CD38 Antibody Drug Conjugate

Abstract

Disclosed herein are antibody drug conjugates (ADCs) having an IgG antibody that binds to a CD38 target, the IgG antibody being conjugated at a Cys site in its hinge region. Also disclosed herein are methods of treating multiple myeloma, the methods comprising providing an effective amount of a CD38 ADC.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 62 / 522,516 filed on June 20, 2017 and U.S. Provisional Patent Application No. 62 / 553,438 filed on September 1, 2017. Technical Field

[0003] The present disclosure provides an antibody drug conjugate (ADC) having an IgG antibody that binds to a CD38 target, the IgG antibody being conjugated at a Cys site in its hinge region. The present disclosure also provides a method for treating multiple myeloma, the method comprising providing an effective amount of a CD38 ADC. Background Art

[0004] CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 protein is a bifunctional exonuclease that can catalyze the + It is converted to cyclic ADP-ribose (cADPR) and can also hydrolyze cADPR to ADP-ribose. During individual development, CD38 appears on CD34 + Committed stem cells and lineage-committed progenitors of lymphoid, erythroid, and myeloid cells. CD38 expression persists primarily in the lymphoid lineage and has different levels at different stages of T- and B-cell development.

[0005] CD38 is upregulated in many hematopoietic malignancies and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple neoplasms (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL) and chronic myeloid leukemia (CML). On the other hand, most primitive pluripotent stem cells in the hematopoietic system are CD38 - The expression of CD38 in hematopoietic malignancies and its association with disease progression make CD38 an attractive target for anti-CD38 antibody therapy.

[0006] It has been reported that CD38 is involved in Ca 2+Mobilization of mitochondria (Morra et al., 1998, FASEB J., 12:581-592; Zilber et al., 2000, Proc. Natl. Acad. Sci. USA, 97:2840-2845), and involved in signal transduction in lymphoid and myeloid cells or cell lines through tyrosine phosphorylation of various signaling molecules, including phospholipase C-γ, ZAP-70, syk and c-cbl (Funaro et al., 1993, Eur. J. Immunol., 23:2407-2411; Morra et al., 1998, FASEB J., 12:581-592; Funaro et al., 1990, J Immunol, 145:2390-2396; Zubiaur et al., 1997, J Immunol, 159:193-205; Deaglio et al., 2003, Blood CD38 is indicated as an important signaling molecule for the maturation and activation of lymphoid and myeloid cells during normal development.

[0007] Evidence for CD38 function comes from CD38 - / -Knockout mice that have innate immune deficiencies and reduced T cell-dependent humoral responses due to defects in dendritic cell migration (Partida-Sanchez et al., 2004, Immunity, 20:279-291; Partida-Sanchez et al., 2001, Nat. Med., 7:1209-1216). However, it is not clear whether the function of CD38 in mice is the same as that in humans, because the expression pattern of CD38 during hematopoiesis differs greatly between humans and mice: a) unlike immature progenitor stem cells in humans, similar progenitor stem cells in mice express high levels of CD38 (Randall et al., 1996, Blood, 87:4057-4067; Dagher et al., 1998, Biol. Blood Marrow Transplant, 4:69-74), b) during human B cell development, high CD38 expression levels were found in germinal center B cells and plasma cells (Uckun, 1990, Blood, 76:1908-1923; Kumagai et al., 1995, J. Exp. Med., 181:1101-1110), while in mice, CD38 expression levels in the corresponding cells are low (Oliver et al., 1997, J. Immunol., 158:108-1115; Ridderstad and Tarlinton 1998, J. Immunol., 160:4688-4695).

[0008] Several anti-human CD38 antibodies with different proliferation characteristics on various tumor cells and cell lines have been described in the literature. For example, in the presence of peripheral blood mononuclear effector cells from MM patients or normal individuals, the chimeric OKT10 antibody with mouse Fab and human IgG1 Fc is very effective in mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against lymphocytes (Stevenson et al., 1991, Blood, 77: 1071-1079). It has been shown that the humanized form of the CDR transplantation of the anti-CD38 antibody AT13 / 5 has effective ADCC activity against CD38-positive cell lines. It has been shown that human monoclonal anti-CD38 antibodies mediate in vitro killing of CD38-positive cell lines by ADCC and / or complement-dependent cytotoxicity (CDC), and delay tumor growth in SCID mice with MM cell line RPMI-8226 (WO2005 / 103083A2). On the other hand, several anti-CD38 antibodies, IB4, SUN-4B7 and OKT10, but not IB6, AT1 or AT2, induced proliferation of peripheral blood mononuclear cells (PBMCs) from normal individuals (Ausiello et al. 2000, Tissue Antigens, 56:539-547).

[0009] It has been shown that some prior art antibodies can trigger CD38 + However, these antibodies can only trigger CD38 apoptosis in the presence of stromal cells or stromal-derived cytokines. + Apoptosis of B cells. An agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis of human germinal center (GC) B cells (Zupo et al., 1994, Eur. J. Immunol., 24: 1218-1222), induce proliferation of KG-1 and HL-60 AML cells (Konopleva et al., 1998, J. Immunol., 161: 4702-4708), but induce apoptosis of Jurkat T lymphoblastoid cells (Morra et al., 1998, FASEB J., 12: 581-592). Another anti-CD38 antibody, T16, induces apoptosis of immature lymphoid cells and leukemic lymphoblasts from ALL patients (Kumagai et al., 1995, J. Exp. Med., 181: 1101-1110), as well as apoptosis of leukemic myeloblasts from AML patients (Todisco et al. 2000, Blood, 95: 535-542), but T16 induces apoptosis only in the presence of stromal cells or stromal-derived cytokines (IL-7, IL-3, stem cell factor).

