were 5 nM for TF-1 cells and 10 nM for KU812 cells. Fab-ZAP only, media only and no cell controls were included on every plate. Cells were incubated with complexes for 72 hours (TF-1) or 96 hours (KU812). At the end of the incubation, CellTiterGlo reagent was added to all wells. Plates were incubated for 10 minutes before luminescence signal was measured using a FLUOstar Omega plate reader (BMG Labtec).AC-SINS (affinity-capture self-interaction nanoparticle spectroscopy) assay

[0238] Briefly, 20 nm gold nanoparticles were coated with a mixture of 80% goat antihuman Fc-specific and 20% non-specific goat polyclonal antibodies that were buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg / ml. After one hour incubation at room temperature, sites unoccupied on the gold nanoparticles were blocked with thiolated polyethylene glycol (2 kD). The coated nanoparticles were thenconcentrated 10-fold using a syringe filter, and 10 pl was added to 100 pl of IgG at 0.05 mg / ml in PBS pH 7.2. The coated nanoparticles were incubated with the antibody of interest for 2 hrs and read on a Tecan M1000 spectrophotometer. The absorbance was read from 450-650 in 2 nm increments. The smoothed max absorbance of the average blank (PBS alone) was subtracted from the smoothed max absorbance of the antibody sample to determine the antibody AC-SINS score.DNA / Insulin polyreactivity ELISA

[0239] ELISA plates were coated overnight at 4°C with DNA (10 pg / ml) and insulin (5 pg / ml) in PBS pH 7.5. Wells were washed with water, blocked with 50 pl of Polyreactivity ELISA Buffer (PEB; PBS containing 0.05% Tween-20, 1 mM EDTA) for 1 hour at room temperature, and rinsed three times with water. Serially-diluted antibodies were added in quadruplicate to the wells and incubated for 1 h at room temperature. Plates were washed three times with water, and goat anti-human IgG-HRP (Fc specific) was added to each well. Plates were incubated for 1 h at room temperature, washed three times with 80 pl of water, and TMB substrate (Sigma-Aldrich, T-0440) was added to each well. Reactions were stopped after approximately 7 min by adding 0.18 M ortho-phosphoric acid to each well, and absorbance was read at 450 nm. DNA- and insulin-binding scores were calculated as the ratio of the ELISA signal of the antibody at 10 pg / ml to the signal of a well containing buffer instead of the primary antibody.Internalization and co-localization studies using the ImageStream® Amnis® imaging flow cytometer platform

[0240] KU812 cells were revived and cultured in suspension in RPMH640 media supplemented with 10% FBS, 100 units / ml penicillin, 100 pg / ml streptomycin and 80 ng / ml SCF. Two days prior to the start of the experiment, KU812 cells were cultured in SCF-free media. KU812 cells (passage 23) were resuspended at 4xl07cells / ml. For the main staining protocol, KU812 cells were plated into 96 well plates (50 pl per well). Duplicate wells were prepared for all variables. At 6 time points prior to harvest (3h, 2h, Ih, 30 mins, 15 mins or 5mins), 2.23-AF594 or Isotype (50 nM final concentration) was added in 50 pl medium. A subset of wells was stained with isotype control antibody (AF594-conjugated) for 3h, Ih or 15 mins to act as negative controls. Two wells wereincubated with medium alone for 3h. To prepare single staining control samples, 50 pl of unstained KU812 cells were transferred to 1.5 ml tubes. Each tube was taken through the identical stages of staining as below but only received a single stain (either Zombie Violet, LAMP2-FITC or CD71-AF594), receiving the appropriate buffer at all other stages. Cells were washed in PBS, then stained with Zombie violet viability dye diluted 1 in 100 in PBS for 5 minutes at 4°C in the dark. An aliquot of cells was heat-killed (2 min at 90°C as a positive control) before zombie staining. These cells were centrifuged at 1000 x g for all steps. Cells were washed with PBS, centrifuged (500 x g, 5 mins) then resuspended in BD Cytofix / Cytoperm fixation solution for 20 mins at 4°C. A IX solution of BD Perm / wash was added and the cells were centrifuged (500 x g, 5 mins) then washed again in BD Perm / Wash. Cells were stained with anti-LAMP2 (1 / 50) diluted in BD Perm / Wash for 30 mins at 4°C. Cells were washed once with BD Perm / Wash then once with PBS. Cells were kept at 4°C until analysis. Immediately before analysis, cells were filtered through a 30 pm cell strainer (Celltrics) and transferred to 1.5ml tubes. Cells were analyzed on an Amnis® ImageStream® imaging flow cytometer, and 1000 events collected per sample.Synthesis of bis-tolylsulfonyl-propanoyl benzamide (BTP)-Glu-(AA-c-kit inhibitor l)-PEG(24u) linker-payload

[0241] To a solution of BTP Bl-Glu(OH)-PEG(24u) (55 mg, 32.3 pmol) and c-Kit inhibitor 1.2HC1 salt (25 mg, 45.5 pmol) in DMF (1.5 mL) was added HATU (16 mg, 42.2 pmol). The reaction mixture was placed in an ice-bath andNMM (18 pl, 162 pmol) was added (FIG. 9). The reaction mixture was stirred in an ice bath for 2 hours. The reaction mixture was then concentrated in vacuum and the residue was purified by preparative HPLC using a gradient method: 10-70% waterMeCN (with 0.05% TFA modifier) over 45 minutes. Fractions containing the desired product were pooled, flash frozen and concentrated by lyophilization to give the desired product (26 mg, 35.6%) as a white solid. The obtained material was 93-94% pure by HPLC analysis (FIG. 10). MS (ESI), m / z: [M+3H]^+calculated: 756.3; observed: 755.4.Synthesis of BTP-Glu-(AA-c-kit inhibitor 2)-PEG(24u) linker-payload