[0010] Therefore, the inventors believe that antibody drug conjugates (ADCs) targeting with anti-CD38 antibodies offer the promise and potential to provide effective anti-tumor activity while having the advantage of reduced side effects. SUMMARY OF THE INVENTION

[0012] The present disclosure provides an antibody drug conjugate (ADC) having an IgG antibody that binds to a CD38 target, the IgG antibody being conjugated at a Cys site in its hinge region. The present disclosure also provides a method for treating multiple myeloma, the method comprising providing an effective amount of a CD38 ADC.

[0013] More specifically, the present disclosure provides an anti-CD38 ADC composition comprising:

[0014] (a) anti-CD38 IgG antibody C38A2 (whose heavy chain / light chain variable region comprises SEQ ID NO.1 / 2) or C38D8 (whose heavy chain / light chain variable region comprises SEQ ID NO.3 / 4);

[0015] (b) a drug or toxic substance moiety, wherein the drug or toxic substance moiety is a tubulin inhibitor or a doxorubicin analog; and

[0016] (c) a conjugated linker moiety, wherein the conjugated linker moiety binds to a single Cys residue in the hinge region of an IgG antibody, and wherein the heavy chain hinge region of the IgG antibody can be mutated such that the heavy chain hinge region contains only one Cys residue.

[0017] Preferably, the drug or toxic substance portion is selected from the group consisting of D1, D2, D3, D4, D5 and a combination thereof, wherein the structures of D1, D2, D3, D4 and D5 are:

[0018]

[0019] In some embodiments, the conjugated linker portion comprises a linker portion and a conjugated portion. In some embodiments, the conjugated linker portion comprises one or more of the following structures:

[0020]

[0021] Wherein the wavy line indicates the site of connection to the drug or toxic substance part and the conjugate part. In some embodiments, the conjugate part is The wavy line indicates the site of attachment to the conjugation linker moiety.

[0022] Preferably, the conjugated linker moiety is selected from:

[0023]

[0024] The wavy lines indicate connection points.

[0025] Preferably, the conjugated moiety is The wavy lines indicate connection points.

[0026] In another aspect, an antibody drug conjugate (ADC) composition is provided, comprising an IgG antibody that binds to CD38, a linker portion, and a toxic substance portion conjugated to the linker portion, wherein the linker portion is conjugated to one Cys residue in the hinge region of an IgG antibody mutated to have only one Cys residue.

[0027] In another aspect, an anti-CD38 ADC composition is provided, comprising:

[0028] (a) anti-CD38 IgG antibody C38A2-SV (whose heavy chain / light chain variable region comprises SEQ ID NO.1 / 3) or C38A2 (whose heavy chain / light chain variable region comprises SEQ ID NO.1 / 2);

[0029] (b) a drug or toxic substance moiety, wherein the drug or toxic substance moiety is a tubulin inhibitor or a doxorubicin analog; and

[0030] (c) a conjugate linker moiety, wherein the conjugate linker comprises a linker and a conjugate moiety, the conjugate moiety is covalently bound to a single Cys residue in the hinge region of an IgG antibody, and wherein the heavy chain hinge region of the IgG antibody can be mutated so that the heavy chain hinge region contains only one Cys residue.

[0031] In some embodiments, the drug or toxic substance portion is selected from the group consisting of D1, D2, D3, D4, D5 and a combination thereof, wherein the structures of D1, D2, D3, D4 and D5 are:

[0032]

[0033] In some embodiments, the linker is selected from:

[0034]

[0035] The wavy lines indicate the sites of attachment to the conjugate moiety and the drug or toxic substance moiety.

[0036] In some embodiments, the conjugated moiety is The wavy line indicates the site of attachment to the linker.

[0037] The present disclosure also provides a method for treating multiple myeloma, the method comprising administering a therapeutically effective amount of an anti-CD38 ADC composition, the anti-CD38 ADC composition comprising:

[0038] (a) anti-CD38 IgG antibody C38A2 (whose heavy chain / light chain variable region comprises SEQ ID NO.1 / 2) or C38D8 (whose heavy chain / light chain variable region comprises SEQ ID NO.3 / 4);

[0039] (b) a drug or toxic substance moiety, wherein the drug or toxic substance moiety is a tubulin inhibitor or a doxorubicin analog; and

[0040] (c) a conjugated linker moiety, wherein the conjugated linker moiety binds to a single Cys residue in the hinge region of an IgG antibody, and wherein the heavy chain hinge region of the IgG antibody can be mutated such that the heavy chain hinge region contains only one Cys residue.

[0041] Preferably, the drug or toxic substance portion is selected from the group consisting of D1, D2, D3, D4, D5 and a combination thereof, wherein the structures of D1, D2, D3, D4 and D5 are:

[0042]

[0043] In some embodiments, the conjugated linker portion comprises a linker portion and a conjugated portion. In some embodiments, the conjugated linker portion comprises one or more of the following structures:

[0044]

[0045] Wherein the wavy line indicates the site of connection to the drug or toxic substance part and the conjugate part. In some embodiments, the conjugate part is The wavy line indicates the site of attachment to the conjugation linker moiety.

[0046] Preferably, the linker moiety of the conjugated linker moiety is selected from:

[0047]

[0048] The wavy lines indicate connection points.

[0049] Preferably, the conjugated moiety is The wavy lines indicate connection points.

[0050] In another aspect, a method of treating multiple myeloma is provided, the method comprising providing a therapeutically effective amount of an anti-CD38 ADC composition, the anti-CD38 ADC composition comprising:

[0051] (a) anti-CD38 IgG antibody C38A2-SV (whose heavy chain / light chain variable region comprises SEQ ID NO.1 / 3) or C38A2 wild type (whose heavy chain / light chain variable region comprises SEQ ID NO.1 / 2);

[0052] (b) a drug or toxic substance moiety, wherein the drug or toxic substance moiety is a tubulin inhibitor or a doxorubicin analog; and

[0053] (c) a conjugate linker moiety, wherein the conjugate linker comprises a linker and a conjugate moiety, the conjugate moiety is covalently bound to a single Cys residue in the hinge region of an IgG antibody, and wherein the heavy chain hinge region of the IgG antibody can be mutated so that the heavy chain hinge region contains only one Cys residue.