[0242] To a solution of Bl-Glu(OH)-PEG(24u) (48 mg, 28.2 pmol) and c-Kit inhibitor2 (25 mg, 39.7 pmol) in DMF (1.5 mL) was added HATU (15 mg, 39.4 pmol). Thereaction mixture was placed in an ice-bath and NMM (18 pl, 85.7 pmol) was added (FIG. 12). The reaction mixture was stirred in an ice bath for 2 hours. The reaction mixture was concentrated in vacuum and the residue was purified by prep. HPLC using a gradient method: 10-70% waterMeCN (with 0.05% TFA modifier) over 45 minutes. Fractions containing the desired product were pooled, flash frozen and concentrated by lyophilization to give the desired product (55 mg, 84.6%) as a white solid. The obtained material was 97-99% pure by HPLC analysis (FIG. 13). MS (ESI), m / z: [M+2H]2+calculated: 1155.5; observed: 1154.9.Conjugation of 2.23 IgG with BTP-Glu-(AA-c-kit inhibitor l)-PEG(24u) linkerpayload

[0243] 2.23 IgG at 7.3 mg / ml in Dulbecco’s PBS, pH 7.5, 5 mM EDTA (17 mg, 2.33 mL, 1.0 eq) was diluted to 5.2 mg / ml with Dulbecco’s PBS, pH 7.5, 5 mM EDTA (936.6 pL). A 5 mM solution of TCEP in endotoxin free water (137.8 pL; 687 nmol; 6.0 eq.) was added to the solution, resulting in a final antibody concentration of 5 mg / mL. The reduction was allowed to proceed at 40°C for 2 h. The reduction mixture was diluted to 4.4 mg / mL with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool to 22°C. A 5.0 mg / mL (2.21 mM) solution of BTP-Glu-(AA-c-kit inhibitor 1)- PEG(24u) was prepared by dissolving 26 mg (11464 nmol) of linker-payload into 5.2 mL of DMF. A portion of the BTP linker-payload solution (343.8 pL, 758.4 nmol, 6.6 eq) was added to the reduced antibody solution, resulting in a final co-solvent concentration of 10% DMF. The conjugation reaction was allowed to proceed at 22°C for 16 h. After 16 h the reaction mixture was diluted with an equal volume of HIC loading buffer (50 mM sodium phosphate, 4 M NaCl, pH 7.0) and purified by preparative HIC on a 5 mL HIC ToyoPearl® Phenyl-650S column. The mobile phase buffers used were Buffer A: 50 mM sodium phosphate, 2 M NaCl, pH 7.0, Buffer B: 50 mM sodium phosphate, 20% isopropanol, pH 7.0. A linear gradient from 0 to 100% buffer B was ran at a flow rate of 2 mL / min over 35 column volumes. Fractions were collected and analyzed by HIC and LC-MS. Fractions containing DAR4 as the majority species were concentrated by ultrafiltration / diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa MWCO). Three rounds of buffer exchange into Dulbecco’s PBS, pH 7.5 was performed. The concentrated sample (8.0 mg; 2.0 mL) was sterile filtered through a 0.22 pm pore size, PVDF membrane filter. 2.23-BTP-Glu-(AA-c-kit inhibitor l)-PEG(24u) conjugate was characterized by HIC, SEC, LC-MS, quantified by UV and endotoxin levels were determined.Conjugation of 2.23 IgG with BTP-Glu-( AA-c-kit inhibitor 2)-PEG(24u) linkerpayload

[0244] 2.23 at 7.3 mg / ml in Dulbecco’s PBS, pH 7.5, 5 mM EDTA (16.5 mg, 2.25 mL, 1.0 eq) was diluted to 5.2 mg / ml with Dulbecco’s PBS, pH 7.5, 5 mM EDTA (915.2 pL). A 5 mM solution of TCEP in endotoxin free water (133.8 pL; 669 nmol; 6.0 eq.) was added to the solution, resulting in a final antibody concentration of 5 mg / mL. The reduction was allowed to proceed at 40°C for 2 h. The reduction mixture was diluted to 4.4 mg / mL with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool to 22°C. A 5.0 mg / mL (2.16 mM) solution of BTP-Glu-(AA-c-kit inhibitor 2)-PEG(24u) was prepared by dissolving 26 mg (11251 nmol) of linker-payload into 5.2 mL of DMF. A portion of the BTP linker-payload solution (309.2 pL, 668 nmol, 6.0 eq) was added to the reduced antibody solution, resulting in a final co-solvent concentration of 10% DMF. The conjugation reaction was allowed to proceed at 22°C for 16 h. After 16 h the reaction mixture was diluted with an equal volume of HIC loading buffer (50 mM sodium phosphate, 4 M NaCl, pH 7.0) and purified by preparative HIC on a 5 mL HIC ToyoPearl® Phenyl-650S column. The mobile phase buffers used were Buffer A: 50 mM sodium phosphate, 2 M NaCl, pH 7.0, Buffer B: 50 mM sodium phosphate, 20% isopropanol, pH 7.0. A linear gradient from 0 to 100% buffer B was ran at a flow rate of 2 mL / min over 35 column volumes. Fractions were collected and analyzed by HIC and LC-MS. Fractions containing DAR4 as the majority species were concentrated by ultrafiltration / diafiltration using a Vivaspin 20 centrifugal concentrator (PES membrane, 30 kDa MWCO). Three rounds of buffer exchange into Dulbecco’s PBS, pH 7.5 was performed. The concentrated sample (11.7 mg; 2.9 mL) was sterile filtered through a 0.22 pm pore size, PVDF membrane filter. 2.23-ThioBridge®-Glu-(AA-c- kit inhibitor 2)-PEG(24u) conjugate was characterized by HIC, SEC, LC-MS, quantified by UV and endotoxin levels were determined.LC-MS analysis