[0054] In some embodiments, the drug or toxic substance portion is selected from the group consisting of D1, D2, D3, D4, D5 and a combination thereof, wherein the structures of D1, D2, D3, D4 and D5 are:

[0055]

[0056] In some embodiments, the linker is selected from:

[0057]

[0058] The wavy lines indicate the sites of attachment to the conjugate moiety and the drug or toxic substance moiety.

[0059] In some embodiments, the conjugated moiety is The wavy line indicates the site of attachment to the linker.

[0060] Preferably, the antibody portion is a variant of the CD38A2 wild-type antibody disclosed and claimed in U.S. Serial No. 15 / 094,384 filed on April 8, 2016, the disclosure of which is incorporated herein by reference. The CD38A2 wild-type variable region sequence is disclosed herein as heavy chain SEQ ID NO.1 and light chain SEQ ID NO.2. More specifically, the variant sequence changes the second and third amino acids from the N-terminus of the light chain variable region. Preferably, the antibody portion comprises CD38A2-SV (SV variant) having heavy chain SEQ ID NO.1 and light chain SEQ ID NO.3. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A shows an in vivo study of an anti-CD38 ADC in a Burkitt lymphoma model. In this study, 10 million Daudi cells were injected subcutaneously into Nu Nu mice. The average tumor volume in tumor-bearing mice reached 200 mm 3 Afterwards, ADC#45 was intravenously injected into tumor-bearing mice.

[0063] Figure 1 B shows body weight measurements of mice treated with three different indicated doses of ADC#45.

[0064] Figure 2 A shows an in vivo study of an anti-CD38 ADC in a Burkitt lymphoma model. In this study, 10 million Ramos cells were injected subcutaneously into Nu Nu mice. The average tumor volume in tumor-bearing mice reached 200 mm 3 Afterwards, ADC#45 was intravenously injected into tumor-bearing mice at the indicated doses.

[0065] Figure 2 B shows body weight measurements of mice treated with anti-CD38-ADC.

[0066] Figure 3 An in vivo study of an anti-CD38 ADC in a Burkitt's lymphoma model is shown. In this study, 10 million Daudi-luc cells were injected intravenously into NOD-SCID mice. 14 days after tumor injection, an anti-CD38 antibody (A2) and two anti-CD38 ADCs made from the same A2 antibody were injected intravenously into tumor-bearing mice.

[0067] Figure 4 In vivo studies of anti-CD38 ADCs in a Burkitt's lymphoma model are shown. In this study, 10 million Daudi-luc cells were injected intravenously into NOD-SCID mice. ADC #45 and ADC #41 were injected intravenously into tumor-bearing mice 14 days after tumor injection.

[0068] Figure 5 An in vivo study of anti-CD38 ADC in a Burkitt's lymphoma model is shown. In this study, 10 million Daudi-luc cells were injected intravenously into NOD-SCID mice. 14 days after tumor injection, anti-CD38 antibodies and anti-CD38-ADC were injected intravenously into tumor-bearing mice. Images of mice were taken once a week.

[0069] Figure 6In vivo studies of anti-CD38 ADCs in a Burkitt's lymphoma model are shown. In this study, 10 million Daudi-luc cells were injected intravenously into NOD-SCID mice. ADC#45 and ADC#41 were injected intravenously into tumor-bearing mice 14 days after tumor injection. Images of mice were taken weekly.

[0070] Figure 7 CD38-expressing cancer cell lines Ramos, Raji and RPMI8226, and CD38-negative cell line PC-3 were seeded in 96-well plates and treated with serial dilutions of ADC#45, ADC#41 and ADC#46 starting at 100 nM. Cells were treated for 3-5 days depending on the nature of the conjugated payload. At the end of treatment, cells were stained with CelltitreGlo from Promega. TM The cells were stained with a luminescence kit and the signal was captured by a luminescence plate reader. The activity of ADC on tumor cell growth inhibition was expressed as the concentration required for 50% cell growth inhibition, the so-called EC50 (in nM). The data showed that ADC#45, ADC#41 and ADC#46 showed selective inhibition of cells expressing CD38 on their surface.

[0071] Figure 8 Shown is an HIC-HPLC overlay of the starting anti-CD38 antibody and the purified ADC46 conjugate with detection at 280 nm.

[0072] Fig. 9 Shown is an HIC-HPLC overlay of the starting anti-CD38 antibody and the purified ADC41 conjugate with detection at 280 nm.

[0073] Fig.10 A shows an in vivo study of anti-CD38 ADC46 in a Burkitt lymphoma model. In this study, 10 million Daudi cells were injected subcutaneously into Nu Nu mice. The average tumor volume of tumor-bearing mice reached 200 mm 3 Afterwards, ADC 46 was intravenously injected into tumor-bearing mice.

[0074] Fig.10 B shows body weight measurements of mice treated with three different indicated doses of ADC 46. DETAILED DESCRIPTION OF THE INVENTION

[0076] The present disclosure provides an antibody drug conjugate containing a novel human anti-CD38 antibody (A2) (described in U.S. Patent Application 2016 / 0297888, Serial No. 15 / 094,384, filed on April 8, 2016, the disclosure of which is incorporated herein by reference) and a toxic moiety as described herein (including a tubulin inhibitor or a DNA damaging agent, such as a doxorubicin analog). The ADC conjugate retains binding affinity and exhibits potent cell killing in a variety of CD38-positive cell lines and in vivo.