[0245] LC-MS (liquid chromatography-mass spectrometry) analysis was carried out using a Waters XEVO G2S TOF mass spectrometer and a POROSHELL 300SB C3 column (2.1 x 12.5 mm, 5 pm) connected to a Waters Acquity H Class UPLC system.The mobile phase was buffer A (0.1 % formic acid in water). A gradient (2.5 min 10% B, 10-80% B gradient in 3.5 min) was applied using Buffer B (acetonitrile, 0.1 % formic acid) at a flow rate of 0.4 mL / min. The column was maintained at 60 °C throughout the analysis. The ADCs were directly analyzed after dilution to 0.05 mg / mL. 10 pL of ADC solution were injected for analysis. Average DAR (drug-to-antibody ratio) was calculated as the weighted average of the observed DAR species based on main glycoform signal intensity (SI) for each DAR species (i.e., from DAR 0 to DAR 8) on the deconvoluted m / z spectra, and average molecular weight for the ADC was calculated based on DAR and linker-payload mass contribution, as follows:MWADC= MWmAb+ DAR x MWLPSEC analysis

[0246] Analytical SEC was carried out using an ACQUITY UPLC BEH 200 SEC column (4.6 mm x 15 cm, 200 A, 1.7 pm) and guard column (4.6 mm x 3 cm), connected to a Dionex Ultimate 3000 UPLC system. The mobile phase was 0.2 M potassium phosphate buffer, pH 6.8, 0.2 M potassium chloride, 15% (v / v) isopropanol. The flow rate was kept constant at 0.35 mL / min. The column was maintained at 30°C throughout the analysis. The analysis was carried out in a 10 min isocratic elution with UV detection at 248, 280 nm and 330 or 365 nm. 10 pg of ADC were injected for analysis. The percentage of high molecular weight (HMW) species was calculated by comparing the peak area corresponding to HMW species at 280 nm to total peak area corresponding to HMW and monomeric species at 280 nm. The percentage of free drug related species was calculated by comparing the peak area corresponding to free drug related species at a wavelength specific for the drug to total peak area corresponding to ADC species at the same wavelength.HIC analysis

[0247] Analytical HIC (hydrophobic interaction chromatography) was carried out using a TOSOH Bioscience TSKgel Butyl-NPR column (4.6 mm x 3.5 cm, 2.5 pm), connected to a Dionex Ultimate 3000 UPLC system. The mobile phase was buffer A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. A linear gradient (0-100% B in 10.5 min) was applied using Buffer B (20 % isopropanol, 50 mM sodium phosphate, pH 7.0) at a flow rate of 1.35 mL / min to elute bound species. The column was maintained at 30 °C throughout the analysis. The analysis was carried with UV detection at 280 nm. 10 pg of ADC were injected per analysis. The percentage of each DAR species (i) was calculated by comparing the peak areas of each assigned peak to total peak area. Average DAR was calculated as the weighted average of the observed DAR species based on peak area under the curve (AUCi) as follows:Concentration determination by UV absorbance

[0248] The concentration of the conjugates was determined by UV absorbance at 280 nm (A280) using a Nanodrop 2000 spectrophotometer. Measurements were taken in triplicate and the average values used for calculations. Molar extinction coefficients used for antibodies and linker-payloads were determined experimentally (Table 25 and Table 26).Table 25. Molar extinction coefficient for 2.23 antibodyTable 26. Molar extinction coefficients for linker-payloads

[0249] The following formula was used to calculate the conjugate concentration:Where: c = concentration ((mg / mL) Acm1)7^280=absorbance at 280 nm^mAb=molar extinction coefficient of antibody at 280 nm (M4.cm-1)£LP°=molar extinction coefficient of linker-payload at 280 nm (M Acm-1)DAR = average DAR l= optical path (cm) antibody MW (g.mol-1) MWLP= linder-payload MW (g.mol-1)Endotoxin level determination method

[0250] The EndoSafe®-PTSTM platform (Charles River) was used to determine the level of endotoxin. EndoSafe®-PTSTM is a chromogenic kinetic test system aligned with USP <85> and Pharm Eur 2.6.14 that provides quantitative Limulus Amebocyte Lysate (LAL) results. The EndoSafe®-PTSTM utilizes LAL reagents in FDA-licensed disposable test cartridges which are pre-loaded with all the reagents required to perform a LAL test. The EndoSafe®-PTSTM mimics licensed LAL kinetic chromogenic methodology by measuring colour intensity directly related to the endotoxin concentration in a sample; the concentrations are calculated against an internal standard curve (0.01-1.00 EU / mL) associated with the lot number of the cartridges.Cytotoxicity / Cell Viability Assay

[0251] KU812 cells (passage 23) were seeded at a density of 4.0 x 10 cells per ml in complete phenol red-free RPMI media without SCF in a volume of 50 pl per well in 3 black walled 96-well tissue culture plates. Cells were incubated at 37°C in a humidified atmosphere for 2 h to allow for the SCF starvation to take effect. Test agents were added to cells at concentrations between 0.0015 nM and 100 nM (9-point, 4-fold dilution series). Vehicle (RPMI), DMSO, and cells only wells were included as controls for the assay. Test antibodies were added in a 50 pl volume at a 2X concentration. Cells were incubated at 37°C, 5% CO2 in a humidified atmosphere for 24, 48 or 72 h (one plate per timepoint). CellTox Green assay components were thawed by immersing in a 37°C water bath. Once thawed, each component was vortexed and equilibrated to room temperature. CellTiter-GLO buffer was thawed and equilibrated to room temperature overnight. 30 min prior to each timepoint, the plate was incubated at room temperature. Lysis solution (25X) was added to the cells (4 pl per well). 20 pl CellTox Green dye was added to 2 ml of Assay buffer to form CellTox Green reagent. 20 pl of CellToxGreen reagent was added to each well. The plate was then placed on an orbital shaker at 700 rpm for 1 min. The plate was incubated for 15 minutes at room temperature, protected from light. Fluorescence was measured on a FLUOStar plate reader at 485 nm excitation and 520 nm emission with a gain of 1000. 10 ml CellTiter-GLO buffer was used to reconstitute a vial of CellTiter-GLO substrate to form CellTiter-GLO Reagent. 100 pl of CellTiter-GLO reagent was added to each well. The plate was then placed on an orbital shaker at 700 rpm for 10 min to facilitate cell lysis and ATP extraction from cells. Luminescence was measured on a FLUOStar plate reader. CellTox Green and CellTiter-GLO data were expressed as a percentage of untreated control cells (Cells alone).C-kit receptor phosphorylation assay

[0252] KU812 cells were cultured in SCF-free RPMI +10% FCS for 48 h. Cells were seeded in 24-well plates at 1 x 106 / mL in a total volume of 1.5 mL for 1 h, followed by treatment with isotype control or test agents for 2 h. Cells were stimulated with SCF at 40 ng / mL for 5 min, washed in PBS and cell lysates were prepared using lysis buffer according to manufacturer’s instructions (Biotechne). Total and phospho-c-kit levels were detected using 6.25 pg and 50 pg total protein, respectively, using ELISA kits from Biotechne.Pharmacokinetic assay

[0253] Rat pharmacokinetics (PK) of naked 2.23 vs 2.23-cKit 2 ADC: Test agents were administered to Sprague-Dawley rats at 3 mg / kg via an intravenous route (IC) on Day 0 (3 animals per group), with blood collected at nine time points post-dosing (5 min, 3, 6, 24, 48, 72, 168, 336 and 504 hours). Samples were diluted 1 / 2000 and antibody levels were quantified using a human IgG ELISA kit (Mabtech) (FIG. 36A). PK parameters shown in the table (FIG. 36B) were calculated using PKsolver.Tumor tolerability study

[0254] In-vivo non-tumor bearing tolerability study of 2.23-cKit2 ADC using female NSG mice: 2.23-cKit2 ADC was dosed at 3 and 10 mg / kg in non-tumor bearing NSG mice to assess any potential toxicity effects (3 mice per group, 6 doses, Q4D). All animals were weighed on Day 0, and then each day post-treatment for the duration ofthe 28-day study. Relative body weights were plotted as a % of body weight on Day 1 of dosing, and data was shown as mean + / - SEM for each group (n=3) (FIG. 37).In vivo efficacy study

[0255] In vivo efficacy of 2.23-cKit2 ADC in a KU812 tumor model in NSG mice: NSG mice were implanted with 5 x 106KU812-luciferin cells IC via the lateral tail vein. Tumor growth was allowed to progress to exponential growth phase and dosing with PBS control or 10 mg / kg 2.23-cKit2 ADC was initiated in therapeutic mode on Day 19 (10 mice / group, Q4D for 4 weeks). Tumor growth was monitored by bioluminescent imaging twice a week from Day 7 and is reported in both linear (FIG. 38A) and log (FIG. 38B) scales.Dose-dependent in vivo efficacy of 2.23-cKit2 ADC in a KU812 tumor model in NSG mice

[0256] NSG mice were implanted with 5xl06KU812-luciferin cells IV via the lateral tail vein. Tumors were allowed to progress to exponential growth phase and dosing with PBS control, 2, 10 & 50 mg / kg 2.23-cKit 2 ADC (10 mice / group, Q4D IP for 4 weeks) or 10 mg / kg dasatinib (QD PO 5 days on, 2 days off for 4 weeks) was initiated in therapeutic mode on Day 25. Tumor growth was monitored by bioluminescent imaging twice a week from Day 7 and is reported in log scale.Multiple dose PK study of 2.23-cKit2 ADC in cynomolgus monkeys

[0257] Test agents were administered to adult male cynomolgus monkeys at 10 or 30 mg / kg (3 animals / group) via an intravenous route (IV) on Day 0 and again on Day 21 with necropsy at Day 42. Blood was collected at multiple time points post-dosing (for Day 0 dose: pre-dose and 0.5, 2, 6, 24, 48, 72, 168, 556 & 504 h post-dose; Day 21 samples were taken at the same time points post-dose). Antibody levels in serum were quantified using a human IgG bioanalytical ELISA method. The data were subjected to a non-compartmental pharmacokinetic analysis using the Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA).Blood parameters measured in cynomolgus monkey after treatment with 2.23- cKit2 ADC

[0258] Test agents were administered to adult male cynomolgus monkeys at 10 or 30 mg / kg (3 animals / group) via an intravenous route (IV) on Day 0 and again on Day 21 with necropsy at Day 42. Blood was collected at multiple time points - Day 0 pre-dose, Day 7, Day 14, Day 21 (pre-dose), Day 28 & Day 42 prior to terminal necropsy. At each time point, 1 mL of whole blood was collected into a Potassium -EDT A tube and mixed. Samples were kept on ice and subjected to complete blood count tests on the same day as collection. This panel included white blood cells, red blood cells, hemoglobin, platelets, neutrophils, lymphocytes, monocytes, eosinophils, and basophils.Direct filter-binding radiometric kinase activity assay

[0259] A direct filter-binding radiometric kinase activity assay (Eurofins) was used to measure the potency of cKit2 free payload in inhibiting a number of tyrosine kinases including Bruton’s Tyrosine Kinase (BTK; FIG. 42A), wild-type c-Kit (FIG. 42B) and variant c-Kit (D816V; FIG. 42C). Each test agent was assayed across a concentration range from 10 mM to 1 nM in triplicate. Calculated IC50 values are summarized in FIG. 42D.Selectivity screening of cKit2 free payload

[0260] A KINOMEscan competition assay (Eurofins) was used to assess the selectivity of the cKit2 free payload compared to other benchmark tyrosine kinase inhibitors. This assay format measures the ability of test agent to complete with an immobilized active site directed ligand. Each test agent was assayed at 12 concentrations ranging from 1 mM to 0.1 nM and dose response curves were used to determine relative binding Kd values.Results and DiscussionLead IgG generation and screening

[0261] Anti-CD203c antibodies (described in Tables 1-23) were readily expressed and purified from CHO cells using Protein-A affinity chromatography. Antibodies are referred to by the number in the table title providing the sequence of that antibody (2.1 through 2.23). Analytical SEC demonstrated that all clones were >95% monomeric and preparations had a low endotoxin content < 1 EU / mg.

[0262] All 23 purified IgGs were initially tested for binding to His-tagged recombinant versions of human and rhesus CD203c (FIG. 3) and all clones (18) that demonstrated measurable binding to both orthologs were brought forward for binding analysis using flow cytometry on KU812 cells, which endogenously express human CD203c. Of the 18 clones tested, only 10 retained robust binding to cell-surface expressed CD203c (FIG. 4A and FIG. 4B) and these were selected for subsequent internalization assays.