[0077] The present disclosure provides an antibody drug conjugate (ADC) composition, the composition comprising an IgG antibody that binds to CD38, a conjugated linker portion (which binds to a single Cys residue in the hinge region of the IgG antibody, wherein the hinge region can be mutated so that the heavy chain hinge region contains only one rather than two Cys residues), and a toxic substance portion selected from anthracyclines and tail sea urin derivatives. Preferably, the toxic substance portion is a tubulin inhibitor or a doxorubicin analog. Preferably, the antibody is an IgG antibody of the family called human C38A2 (heavy / light chain SEQ ID NO 3 / 4 in U.S. patent application 2016 / 0297888 or heavy chain / light chain variable region SEQ ID NO.1 / 2 herein), or C38D8 (heavy / light chain SEQ ID NO 21 / 22 in U.S. patent application 2016 / 0297888 or heavy chain / light chain variable region SEQ ID NO.3 / 4 herein). Preferably, the conjugated toxic substance with a linker structure is selected from:

[0078]

[0079]

[0080] The present disclosure provides a method for treating multiple myeloma, the method comprising administering an effective amount of an antibody drug conjugate (ADC) composition, the composition comprising an IgG antibody bound to CD38, a conjugated linker portion bound to a Cys residue in the hinge region of the IgG antibody, and a toxic substance portion. "Binding to a Cys residue" means that the conjugated linker portion can be covalently bound to the sulfur atom of the Cys residue in the hinge region of the IgG antibody. Preferably, the toxic substance portion is a tubulin inhibitor or a doxorubicin analog. Preferably, the antibody is an IgG antibody of the family called human C38A2 (heavy / light chain SEQ ID NO 3 / 4 in U.S. patent application 2016 / 0297888 or heavy chain / light chain variable region SEQ ID NO.1 / 2 herein), or C38D8 (heavy / light chain SEQ ID NO 21 / 22 in U.S. patent application 2016 / 0297888 or heavy chain / light chain variable region SEQ ID NO.3 / 4 herein). Those skilled in the art will recognize that the toxic substance moiety disclosed herein conjugated to the linker and conjugated moiety disclosed herein represents an intermediate toxic substance linker conjugate, which, when covalently bound (conjugated to) the IgG antibody disclosed herein, is an ADC disclosed herein. Preferably, the conjugated toxic substance with a linker structure is selected from (the number of each compound is indicated):

[0081]

[0082]

[0083]

[0084]

[0085] Toxic substances (D): D1 and D2 are anthracycline derivatives. D3, D4 and D5 are tubulin inhibitors.

[0086]

[0087] Connector part (L2):

[0088]

[0089] Conjugation method (L1)

[0090]

[0091] The wavy line indicates the site of attachment to the linker.

[0092] In some embodiments, the drug-linker conjugate comprises a linker L2 and a conjugated moiety, wherein the linker L2 is covalently bound to the conjugated moiety; the conjugated moiety is capable of reacting with a free cysteine ​​thiol group in the hinge region of an IgG antibody. In some embodiments, the conjugated moiety has a structure ("Conjugation method L1").

[0093] Examples of Drug-Linker Conjugates:

[0094]

[0095]

[0096] Examples of anti-CD38 ADCs

[0097]

[0098]

[0099] definition

[0100] As used herein, common organic abbreviations are defined as follows:

[0101] Ac Acetyl

[0102] ACN Acetonitrile

[0103] Ala Alanine

[0104] Asn Asparagine

[0105] aq. containing water

[0106] BOC or Boc tert-butyloxycarbonyl

[0107] ℃ Celsius temperature

[0108] Citrulline

[0109] DCM Dichloromethane

[0110] DIEA Diisopropylethylamine

[0111] DMF N,N'-Dimethylformamide

[0112] EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0113] Et Ethyl

[0114] EtOAc Ethyl acetate

[0115] Eq equivalent

[0116] Fmoc 9-Fluorenylmethoxycarbonyl

[0117] g

[0118] h hour

[0119] HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

[0120] HOBt N-Hydroxybenzotriazole

[0121] HPLC High Performance Liquid Chromatography

[0122] LC / MS Liquid chromatography-mass spectrometry

[0123] Me Methyl

[0124] mg milligram

[0125] MeOH Methanol

[0126] mL milliliters

[0127] μL / μL microliter

[0128] mol mole

[0129] mmol millimole

[0130] μmol / umol micromole

[0131] MS

[0132] NHS N-Hydroxysuccinimide

[0133] PAB p-Aminobenzyl

[0134] Pip Piperidine

[0135] RP-HPLC Reverse Phase HPLC

[0136] rt room temperature

[0137] t-Bu tert-butyl

[0138] Tert, Uncle

[0139] TFA Trifluoroacetic acid

[0140] THF Tetrahydrofuran

[0141] Val

[0142] Examples of Drug-Linker Conjugates:

[0143]

[0144]

[0145] Examples of anti-CD38 ADCs (the antibody component is referred to as "Ab"):

[0146]

[0147]

[0148] Synthesis Example 1

[0149] Synthesis of compound 18:

[0150]

[0151] Synthesis of compound 11:

[0152] Compound 10 (30 mg, 46.8 μmol) was dissolved in 3 mL of anhydrous DCM under nitrogen. DIEA (24 μL, 140 μmol) was subsequently added and the reaction mixture was cooled with an ice bath. Methanesulfonyl chloride (7.2 μL, 93.6 μmol) was subsequently added and the mixture was stirred for 30 minutes. The reaction was diluted with 3 mL of DCM and washed with 4 mL of water, dried over anhydrous Na2SO4 and evaporated to give compound 11 (33 mg, 98%) as a red solid. MS m / z=720.5 (M+H).

[0153] Synthesis of compound 13:

[0154] Compound 11 (20 mg, 27.8 μmol) was dissolved in 3 mL of anhydrous ethanol under nitrogen. Thioamide 12 (45 mg, 139 μmol) was then added and the mixture was heated at 40° C. for 24 hours. The mixture was purified by HPLC to give compound 13 (15 mg, 59%) as a red solid. MS m / z=932.6 (M+H).