[0263] In order to assess internalization capacity of anti-CD203c lead IgGs, 2 cell lines were selected for analysis. The TF1 cell line expresses low levels of CD203c as determined by flow cytometry (FIG. 5A), while the KU812 cell lines expresses approximately 10-fold higher levels of CD203c (FIG. 5B). The 10 prioritized anti- CD203c IgGs were pre-incubated with Fab-ZAP reagent to enable complex formation, and then incubated with each of the cell lines. FIG. 3 demonstrates that all 10 antibodies induced cell death via intracellular delivery of the cytotoxic ZAP reagent, and the potency of cell killing was proportional to the level of CD203c expression. Relative to the isotype control, the antibodies achieved between 20-40% cell kill on low-expressing TF1 cells compared to up to 90% cell kill on higher-expressing KU812 cells. Based on this characterization, all internalizing antibodies were moved forward to biophysical characterization to enable the selection of well-behaved molecules for the immunoconjugate preparation.

[0264] Increasingly, developability has become an important characteristic for successful therapeutic progression. In antibody drug conjugates, this is even more important, as both the antibody and conjugated drug moieties can each exhibit prohibitively poor biophysical behavior to be commercially or clinically viable. In particular, the solubility and monospecificity of the antibody moiety is critical. The lead panel of anti-CD203c IgGs demonstrated similar binding to cell-surface target, and similar potency in an internalization assay. In order to differentiate between lead molecules, and enable smooth downstream immunoconjugate generation, the lead panel were subjected to assays characterizing potential for aggregation and specificity. The AC-SINS assay was used to look at propensity of each IgG to self-interact compared to the well-understood comparators Alirocumab, which is well behaved, and Bococizumab, which displays high aggregation via self-association. In addition, antibodies were assessed for binding to DNA and insulin, which are highly negatively charged molecules and predict polyreactivity and potential for off-target binding. In this case, Bevacizumab and Ustekinumab were used as well-behaved comparators andBococizumab and Briakinumab as benchmarks for antibodies with known polyreactivity. FIG. 6 A and FIG. 6B show that IgGs 2.7, 2.12, 2.13, 2.14 and 2.23 performed well in these assays with no indication of aggregation propensity or polyreactivity. Based on combined characterization, clone 2.23 was taken forward for conjugation studies.Internalization and lysosomal tracking of covalently-labelled antibody 2.23

[0265] 2.23 IgG was covalently coupled to the AF594 fluorophore and tracked for internalisation using a high content Amnis® imaging platform. Exemplar images shown in FIG. 7A show 2.23-AF594 binding to cell surface expressed CD203c on KU812 cells at 4°C. When cells are subsequently incubated at 37°C, the labelled antibody is internalized and co-localizes with lysosomes. The rate of internalization is shown in FIG. 7B and co-localisation with LAMP2 is quantified over time in FIG. 7C. This demonstrates time-dependent lysosomal accumulation of the conjugate and productive delivery of a conjugated payload to enable lysosomal processing and release.Linker-payload design for dasatinib derivatives

[0266] FIG. 8A and FIG. 8B illustrate the linker-payload design elements. A bis-thiol alkylating reagent (BTP) is first conjugated via a stable amino acid linker to dasatinib and ultimately links to both cysteine thiols from the reduced disulfide of 2.23 resulting in a controlled and homogenous DAR of 4. Incorporation of a hydrophilic 24u PEG spacer aids in conjugate solubility, linker masking and half-life extension. Two versions of the 2.23 conjugate are shown cKitl (FIG. 8A) and cKit2 (FIG. 8B), which has a slightly longer amino acid linker. The cKit2 version has an additional amino ethyl group between the piperazine and the alanine moieties. The piperazine therefore no longer forms the amide bond directly with the alanine amino acid (as it does in the cKitl version), but the amino group within the amino ethyl moiety does this instead.Linker-payload synthesis and characterization

[0267] FIG. 9 - FIG. 11 describe the synthesis and analytical characterization of the BTP-cKitl-PEG linker payload by RPLC and LC / MS. The RPLC analysis shows a single peak with LC / MS confirming the expected mass. This is also true for BTP-cKit2- PEG, the data for which is summarized in FIG. 12 - FIG. 14.ADC generation and characterization

[0268] Antibody 2.23 was conjugated to both BTP-cKit-PEG linker payloads and analytical characterization was performed post-purification. FIG. 15 shows the Yi mAb analysis by LC / MS confirming a DAR of 3.9 for 2.23-cKitl ADC. FIG. 16A estimates an average DAR of 3.9 by HIC, and a single peak corresponding to >99.1% monomer by SEC (FIG. 16B). The same data was generated for 2.23-cKit2 ADC, summarized in FIG. 17 and FIG. 18A - FIG. 18B. A summary for both ADC molecules is presented in Table format in FIG. 19 (2.23-cKitl-ADC) and FIG. 20 (2.23-cKit2-ADC).Conjugate potency testing in CD203c+ granulocyte and non-granulocyte cell lines

[0269] Functional characterization of 2.23-c-Kit 1 and 2-ADCs demonstrated the ability of both conjugates to reduce KU812 viability in a concentration-dependent manner, with 2 being significantly more potent than 1 (FIG. 21A). 2.23-c-Kit 2-ADC activity correlated directly with c-Kit phosphorylation inhibition, with potency lower than the free dasatinib at this time point (FIG. 21B).

[0270] Running a time course of the same experiment shows naked 2.23 has no effect on cell viability or cytoxicity, 2.23-c-Kit2 is highly potent, but requires 72h to achieve maximal potency, while free dasatinib potency is already at maximum after 24 / 48h (FIG. 22). This shows that the effect of 2.23-c-Kit2 is likely specific receptor-driven internalization delivery of payload. A repeat was performed where isotype IgGl-c-Kit2 conjugate was added, and all compounds tested on KU812 cells after 72h. Potency of 2.23-c-Kit2 was reproducibly observed on KU812 cells while the isotype control conjugate showed no significant impact on cell viability (FIG. 23). This confirms that the potency of 2.23-c-Kit2 is due to target-mediated internalization and payload delivery and not payload drop-off in culture.