[0155] Synthesis of compound 14:

[0156] Compound 13 (15 mg, 16.1 μmol) was dissolved in 2 mL of anhydrous DMF under nitrogen. 60 μL of piperidine was then added and the mixture was stirred at ambient temperature for 10 minutes. The mixture was purified by HPLC to give compound 14 (6.9 mg, 60%) as a red solid. MS m / z=710.4 (M+H).

[0157] Synthesis of compound 16:

[0158] Compound 15 (8.9 mg, 16.9 μmol) was dissolved in 2 mL DMF, followed by the addition of HATU (6.4 mg, 16.8 μmol) and DIEA (9 μL, 51.8 μmol). After 2 minutes, compound 14 (10 mg, 14.1 μmol) was added, and the mixture was stirred at ambient temperature for 1 hour. 40 μL DBU was added to the mixture and stirred for 10 minutes. The mixture was then purified by HPLC to give compound 16 (12.2 mg, 87%) as a red solid. MS m / z=995.4 (M+H).

[0159] Synthesis of compound 18:

[0160] Compound 17 (12.5 mg, 24.2 μmol) was dissolved in 2 mL DCM, followed by the addition of DIC (1.6 mg, 12.7 μmol). After 10 minutes, compound 16 (12 mg, 12.1 μmol) dissolved in 0.5 mL DMF was added, and the mixture was stirred at ambient temperature for 10 minutes. The mixture was then purified by HPLC to give compound 18 (12.8 mg, 71%) as a red solid. MS m / z=1494.4 (M+H).

[0161] Synthesis of compound 22:

[0162]

[0163] Synthesis of compound 21:

[0164] To a solution of acid 19 (51 mg, 81 μmol) in DCM (6 mL) was added N-hydroxysuccinimide (46 mg, 400 μmol) and EDC (100 mg, 523 μmol). After 30 minutes, the mixture was washed with water (2x6 mL), dried over Na2SO4 and evaporated. The residue was dissolved in 2 mL DMF. Amine 20 (55 mg, 81 μmol, TFA salt form) was then added, followed by DIEA (50 μL). The mixture was stirred for 1 hour. Piperidine (40 μL) was then added and stirred for 20 minutes. The mixture was purified by HPLC to give compound 21 (34 mg, 44%) as a red solid; MS m / z 955.2 (M+H).

[0165] Synthesis of compound 22:

[0166] Compound 17 (12.5 mg, 24.2 μmol) was dissolved in 2 mL DCM, followed by the addition of DIC (1.6 mg, 12.7 μmol). After 10 minutes, compound 21 (11.5 mg, 12.1 μmol) dissolved in 0.5 mL DMF was added, and the mixture was stirred at ambient temperature for 10 minutes. The mixture was then purified by HPLC to give compound 22 (7.0 mg, 42%) as a red solid. MS m / z=1453.6 (M+H).

[0167] Synthesis of compound 27:

[0168]

[0169] Synthesis of compound 26:

[0170] Compound 24 (120.6 mg, 96.2 μmol), DIEA (50 μL), and HOBt (13 mg, 96.3 μmol) were added to a solution of the TFA salt of compound 23 (84.2 mg, 96.3 μmol) in DMF (2 mL). After 24 hours, the reaction was complete, followed by the addition of piperidine (60 μL) and stirring for 10 minutes. The mixture was purified by HPLC to give compound 26 (134 mg, 80%) as a white solid; MS m / z 1635.3 (M+H).

[0171] Synthesis of compound 27:

[0172] Compound 17 (25 mg, 48.4 μmol) was dissolved in 2 mL DCM, followed by the addition of DIC (3.2 mg, 25.4 μmol). After 10 minutes, compound 26 (39.5 mg, 24.2 μmol) dissolved in 0.5 mL DMF was added, and the mixture was stirred at ambient temperature for 10 minutes. The mixture was then purified by HPLC to give compound 27 (32.0 mg, 62%) as a white solid. MS m / z=2134.1 (M+H).

[0173] Synthesis of compound 31:

[0174]

[0175] Synthesis of compound 30:

[0176] Compound 24 (45 mg, 36 μmol), DIEA (20 μL), and HOBt (5 mg, 37 μmol) were added to a solution of the TFA salt of compound 28 (30 mg, 36 μmol) in DMF (1 mL). After 24 hours, the reaction was complete, and piperidine (20 μL) was subsequently added and stirred for 30 minutes. The mixture was purified by HPLC to give compound 30 (46 mg, 79%) as a white solid; MS m / z 1635.3 (M+H).

[0177] Synthesis of compound 31:

[0178] Compound 17 (30 mg, 57.1 μmol) was dissolved in 2 mL DCM, followed by the addition of DIC (3.6 mg, 28.6 μmol). After 10 minutes, compound 30 (46 mg, 28.6 μmol) dissolved in 0.5 mL DMF was added, and the mixture was stirred at ambient temperature for 10 minutes. The mixture was then purified by HPLC to give compound 31 (44.8 mg, 59%) as a white solid. MS m / z=2109.2 (M+H).

[0179] Synthesis of compound 34:

[0180]

[0181] Synthesis of compound 33:

[0182] Compound 15 (17 mg, 33 μmol) was dissolved in 2 mL DMF, followed by the addition of HATU (12.5 mg, 32.9 μmol) and DIEA (23 μL). After 2 minutes, the TFA salt of compound 32 (24 mg, 27.6 μmol) was added, and the mixture was stirred at ambient temperature for 1 hour. 40 μL DBU was added to the mixture and stirred for 10 minutes. The mixture was then purified by HPLC to give compound 33 (30.2 mg, 85%) as a white solid. MS m / z=1057.8 (M+H).

[0183] Synthesis of compound 34:

[0184] Compound 17 (12.5 mg, 24.2 μmol) was dissolved in 2 mL DCM, followed by the addition of DIC (1.6 mg, 12.7 μmol). After 10 minutes, compound 33 (12.8 mg, 12.1 μmol) dissolved in 0.5 mL DMF was added, and the mixture was stirred at ambient temperature for 10 minutes. The mixture was then purified by HPLC to give compound 34 (14.5 mg, 77%) as a white solid. MS m / z=1556.8 (M+H).

[0185] Synthesis of compound 39:

[0186]

[0187] Synthesis of compound 36:

[0188] Into a round bottom flask were added the TFA salt of compound 33 (88.6 mg, 0.1 mmol), compound 35 (84 mg, 0.1 mmol), HOAt (41 mg, 0.3 mmol), DCM (5 mL), DIEA (104 μL) and DIC (25 mg, 0.2 mmol). After stirring for 16 hours, the reaction mixture was diluted with 5 mL of DCM, then washed with 5 mL of water, dried over Na2SO4, and the solvent was evaporated under vacuum to obtain a crude glassy solid, which was used in the next step. The obtained solid was dissolved in a mixture consisting of 2 mL of DCM, 2 mL of TFA and 0.2 mL of triisopropylsilane and stirred for 1 hour. The solvent was evaporated under vacuum and purified by HPLC to obtain compound 36 (81 mg, 60%), MS m / z 1351.5 (M+H).

[0189] Synthesis of compound 37:

[0190] Compound 36 (81 mg, 0.06 mmol), 2 mL CAN, 1 mL water and 1 mL saturated NaHCO3 aqueous solution were added to a round-bottom flask, followed by Na2S2O4 (42 mg, 0.24 mmol) and continued stirring for 20 minutes. The mixture was purified by HPLC to give compound 37 (55 mg, 70%), MS m / z 1321.7 (M+H).

[0191] Synthesis of compound 39:

[0192] Compound 37 (53 mg, 0.04 mmol) was dissolved in 2 mL of CAN, and 1,4-dibromo-2,3-butanedione (38) (29 mg, 0.12 mmol) was added. After stirring for 20 minutes, the mixture was purified by HPLC to obtain compound 39 (40 mg, 65%), MS m / z 1527.6 (M+H).

[0193] Synthesis of compound 52:

[0194]

[0195] Synthesis of compound 51:

[0196] To a solution of amine 32 (875 mg, 1.13 mmol) and acid 50 (1000 mg, 1.13 mmol) in DMF (10 mL) was added ethyl 2-oximecyanoacetate (Oxima-pure) (160 mg, 1.13 mmol) and DIC (428 mg, 2.74 mmol). After 2 hours, the coupling was complete, followed by the addition of 1 mL of piperidine and stirring for 20 minutes. The mixture was purified by HPLC to give the TFA salt of compound 51 as a white solid (1020 mg, 69%); MS m / z 1191.7 (M+H).

[0197] Synthesis of compound 52:

[0198] The TFA salt of compound 51 (200 mg, 141 μmol) was dissolved in 2 mL ACN and 1 mL water. Then, a solution of 1,4-dibromo-2,3-butanedione 38 (69 mg, 282 μmol) in ACN (1 mL) was added. After stirring for 15 minutes, the mixture was purified by HPLC to obtain compound 52 (166 mg, 84%) as a white solid. MS m / z=1397.6 (M+H).

[0199] ADC Preparation Example 1

[0200] Preparation of ADC46

[0201] The affinity purified anti-CD38 antibody was buffer exchanged to 50mM sodium phosphate buffer (pH 7.0-7.2) with 4mM EDTA at a concentration of 5-10mg / mL. A fresh 10mM aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) was added to a portion of the antibody stock solution until a 20-fold molar excess. The resulting mixture was incubated overnight at 4-8°C. Excess TCEP was removed by gel filtration chromatography or several rounds of centrifugal filtration. The recovered reduced antibody material was subjected to UV-Vis quantitative analysis, followed by confirmation of sufficient free thiol / antibody ratio. In brief, a 1mM aliquot of a freshly prepared (5,5'-dithiobis-(2-nitrobenzoic acid) in 50mM sodium phosphate, pH 7.0-7.2, 4mM EDTA was mixed with an equal volume of purified antibody solution. The resulting absorbance was measured at 412nm and 14,150M was used. -1 cm -1 The extinction coefficient of was used to determine the reduced cysteine ​​content.

[0202] To initiate the conjugation of compound 52 with anti-CD38 antibody, L014-077 was first dissolved in a 3:2 acetonitrile / water mixture at a concentration of 5 mM. Then an aliquot of the newly prepared toxic substance-linker solution was added to a portion of the reduced, purified anti-CD38 antibody intermediate with a 4.5-5 times molar excess. After fully mixing and incubating for ≥1 hour at ambient temperature, the crude conjugation reaction was analyzed by HIC-HPLC chromatography, and the reaction was confirmed to be complete (the initial antibody peak disappeared) under 280 nm wavelength detection. Subsequently, the AKTA system of a Superdex 200 pg column (GE Healthcare) equipped with PBS balance was used to purify ADC46 by gel filtration chromatography. Drug / antibody ratio (DAR) was calculated based on UV-VIS and HIC-HPLC. Figure 8 A representative HIC-HPLC comparison of the starting anti-CD38 antibody and purified ADC46 is shown. Analytical SEC-HPLC was used to determine that the resulting ADC46 was a low percentage (<5%) of high molecular weight (HMW) aggregates.

[0203] Preparation of ADC41

[0204] The reduction and analysis of the anti-CD38 antibody of ADC41 were performed in the same manner as the steps for producing ADC46. To initiate the final drug-linker conjugation with the antibody, compound 22 was first dissolved in a 2:3 acetonitrile / water mixture at a concentration of 5 mM. Propylene glycol (PG) was then added to an aliquot of the reduced, purified anti-CD38 antibody to obtain a final PG concentration of 10-30% (v / v), followed by the addition of a freshly prepared solution of compound 22 in 4.5-5-fold molar excess. The analysis and purification of ADC41 were then performed in the same manner as the steps in ADC46. Fig. 9 Representative HIC-HPLC comparisons of starting anti-CD38 antibody and purified ADC41 are shown.