[0271] Further testing on several renal cell lines with varying levels of CD203c expression and lacking c-Kit (RKO, RXF393 and HepG2) confirmed that the potency of the dasatinib payload on granulocytes is not simply due to inherent toxicity, as 2.23- cKit2-ADC has no impact on non-granulocyte cell viability even at 100 nM (FIG. 24 - FIG. 26) This demonstrates that the 2.23-cKit2 mechanism is driven by two properties (i) expression of CD203c on the cell surface combined with (ii) dependency on c-Kit signaling for survival.

[0272] A series of modified cKit2 linker-payloads were synthesized to explore the impact of using unnatural amino acids in the linker (shown in FIG. 27A). Instead of the parental L-Ala-L-Ala linker, D-Ala-D-Ala, D-Ala-L-Ala and L-Ala-D-Ala variants were compared to the parental and its cleavage products, cKit2-dAla (with one Ala cleaved from the linker) and cKit2-dAlaAla (with both Ala now cleaved from the linker to release the free cKit2 payload) in viability and cytoxicity assays in KU812 cells (FIG. 27C and FIG. 27D, respectively). The comparative potencies are described in FIG. 27B and show that incorporation of unnatural amino acids into the linker have a significant negative impact on potency.

[0273] A number of alternative tyrosine kinase inhibitors were tested as potential payloads for the CD203c-ADC. These were all synthesized as free payload, a dAla version (with a single L-Ala linker) and an L-Ala-L-Ala version to mimic the parental cKit2 molecule and its respective cleavage products. These potential payload variants were subsequently compared to cKit2dAlaAla in cytoxicity and viability assays at 24h, 48h and 72h time points. Anlotinib variants (FIG. 28A) had no potency in KU812 cell killing (FIG. 28B) compared to cKit2dAlaAla. The same was true of Crenolanib (FIG. 30), Avapritinib (Fig 32), Elenestinib (FIG. 33) and Midostaurin (FIG. 35). Imatinib (FIG. 29) showed some potency at the highest concentration tested, 100 nM, and this became apparent after 48h of treatment. Ponatinib free payload (FIG. 31) showed almost equivalent potency to cKit2, with Bezuclastinib (FIG. 34) showing minimal toxicity at concentrations > 100 nM.2.23-cKit2 ADC retains favorable PK properties

[0274] Rats were administered with a single dose of 3 mg / kg 2.23 antibody or 2.23- cKit2 ADC to explore the impact of payload conjugation on the antibody PK. A standard human IgG ELISA was used to measure antibody levels in blood samples taken from rats at a series of time points post-administration. Analysis of PK samples shows a two-phase decay of both antibody and ADC over time, with half lives of 326 and 285 hours, respectively (FIG. 36A and FIG. 36B). ADCs often suffer with poor solubility and hydrophobicity which can have negative impacts on subsequent biodistribution and PK. 2.23-cKit2 ADC behaves very similarly to the naked antibody, suggesting a well-behaved molecule with a standard half-life.2.23-cKit2 ADC is well tolerated in mice

[0275] The 2.23-cKit2 ADC was dosed in non-tumor bearing NSG mice every 4 days for a total of 6 doses at 3 and 10 mg / kg to explore any potential safety concerns. Animals were monitored for body weight before dosing and body weight compared over time after dosing for the duration of the 28-day study. FIG. 37 shows that there was no impact on body weight at either dose with animals feeding normally and experiencing no weight loss. There was no impact on behavior over the course of the study, and no overt clinical signs. No follow up histopathological analysis was required. 10 mg / kg dosing was selected as a safe dose for the subsequent KU812 in vivo efficacy model.2.23-cKit2 ADC ablates tumor growth in the KU812 model

[0276] A luciferin variant of the KU812 cell line was generated to enable the use of bioluminescent imaging (BLI) to dynamically monitor tumor growth in vivo. NSG mice were implanted with KU812 cells and animals monitored until a stage of exponential tumor growth was reached. At this stage, therapeutic dosing with 2.23-cKit2 ADC was initiated (Day 19) at 10 mg / kg, with dosing repeated every 4 days over a 4-week period. BLI measurements were taken twice-weekly over the course of the study and data is summarized in FIG 38. 2.23-cKit2 ADC significantly ablates tumor growth compared to the PBS control (FIG. 38A), despite the fact that dosing was only initiated after tumors were already established and in exponential growth phase (FIG. 38B).2.23-cKit2 ADC demonstrates dose-dependent tumor ablation in the KU812- luciferin model

[0277] A luciferin variant of the KU812 cell line was generated to enable the use of bioluminescent imaging (BLI) to dynamically monitor tumor growth in vivo. NSG mice were implanted with KU812 cells and animals monitored until a stage of exponential tumor growth was reached. At this stage, therapeutic dosing with 2.23-cKit2 ADC was initiated (Day 25) across a range of concentrations from 2-50 mg / kg, with dosing repeated every 4 days over a 4-week period. BLI measurements were taken twice- weekly over the course of the study, and data are summarized in FIG. 39. 2.23-cKit2 ADC significantly ablates tumor growth compared to the PBS control in a dosedependent manner, with 98% inhibition observed at the highest dose of 50 mg / kg compared to 96% at 10 mg / kg and 83% at 2 mg / kg by study end. 2.23-cKit2 ADCshows similar potency to the benchmark small molecule dasatinib, which was also included in the study (10 mg / kg daily dosing by the oral route).2.23-cKit2 ADC demonstrates standard antibody-like PK in cynomolgus monkeys

[0278] The 2.23-cKit2 ADC was dosed in cynomolgus monkeys at 10 or 30 mg / kg on Days 0 & 21 with monitoring to study end at Day 42. The serum antibody concentrations are shown in FIG. 40. The antibody has a half-life of 8-11 days with no evidence of TMDD and a Ctrough of > 20 pg / mL, providing strong support for an infrequent dosing regimen in patients.2.23-cKit2 ADC has no impact on hematological parameters in cynomolgus monkeys

[0279] C-kit targeting antibodies and small molecules suffer from hematological adverse events due to the broad global expression of c-Kit across a number of cell types, including hematopoietic stem cells (HSCs) and myeloid / erythroid precursors. 2.23- cKit ADC aims to avoid these off-target cell events through selective delivery of the tyrosine kinase inhibitor to activated mast cells, avoiding HSCs. In line with this desired mechanism, 2.23-cKit2 ADC had no significant impact on blood parameters after 6- week dosing at 10 and 30 mg / kg in cynomolgus monkeys. Blood samples taken from dosed animals across multiple time points were subjected to complete blood cell counts and the results are summarized in FIG. 41A - FIG. 411. All counts remained in the normal range with no significant or dose-dependent decreases observed over time. cKit2 free payload exhibits potent inhibition across multiple tyrosine kinases

[0280] The potency of the cKit2 free payload was measured using a radiometric kinase activity assay across a broad concentration range (1 nM - 10 pM). While the cKit2 payload effectively inhibited the kinase activity of wild-type cKit (270 nM), it showed higher potency against the D816V variant form (71 nM). This mutant form of c-Kit is particularly prevalent in patients with systemic mastocytosis. In addition, cKit2 also potently inhibited Bruton’ s Tyrosine Kinase (4 nM), which is critically involved in mast cell activation and signaling. Representative dose-response inhibition curves are shown in FIG. 42A - FIG. 42C with associated IC50 values summarized in FIG. 42D.

[0281] A high throughput competition assay format was also used to compare cKit2 potency against c-Kit D816V to other well-described c-Kit inhibitors which are currently in clinical use or clinical testing, including avapritinib and bezuclastinib. cKit2 potency against the disease relevant D816V compared favorably to benchmarks and was the only inhibitor tested that demonstrated dual activity against c-Kit and BTK, which are both critical for mast cell function and represent validated clinical targets for mast cell modulation. In fact, cKit2 had similar sub-nM potency to Remibrutinib in binding to BTK, in this assay format. Comparative Kd values are summarized in FIG. 43. Combined with the data in FIG. 42A - FIG. 42D, these observations suggest that cKit2 has the potential to mediate multi-pathway inhibition of mast cell activation.

Claims

CLAIMS1. An antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor.

2. The antibody-drug conjugate of claim 1, wherein the antibody or antigenbinding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:(a) the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;(b) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;(c) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 18; a HCDR2 comprising SEQ ID NO: 19; and a HCDR3 comprising SEQ ID NO: 20; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 22; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 23;(d) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 26; and a HCDR3 comprising SEQ ID NO: 27; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 29; a LCDR2 comprising SEQ ID NO: 30; and a LCDR3 comprising SEQ ID NO: 31;(e) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 34; and a HCDR3 comprising SEQ IDNO: 35; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 37; a LCDR2 comprising SEQ ID NO: 38; and a LCDR3 comprising SEQ ID NO: 39;(f) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;(g) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;(h) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 57; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 59; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 60;(i) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 66; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(j) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;(k) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 76; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 77;(l) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 79; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 81; a LCDR2 comprising SEQ ID NO: 82; and a LCDR3 comprising SEQ ID NO: 83;(m) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 85; a HCDR2 comprising SEQ ID NO: 86; and a HCDR3 comprising SEQ ID NO: 87; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 89; a LCDR2 comprising SEQ ID NO: 90; and a LCDR3 comprising SEQ ID NO: 91;(n) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;(o) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO: 103; and the VL domain amino acid sequence comprises a LCDRl comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;(p) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;(q) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;(r) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 121; and the VL domain amino acid sequence comprises a LCDRl comprisingSEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 123; and a LCDR3 comprising SEQ ID NO: 124;(s) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDRl comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;(t) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;(u) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 139; a HCDR2 comprising SEQ ID NO: 140; and a HCDR3 comprising SEQ ID NO: 141; and the VL domain amino acid sequence comprises a LCDRl comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 143;(v) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 145; and the VL domain amino acid sequence comprises a LCDRl comprising SEQ ID NO: 147; a LCDR2 comprising SEQ ID NO: 148; and a LCDR3 comprising SEQ ID NO: 149; or(w) the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDRl comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and aLCDR3 comprising SEQ ID NO: 83.

3. The antibody-drug conjugate of claim 2, wherein:(a) the VH domain amino acid sequence comprises SEQ ID NO: 1, and the VL domain amino acid sequence comprises SEQ ID NO: 5;(b) the VH domain amino acid sequence comprises SEQ ID NO: 9, and the VL domain amino acid sequence comprises SEQ ID NO: 13;(c) the VH domain amino acid sequence comprises SEQ ID NO: 17, and the VL domain amino acid sequence comprises SEQ ID NO: 21;(d) the VH domain amino acid sequence comprises SEQ ID NO: 24, and the VL domain amino acid sequence comprises SEQ ID NO: 28;(e) the VH domain amino acid sequence comprises SEQ ID NO: 32, and the VL domain amino acid sequence comprises SEQ ID NO: 36;(f) the VH domain amino acid sequence comprises SEQ ID NO: 40, and the VL domain amino acid sequence comprises SEQ ID NO: 44;(g) the VH domain amino acid sequence comprises SEQ ID NO: 47, and the VL domain amino acid sequence comprises SEQ ID NO: 51;(h) the VH domain amino acid sequence comprises SEQ ID NO: 55, and the VL domain amino acid sequence comprises SEQ ID NO: 58;(i) the VH domain amino acid sequence comprises SEQ ID NO: 61, and the VL domain amino acid sequence comprises SEQ ID NO: 65;(j) the VH domain amino acid sequence comprises SEQ ID NO: 68, and the VL domain amino acid sequence comprises SEQ ID NO: 70;(k) the VH domain amino acid sequence comprises SEQ ID NO: 72, and the VL domain amino acid sequence comprises SEQ ID NO: 75;(l) the VH domain amino acid sequence comprises SEQ ID NO: 78, and the VL domain amino acid sequence comprises SEQ ID NO: 80;(m) the VH domain amino acid sequence comprises SEQ ID NO: 84, and the VL domain amino acid sequence comprises SEQ ID NO: 88;(n) the VH domain amino acid sequence comprises SEQ ID NO: 92, and the VL domain amino acid sequence comprises SEQ ID NO: 96;(o) the VH domain amino acid sequence comprises SEQ ID NO: 100, and the VL domain amino acid sequence comprises SEQ ID NO: 104;(p) the VH domain amino acid sequence comprises SEQ ID NO: 108, and the VL domain amino acid sequence comprises SEQ ID NO: 112;(q) the VH domain amino acid sequence comprises SEQ ID NO: 116, and the VL domain amino acid sequence comprises SEQ ID NO: 117;(r) the VH domain amino acid sequence comprises SEQ ID NO: 120, and the VL domain amino acid sequence comprises SEQ ID NO: 122;(s) the VH domain amino acid sequence comprises SEQ ID NO: 125, and the VL domain amino acid sequence comprises SEQ ID NO: 129;(t) the VH domain amino acid sequence comprises SEQ ID NO: 133, and the VL domain amino acid sequence comprises SEQ ID NO: 134;(u) the VH domain amino acid sequence comprises SEQ ID NO: 138, and the VL domain amino acid sequence comprises SEQ ID NO: 142;(v) the VH domain amino acid sequence comprises SEQ ID NO: 144, and the VL domain amino acid sequence comprises SEQ ID NO: 146; or(w) the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.