[0205] Test Example 1

[0206] Upon receipt of the animals, they were housed in an environmentally controlled room with 5 mice per cage. Rodent chow and water were provided to the animals ad libitum. Mice were acclimated to laboratory conditions at least 72 hours prior to the start of cell administration and dosing. During the acclimation period, the health status of the animals was determined. Only animals that were observed to be healthy before the start of the study were used.

[0207] This example provides an in vivo experiment comparing treatment of mice with a control (PBS), anti-CD38 IgG1 antibodies (STI-0602 and STI-0607), and ADC variants of both antibodies. The procedure begins with tumor cell inoculation and tumor establishment:

[0208] a. U87 cells were cultured with 10% FBS U87 medium (EMEM) and harvested with 0.05% trypsin. The cells were washed twice with serum-free EMEM, counted, and counted at 5x10 6 cells / 0.2mL or 25x10 6 Cells were resuspended at a concentration of 1:1 in a 1:1 mixture of serum-free EMEM and Matrigel and injected subcutaneously into the right upper abdomen of each mouse.

[0209] b. Tumor growth was monitored by measuring tumor volume dimensions using a digital caliper starting on days 6-9 after inoculation and twice weekly thereafter until study termination.

[0210] c. Measure the tumor using a digital caliper. Measure the length (longest dimension) and width (the distance perpendicular to the length and in the same plane). The formula used to calculate tumor volume is TV (mm 3 )=1 / 2x L x W 2 .

[0211] treat:

[0212] a. Once the tumor has been staged to the desired volume (average 200 to 300 mm 3 ), the animals were randomly divided into groups and mice with very large or very small tumors were excluded. Mice were randomly divided into 8 groups according to tumor volume, with 10 mice in each group.

[0213] b.According to Figure 4 , mice were treated with vehicle or test article. Mice received a total of 5 doses.

[0214] c. Monitor tumor response once a clear therapeutic trend is established and / or when tumor burden in vehicle-treated mice reaches IACUC protocol limits (2000 mm 3 ), the study was terminated.

[0215] Test Example 2

[0216] This example is an in vivo experiment comparing two disclosed CD38 ADCs in mice. In the in vivo study, 10 million Daudi-fluc cells were intravenously injected into NOD-SCID mice. Four days after the mouse tumors were formed, the anti-CD38 antibody and ADC were injected intravenously (IV) into the mice. The inhibition of tumor growth by the antibody or ADC was monitored by the changes in the luminescence intensity of the tumor ( Figure 3 , 4 , 5 and 6).

[0217] ADC#45 and ADC#41 were tested. Both ADCs used the same A2 antibody. Daudi and Ramos cell lines were obtained from ATCC. Cells were cultured in RPMI 1640 1X medium containing 10% FBS at 37°C and a humidified environment with 5% carbon dioxide. The cells were cultured for 2 weeks and passaged 3 times before harvesting. Prior to injection, Daudi or Ramos cells were resuspended in a 1:1 ratio of HBSS (Hank's Balanced Salt Solution) and Matrigel, and 10 million cells / 0.2 ml were injected subcutaneously into the right upper abdomen of each mouse.

[0218] Daudi-luc cells were cultured in RPMI 1640 1X medium containing 10% FBS and 0.2ug / ml puromycin at 37°C and 5% CO2 humidified environment. The cells were cultured for 2 weeks and passaged 3 times before harvesting. Before injection, Daudi-luc cells were resuspended in HBSS. 10 million cells / 0.2ml were injected intravenously into the tail vein of each mouse.

[0219] Female NOD SCID mice (Charles River) at 6 weeks of age were used for Daudi subcutaneous xenografts and Daudi-luc intravenous xenografts. In the study, female Nu / Nu mice (Charles River) at 6 weeks of age were used for Ramos subcutaneous xenografts. Upon receipt, mice were housed in an environmentally controlled room with 5 mice per cage. Rodent food and water were provided ad libitum. Before dosing began, mice were acclimated to laboratory conditions for 72 hours. During the acclimation period, the health of the animals was monitored. Each cage was identified by group number and study number, and mice were identified by ear tags.

[0220] The table below shows the study design and dosing schedule.

[0221]

[0222] Tumor growth was monitored starting on days 5–7 post-inoculation by measuring tumor width and length using digital calipers and twice weekly thereafter until tumor volumes reached ∼100–250 mm 3 Tumor volume was calculated using the following formula: Volume (mm 3 )=[length (mm) x width (mm) 2 ] / 2.

[0223] Once the tumors were staged to the desired size, the animals were randomized and mice with very large or very small tumors were removed. The mice were divided into groups with the number of animals in each group as indicated in the study design. The mice were then treated intravenously with PBS, Ab, ADC#45, or ADC#41 (0.2 ml / animal). After treatment, tumor growth, animal health, and body weight were monitored. When the mean subcutaneous tumor burden in the group exceeded 2000 mm 3 , when the body weight loss of the animals exceeded 20% or at the end of the study, the test animals were sacrificed.

[0224] During the whole experiment, tumor volume was measured twice a week. TGI (tumor growth inhibition %) was calculated using the following formula: TGI = [(the volume of the treatment group on the same day of the last volume size of the PBS group - PBS control) / (the last volume size of the PBS group)] x 100. The body weight of each mouse was measured twice a week by electrical balance.

[0225] The raw data of individual body weight and tumor volume were calculated. The mean and standard deviation of the group were calculated and statistical analysis was performed (using one-way ANOVA and Dunnett's multiple comparison test; GraphPad Prism 6.0). All treatment groups were compared with the PBS group. P < 0.05 was considered statistically significant.