4. The antibody-drug conjugate of claim 1, wherein the antibody or antigenbinding portion is human, humanized, or chimeric.

5. The antibody-drug conjugate of any one of claims 1-4, wherein the antibody or antigen-binding portion comprises an immunoglobulin constant region.

6. The antibody-drug conjugate of claim 5, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

7. The antibody-drug conjugate of claim 6, wherein the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2.

8. The antibody-drug conjugate of claim 5, wherein the immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region.

9. The antibody-drug conjugate of claim 5, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgGl constant region, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A or a wild-type human IgG2 constant region, wherein numbering is according to the EU index as in Kabat.

10. The antibody-drug conjugate of claim 5, wherein the immunoglobulin constant region comprises the amino acid sequence of any one of SEQ ID NOs: 155-160.

11. The antibody-drug conjugate of any one of claims 1-10, wherein the antibody or antigen-binding portion is an Fab, an Fab', an F(ab')2, an Fv, an scFv, a maxibody, a minibody, a diabody, a triabody, a tetrabody, or a bis-scFv.

12. The antibody-drug conjugate of any one of claims 1-10, wherein the antibody or antigen-binding portion is tetrameric, tetravalent, or multispecific.

13. The antibody-drug conjugate of any one of claims 1-12, wherein the c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib.

14. The antibody-drug conjugate of any one of claims 1-12, wherein the c-Kit inhibitor is dasatinib.

15. The antibody-drug conjugate of any one of claims 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a peptide linker.

16. The antibody-drug conjugate of claim 15, wherein the peptide linker comprises two amino acid residues independently selected from glycine, alanine, phenylalanine, lysine, arginine, valine, and citrulline.

17. The antibody-drug conjugate of claim 15, wherein the peptide linker is an alanine-alanine (Ala-Ala) linker.

18. The antibody-drug conjugate of claim 15, wherein the peptide linker is L-Ala- L-Ala, D-Ala-D-Ala, D-Ala-L-Ala, or L-Ala-D-Ala.

19. The antibody-drug conjugate of claim 15, wherein the peptide linker is L-Ala- L-Ala.

20. The antibody-drug conjugate of any one of claims 1-19, wherein the peptide linker is L-Ala-L-Ala; and wherein the antibody-drug conjugate comprises a PEG spacer.

21. The antibody-drug conjugate of any one of claims 1-14, wherein the antibody or antigen-binding portion comprises the c-Kit inhibitor and a linker comprising the structure:(a)22. The antibody-drug conjugate of any one of claims 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via an acid cleavable linker, a glutathione (GSH) cleavable linker, a Fe(II) cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, a phosphatase cleavable linker, a sulfatase cleavable linker, a photo-responsive cleavable linker, or a bioorthogonal cleavable linker.

23. The antibody-drug conjugate of any one of claims 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a hydrazone linker.

24. The antibody-drug conjugate of any one of claims 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a non-cleavable linker.

25. The antibody-drug conjugate of any one of claims 1-19 and 21-24, further comprising a PEG spacer.

26. The antibody-drug conjugate of any one of claims 1-25, wherein the drug-to- antibody ratio (DAR) is about 2 to about 5.

27. The antibody-drug conjugate of any one of claims 1-26, wherein the DAR is about 4.

28. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-27, and a pharmaceutically acceptable carrier.

29. A method for treating a proliferative disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of claims 1-27 or the pharmaceutical composition of claim 28.

30. A method for ameliorating a symptom of a proliferative disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of claims 1-27 or the pharmaceutical composition of claim 28.

31. The method of claim 29 or 30, wherein the proliferative disorder is a mast cell proliferative disorder.

32. The method of any one of claims 29-31, wherein the proliferative disorder is c- Kit-reliant.

33. The method of any one of claims 29-32, wherein the proliferative disorder is systemic mastocytosis, mast cell leukemia, or basophil leukemia.

34. A method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of claims 1-27 or the pharmaceutical composition of claim 28.

35. A method for ameliorating a symptom of an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of claims 1-27 or the pharmaceutical composition of claim 28.

36. The method of claim 34 or 35, wherein the inflammatory disorder is a mast cell inflammatory disorder.

37. The method of any one of claims 34-36, wherein the inflammatory disorder is c-Kit-reliant.

38. The method of any one of claims 34-37, wherein the inflammatory disorder is a chronic inflammatory disorder.

39. The method of any one of claims 34-38, wherein the inflammatory disorder is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

40. Use of the antibody-drug conjugate of any one of claims 1-27 or the pharmaceutical composition of claim 28 in the preparation of a medicament.

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