[0226] ADC#45 at 10mg / kg significantly inhibited Daudi tumor growth compared to the PBS-treated control group. Although tumor growth resumed after 3 weeks, a single 10mg / kg treatment significantly delayed tumor growth. In this setting, multiple treatments can be tested to achieve sustained tumor inhibition. A single dose of ADC#45 at 3mg / kg or 1mg / kg did not significantly inhibit tumor growth. However, although the difference was not significant, a single dose of ADC#45 did show a slight inhibition of tumor growth compared to the PBS-treated control group. A dose response was observed in this study, with higher doses showing better tumor growth inhibition ( Figure 1 A). Test animals administered a single intravenous dose of ADC#45 at 10 mg / kg or lower did not lose weight ( Figure 1 B).

[0227] Similarly, ADC#45 at 10 mg / kg significantly inhibited the growth of Ramos tumors compared to the PBS-treated control group, with sustained tumor inhibition for up to 60 days. A single dose of ADC#45 at 3 mg / kg or 1 mg / kg did not significantly inhibit tumor growth. However, although the difference was not significant, a single dose of ADC#45 at 3 mg / kg or 1 mg / kg did show a slight inhibition of tumor growth compared to the PBS-treated control group. A dose response was observed in this study, with higher doses showing better tumor growth inhibition ( Figure 2 A). Test animals administered a single intravenous dose of ADC#45 at 10 mg / kg or lower did not lose weight ( Figure 2 B).

[0228] A single dose of 3 mg / kg of ADC#45 completely inhibited tumor growth, and the survival rate was 100% until day 48 after treatment. Compared with the PBS control group, a single dose of 3 mg / kg of ADC#41 significantly inhibited tumor growth and significantly prolonged the survival of mice ( Figure 3 and 4 ).

[0229] A single dose of 10 mg / kg of ADC#45 and ADC#41 significantly inhibited tumor growth, while at doses of 3 mg / kg or 1 mg / kg, neither showed significant tumor inhibition in the Daudi and Ramos subcutaneous xenograft tumor mouse models. In the Daudi-luc intravenous tumor model in female NOD SCID mice, a single dose of 3 mg / kg of ADC#45 completely inhibited tumor growth, and the survival rate was 100% until 48 days. In the Daudi-luc intravenous tumor model in female NOD SCID mice, a single dose of 3 mg / kg of ADC#41 significantly inhibited tumor growth and prolonged survival. A dose response of ADC#45 and ADC#41 was observed in this study. At the same (10 mg / kg or 3 mg / kg) dose regimen, ADC#45 showed better tumor growth inhibition than ADC#41. No treatment-related weight loss was observed in all treatment groups during the study.

[0230] Sequence Listing

[0231]

Claims

1. An anti-CD38 ADC composition, comprising: (a) an anti-CD38 IgG antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO.1 and the light chain variable region has the sequence shown in SEQ ID NO.3, or wherein the heavy chain variable region has the sequence shown in SEQ ID NO.1 and the light chain variable region has the sequence shown in SEQ ID NO.2; (b) a drug or toxic substance portion, wherein the drug or toxic substance portion is a tubulin inhibitor or a doxorubicin analog, and the drug or toxic substance portion is selected from the group consisting of D1, D2, D3, D4, D5 and a combination thereof; and (c) a conjugate linker moiety, wherein the conjugate linker comprises a linker and a conjugate moiety, the conjugate moiety being covalently bound to a single Cys residue in the hinge region of each of the two heavy chains of the antibody, wherein the heavy chain hinge region of the IgG antibody is mutated so that each of the heavy chain hinge regions contains only one Cys residue; wherein the conjugated moiety is wherein the wavy line indicates the site of attachment to the linker, and the asterisk (*) indicates the site of attachment to the sulfur atom of the Cys residue in the hinge region of the antibody; The structures of D1, D2, D3, D4 and D5 are as follows: and Wherein the linker is selected from: and any combination thereof, wherein the wavy lines indicate the sites of attachment to the conjugate moiety and the drug or toxic substance moiety.

2. The ADC composition according to claim 1, wherein the ADC is selected from 3. The ADC composition according to claim 1, wherein the ADC is 4. The ADC composition according to claim 1, wherein the ADC is 5. The ADC composition according to claim 1, wherein the ADC is 6. The ADC composition according to claim 1, wherein the ADC is 7. The ADC composition according to claim 1, wherein the ADC is 8. The ADC composition according to claim 1, wherein the ADC is 9. Use of an anti-CD38 ADC composition in the preparation of a medicament for treating multiple myeloma, the treatment comprising providing a therapeutically effective amount of an anti-CD38 ADC composition, the anti-CD38 ADC composition comprising: (a) an anti-CD38 IgG antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO.1 and the light chain variable region has the sequence shown in SEQ ID NO.3, or wherein the heavy chain variable region has the sequence shown in SEQ ID NO.1 and the light chain variable region has the sequence shown in SEQ ID NO.2; (b) a drug or toxic substance portion, wherein the drug or toxic substance portion is a tubulin inhibitor or a doxorubicin analog, and the drug or toxic substance portion is selected from the group consisting of D1, D2, D3, D4, D5 and a combination thereof; and (c) a conjugate linker moiety, wherein the conjugate linker comprises a linker and a conjugate moiety, the conjugate moiety being covalently bound to a single Cys residue in the hinge region of each of the two heavy chains of the antibody; wherein the heavy chain hinge region of the IgG antibody is mutated so that each of the heavy chain hinge regions contains only one Cys residue; wherein the conjugated moiety is wherein the wavy line indicates the site of attachment to the linker, and the asterisk (*) indicates the site of attachment to the sulfur atom of the Cys residue in the hinge region of the antibody; The structures of D1, D2, D3, D4 and D5 are as follows: and Wherein the linker is selected from: and any combination thereof, wherein the wavy lines indicate the sites of attachment to the conjugate moiety and the drug or toxic substance moiety.

10. The use according to claim 9, wherein the ADC is selected from

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